3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

  • Sarah C Grünert1, 2,

    Affiliated with

    • Martin Stucki1, 3,

      Affiliated with

      • Raphael J Morscher1,

        Affiliated with

        • Terttu Suormala1, 4,

          Affiliated with

          • Celine Bürer1,

            Affiliated with

            • Patricie Burda1,

              Affiliated with

              • Ernst Christensen5,

                Affiliated with

                • Can Ficicioglu6,

                  Affiliated with

                  • Jürgen Herwig7,

                    Affiliated with

                    • Stefan Kölker8,

                      Affiliated with

                      • Dorothea Möslinger9,

                        Affiliated with

                        • Elisabetta Pasquini10,

                          Affiliated with

                          • René Santer11,

                            Affiliated with

                            • K Otfried Schwab2,

                              Affiliated with

                              • Bridget Wilcken12,

                                Affiliated with

                                • Brian Fowler1, 4,

                                  Affiliated with

                                  • Wyatt W Yue13 and

                                    Affiliated with

                                    • Matthias R Baumgartner1, 3Email author

                                      Affiliated with

                                      Orphanet Journal of Rare Diseases20127:31

                                      DOI: 10.1186/1750-1172-7-31

                                      Received: 2 February 2012

                                      Accepted: 10 April 2012

                                      Published: 29 May 2012

                                      Abstract

                                      Background

                                      Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the α and β subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults.

                                      Methods

                                      We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby.

                                      Results

                                      Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date.

                                      Conclusions

                                      Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.

                                      Keywords

                                      3-Methylcrotonyl-CoA carboxylase MCCC1 MCCC2 Biotin Inborn error Organic aciduria Newborn screening

                                      Background

                                      Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency (MIM#s 210200 and 210210) is an autosomal recessive disorder of leucine metabolism [1]. The mitochondrial enzyme MCC (EC 6.4.1.4) catalyzes the fourth step in the leucine catabolic pathway and belongs to the family of biotin-dependent carboxylases, including acetyl-CoA carboxylase (ACC), propionyl-CoA carboxylase (PCC) and pyruvate carboxylase (PC) [1]. MCC consists of an alpha and a beta subunit assembled into a α6β6 dodecamer. The larger α subunit harbours the biotin carboxylase (BC) domain and the biotin carboxyl carrier protein domain covalently bound with a biotin prosthetic group, while the smaller β subunit contains the carboxyltransferase (CT) domain.

                                      Isolated MCC deficiency is caused by mutations in the MCCC1 (formerly MCCA) or the MCCC2 (formerly MCCB) gene coding for the α and β subunit, respectively [24]. Human MCCC1 has 19 exons and maps to chromosome region 3q25-q27, MCCC2 consists of 17 exons and has been located to chromosome region 5q12-q13 [24]. A total of 49 MCCC1 and 52 MCCC2 mutations have been reported so far with the majority being missense mutations along with small insertions/deletions, nonsense, frameshift, and splice site mutations [212].

                                      Increased urinary levels of 3-hydroxyisovaleric acid (3-HIVA) and 3-methylcrotonylglycine (3-MCG) are usually found in isolated MCC deficiency. Additionally, 3-hydroxyisovalerylcarnitine (C5OH) is characteristically present in blood and urine. Many patients also develop a severe secondary carnitine deficiency [1]. Surprisingly, MCC deficiency was found to be the most frequent organic aciduria detected in tandem mass spectrometry based newborn screening (NBS) programs in North America [13, 14], Europe [15, 16] and Australia [17].

                                      The clinical picture of MCC deficiency is heterogeneous and often highly variable even within the same family [10, 18]. The phenotype ranges from neonatal onset with severe neurological involvement and even lethal cases [1921] to asymptomatic adults [3, 6, 11, 22]. Some patients develop an acute metabolic crisis usually triggered by intercurrent infections or introduction of a protein-rich diet in early childhood. Symptoms include vomiting, opisthotonus, involuntary movements, seizures, coma and apnoea typically associated with metabolic acidosis, hypoglycemia and in some cases mild hyperammonemia [3, 7, 2327]. Others present with neurological abnormalities such as seizures, muscular hypotonia or developmental delay [6, 18, 2831].

                                      In contrast, the majority of children diagnosed by NBS have been reported to have remained asymptomatic so far [6, 7, 11, 32]. Moreover, several asymptomatic MCC-deficient mothers have been identified only by detection of abnormal metabolites in the neonatal-screening sample from their healthy babies [3, 6, 11, 22] and a number of asymptomatic affected siblings have been identified by family screening [3335]. The comparative analysis of published case reports with German NBS data indicated that probably less than 10% of affected individuals develop symptoms [6]. Therefore, MCC deficiency may be considered to be a genetic condition with low penetrance.

                                      Therapeutic approaches comprise supplementation with oral L-carnitine and a diet modestly restricted in leucine but the efficacy of these approaches is unproven [36].

                                      Here we summarize clinical, biochemical, enzymatic and molecular genetic data of 88 MCC-deficient individuals, present an update of all MCCC1 and MCCC2 mutations reported to date including 15 novel MCCC1 and 16 novel MCCC2 mutations and show expression studies of 10 missense mutations and one small deletion.

                                      Patients and methods

                                      Patients

                                      Eighty-eight subjects with MCC deficiency from 78 families (10 sib-pairs) were included in this study (Tables 1, 2, 3 and 4). Cultured fibroblasts (n = 69) or genomic DNA (n = 16) from 85 individuals were sent to our laboratory for confirmation of MCC deficiency. In the remaining 3 subjects mutation analysis was performed in a genetic laboratory in the USA. Forty-five subjects were male, 40 were female; the sex of 3 individuals was not reported. Of the 88 individuals 45 (51%) were Caucasian, 27 (31%) Turkish, 8 (9%) Arab, 6 (7%) Asian, one (1%) African-American, and one patient (1%) was of mixed African Caucasian ancestry.
                                      Table 1

                                      Sociodemographic, biochemical, enzymatic, genetic and clinical information on 88 patients with MCC deficiency 53 Individuals identified by newborn screening without (n = 36) and with (n = 13) reported symptoms (n = 4 without clinical details)

                                      Pt #

                                      Sex

                                      Ethnic origin

                                      Current age (y)

                                      Biochemical phenotype

                                      Carboxylase activities in fibroblasts (pmol/ min/mg protein)1

                                      Genotype

                                      Clinical phenotype§

                                      DBS/ plasma

                                      urine

                                      affected gene

                                      Nucleotide change (at RNA level)

                                      Amino acid change (predicted from RNA)

                                      C5OH

                                      3-HIVA

                                      3MCG

                                      MCC

                                      PCC

                                      Allele 1 Allele 2

                                      20

                                      f

                                      Caucasian

                                      10

                                      ++

                                      ++

                                      ++

                                      15.4

                                      812

                                      MCCC1

                                      c.1155A>C

                                      p.R385S

                                      asymptomatic (fr)

                                                

                                      c.559T>C

                                      p.S187P

                                       

                                      21

                                      f

                                      Turkish

                                      11

                                      ++

                                      ++

                                      ++

                                      0

                                      530

                                      MCCC2

                                      c.803G>C

                                      p.R268T

                                      asymptomatic (ltf, 0.3 y)

                                                

                                      (r.785_803del)

                                      (p.G262_R268delfs*5)

                                       
                                                

                                      c.803G>C

                                      p.R268T

                                       
                                                

                                      (r.785_803del)

                                      (p.G262_R268delfs*5)

                                       

                                      22

                                      f

                                      Turkish

                                      12

                                      ++

                                      ++

                                      ++

                                      5.7

                                      587

                                      MCCC2

                                      c.464G>A

                                      p.R155Q

                                      asymptomatic (fr)

                                                

                                      c.464G>A

                                      p.R155Q

                                       

                                      23

                                      f

                                      Arab

                                      12

                                      ++

                                      ++

                                      ++

                                      0

                                      594

                                      MCCC2

                                      c.469C>T

                                      p.Q157*

                                      asymptomatic (ltf, 1y)

                                                

                                      c.469C>T

                                      p.Q157*

                                       

                                      25

                                      m

                                      Caucasian

                                      11

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC1

                                      c.872C>T◊ -

                                      p.A291V -

                                      asymptomatic (ltf)

                                      26

                                      f

                                      Caucasian

                                      9

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC2

                                      c.1690T>C◊ -

                                      p.X564QLE -

                                      asymptomatic (fr)

                                      27

                                      f

                                      Caucasian

                                      10

                                      ++

                                      ++

                                      ++

                                      1.1

                                      305

                                      MCCC1

                                      c.1155A>C◊ -

                                      R385S -

                                      asymptomatic, but facial dysmorphies with hypertelorism, mongoloid palpebral fissures, low set ears, mild macroglossia, normal karyotype 46XX (ltf, 0.3 y)

                                      29

                                      m

                                      Turkish

                                      10

                                      ++

                                      ++

                                      ++

                                      1.5

                                      723

                                      MCCC2

                                      c.295G>C

                                      p.E99Q

                                      asymptomatic (ltf, 6y)

                                                

                                      c.1574+1G>A

                                      (p.F497Gfs*4)

                                       

                                      34

                                      m

                                      Caucasian

                                      9

                                      ++

                                      ++

                                      ++

                                      16.2

                                      542

                                      MCCC2

                                      c.845A>G

                                      p.H282R

                                      asymptomatic (fr)

                                                

                                      c.845A>G

                                      p.H282R

                                       

                                      39

                                      f

                                      Caucasian

                                      9

                                      ++

                                      ++

                                      ++

                                      0.7

                                      696

                                      MCCC2

                                      c.517dupT

                                      p.S173Ffs*25

                                      asymptomatic (fr)

                                                

                                      c.1123G>T

                                      p.V375F

                                       

                                      40

                                      m

                                      Caucasian

                                      9

                                      ++

                                      ++

                                      ++

                                      5.1

                                      620

                                      MCCC2

                                      c.214C>T

                                      p.R72*

                                      asymptomatic (fr)

                                                

                                      c.416_ 427del12ins16

                                      p.T139_G143 >RWVPGEfs*35

                                       

                                      41

                                      m

                                      Caucasian

                                      8

                                      ++

                                      ++

                                      ++

                                      0

                                      595

                                      MCCC1

                                      c.694C>T◊ -

                                      p.R232W -

                                      asymptomatic, mild developmental delay within the first years of life, normal development at present (fr)

                                      43a

                                      f

                                      Caucasian

                                      8

                                      ++

                                      ++

                                      ++

                                      8.1

                                      704

                                      MCCC1

                                      c.640_641delGG

                                      p.G214Nfs*5

                                      asymptomatic (ltf)

                                                

                                      c.1930G>T

                                      p.E644*

                                       

                                      43b

                                      f

                                      Caucasian

                                      7

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC1

                                      c.640_641delGG

                                      p.G214Nfs*5

                                      asymptomatic (ltf)

                                                

                                      c.1930G>T

                                      p.E644*

                                       

                                      52a

                                      m

                                      Turkish

                                      9

                                      +

                                      +

                                      ++

                                      1.6

                                      603

                                      MCCC2

                                      c.803G>C

                                      p.R268T

                                      asymptomatic (ltf)

                                                

                                      (r.785_803del)

                                      (p.G262_ R268delfs*5)

                                       
                                                

                                      c.803G>C

                                      p.R268T

                                       
                                                

                                      (r.785_803del)

                                      (p.G262_R268delfs*5)

                                       

                                      55

                                      m

                                      Asian

                                      7

                                      ++

                                      ++

                                      na

                                      18.3

                                      634

                                      MCCC2

                                      c.351_353delTGG

                                      p.G118del

                                      asymptomatic (ltf, 0.6y)

                                                

                                      c.659G>A

                                      p.G220E

                                       

                                      56

                                      m

                                      Turkish

                                      7

                                      +

                                      +

                                      ++

                                      9.6

                                      359

                                      MCCC2

                                      c.1567A>G

                                      p.S523G

                                      asymptomatic (fr)

                                                

                                      (exon 6 skipping)

                                      (p.V171Dfs*20)

                                       

                                      57

                                      m

                                      Asian

                                      7

                                      ++

                                      ++

                                      ++

                                      10.2

                                      541

                                      MCCC1

                                      c.863A>G

                                      p.E288G

                                      asymptomatic (fr)

                                                

                                      c.863A>G

                                      p.E288G

                                       

                                      58

                                      m

                                      Turkish

                                      7

                                      ++

                                      ++

                                      ++

                                      5.2

                                      1046

                                      MCCC2

                                      c.538C>T

                                      p.R180*

                                      asymptomatic (fr)

                                                

                                      c.538C>T

                                      p.R180*

                                       

                                      62

                                      m

                                      Turkish

                                      7

                                      ++

                                      ++

                                      ++

                                      9.5

                                      856

                                      MCCC1

                                      c.873+4524_ 6787del2264

                                      p.?

                                      asymptomatic (fr)

                                                

                                      c.873+4524_ 6787del2264

                                      p.?

                                       

                                      64

                                      f

                                      Turkish

                                      8

                                      ++

                                      ++

                                      ++

                                      9.9

                                      762

                                      MCCC2

                                      c.803G>C

                                      p.R268T

                                      asymptomatic (ltf, 5y)

                                                

                                      (r.785_803del)

                                      (p.G262_R268delfs*5)

                                       
                                                

                                      c.803G>C

                                      p.R268T

                                       
                                                

                                      (r.785_803del)

                                      (p.G262_R268delfs*5)

                                       

                                      67

                                      f

                                      Turkish

                                      10

                                      ++

                                      na

                                      na

                                      48.4

                                      1065

                                      MCCC2

                                      c.464G>A

                                      p.R155Q

                                      asymptomatic (fr)

                                                

                                      c.1015G>A

                                      p.V339M

                                       

                                      70a

                                      f

                                      Caucasian

                                      6

                                      +

                                      ++

                                      ++

                                      0

                                      520

                                      MCCC1

                                      c.2079delA◊ -

                                      p.V694* -

                                      asymptomatic (fr)

                                      72

                                      m

                                      Caucasian

                                      7

                                      ++

                                      ++

                                      ++

                                      11.5

                                      451

                                      MCCC2

                                      c.455A>C

                                      p.K152T

                                      asymptomatic (ltf)

                                                

                                      c.903+6_ 903+9delTACG

                                      p.?

                                       

                                      78

                                      f

                                      Caucasian

                                      6

                                      ++

                                      ++

                                      ++

                                      4.8

                                      416

                                      MCCC2

                                      c.671C>T

                                      p.P224L

                                      asymptomatic (fr)

                                                

                                      c.671C>T

                                      p.P224L

                                       

                                      82a

                                      f

                                      Caucasian

                                      5

                                      ++

                                      ++

                                      ++

                                      1.6

                                      783

                                      MCCC2

                                      c.512-1G>A

                                      p.?

                                      asymptomatic (ltf, 1y)

                                                

                                      c.512-1G>A

                                      p.?

                                       

                                      82b

                                      f

                                      Caucasian

                                      5

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC2

                                      c.512-1G>A

                                      p.?

                                      asymptomatic (ltf, 1y)

                                                

                                      c.512-1G>A

                                      p.?

                                       

                                      91

                                      m

                                      Turkish

                                      7

                                      ++

                                      +

                                      +

                                      35.5

                                      513

                                      MCCC2

                                      c.295G>C

                                      p.E99Q

                                      asymptomatic (fr)

                                                

                                      c.295G>C

                                      p.E99Q

                                       

                                      93b

                                      m

                                      Caucasian

                                      8

                                      ++

                                      na

                                      +

                                      na

                                      na

                                      na

                                      na

                                      na

                                      asymptomatic (ltf)

                                      107

                                      m

                                      Caucasian

                                      2

                                      ++

                                      ++

                                      ++

                                      4.0

                                      613

                                      MCCC2

                                      c.1073-12C>G

                                       

                                      asymptomatic (fr)

                                                

                                      (r.1073_1216del+ r.1073insr.1073-48_ r.1073-1)

                                      (p.G358Vfs*6+ p.G358Afs*12)

                                       
                                                

                                      c.1073-12C>G

                                        
                                                

                                      (r.1073_1216del+ r.1073insr.1073-48_ r.1073-1)

                                      (p.G358Vfs*6+ p.G358Afs*12)

                                       

                                      112

                                      m

                                      Turkish

                                      0.8

                                      ++

                                      ++

                                      ++

                                      0

                                      797

                                      MCCC2

                                      c.658_662delTCAGA c.658_662delTCAGA

                                      p.S220Tfs*15 p.S220Tfs*15

                                      asymptomatic, however hyperammonemia of 270 umol/l under leucine loading test (fr)

                                      115

                                      f

                                      Caucasian

                                      0.7

                                      ++

                                      ++

                                      ++

                                      5.0

                                      864

                                      MCCC1

                                      c.803C>A

                                      p.A268D

                                      asymptomatic (fr)

                                                

                                      c.1155A>C

                                      p.R385S

                                       

                                      125

                                      f

                                      Arab

                                      3

                                      +

                                      n

                                      na

                                      102

                                      726

                                      MCCC2

                                      c.1423G>A

                                      p. G475R

                                      asymptomatic (fr)

                                                

                                      c.1423G>A

                                      p. G475R

                                       

                                      126

                                      m

                                      Caucasian

                                      3

                                      +

                                      +

                                      na

                                      60.6

                                      791

                                      MCCC2

                                      c.1300G>C

                                      p.V434L

                                      asymptomatic (fr)

                                                

                                      c.1300G>C

                                      p.V434L

                                       

                                      137

                                      m

                                      Caucasian

                                      5

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC2

                                      c.518C>T

                                      p.S173L

                                      asymptomatic (fr)

                                                

                                      c.518C>T

                                      p.S173L

                                       

                                      138

                                      m

                                      Caucasian

                                      1.5

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC1

                                      c.1155A>C

                                      p.R385S

                                      asymptomatic (fr)

                                                

                                      c.2009_2043del35

                                      p.A670Dfs*34

                                       

                                      24

                                      f

                                      Turkish

                                      10

                                      ++

                                      ++

                                      ++

                                      1.8

                                      390

                                      MCCC2

                                      c.295G>C c.295G>C

                                      p.E99Q p.E99Q

                                      attention deficit hyperactivity disorder (fr)

                                      28

                                      m

                                      Caucasian

                                      10

                                      ++

                                      ++

                                      ++

                                      1.1

                                      318

                                      MCCC1

                                      c.1155A>C (exon 15 skipping)

                                      p.R385S (p.V562*)

                                      attention deficit hyperactivity disorder (fr)

                                      46

                                      m

                                      Caucasian/ African American

                                      9

                                      ++

                                      ++

                                      ++

                                      20.0

                                      1054

                                      MCCC1

                                      c.2088dupA c.1526delG

                                      p.V697Sfs*19 p.C509Sfs*14

                                      3 metabolic decompensations with vomiting, hypoglycaemia and ketonuria (ltf, 7.5y)

                                      53

                                      f

                                      Caucasian

                                      7

                                      +

                                      ++

                                      ++

                                      28.4

                                      773

                                      MCCC1

                                      c.1155A>C c.1315G>A

                                      R385S p.V439M

                                      at the age of 6 months minor psychomotor delay (ltf)

                                      59

                                      m

                                      Faroe Islands

                                      7

                                      ++

                                      ++

                                      ++

                                      7.4

                                      1051

                                      MCCC1

                                      c.1526delG c.1526delG

                                      p.C509Sfs*14 p.C509Sfs*14

                                      muscular hypotonia, muscle wakness, impaired physical performance (fr)

                                      71

                                      f

                                      Turkish

                                      died at 5 weeks

                                      ++

                                      ++

                                      ++

                                      1.7

                                      597

                                      MCCC1

                                      c.1136G>A c.1136G>A

                                      p.G379D p.G379D

                                      metabolic crisis, floppy infant, myoclonic jerks, respiratory insufficiency requiring mechanical ventilation, deceased at age 6 weeks

                                      74

                                      m

                                      African American

                                      6

                                      ++

                                      +

                                      +

                                      23.0

                                      749

                                      MCCC1

                                      c.1302T>G c.2123dupA

                                      p.I434M p.H708Qfs*8

                                      several metabolic decompensations, mild speech delay, immunodeficiency due to CD 16 deficiency (fr)

                                      81

                                      f

                                      Caucasian

                                      5

                                      +

                                      +

                                      n

                                      21.5

                                      233

                                      MCCC2

                                      c.1015G>A◊ -

                                      p.V339M -

                                      Trisomy 21, psychomotor retardation, muscular hypotonia (fr)

                                      90

                                      m

                                      Turkish

                                      7

                                      +

                                      +

                                      +

                                      23.8

                                      483

                                      MCCC2

                                      c.295G>C c.1015G>A

                                      p.E99Q p.V339M

                                      truncal and perioral hypotonia (fr)

                                      105

                                      m

                                      Caucasian

                                      3

                                      ++

                                      ++

                                      ++

                                      0

                                      412

                                      MCCC1

                                      c.1155A>C c.1820delG

                                      p.R385S p.S607Ifs*5

                                      unpleasant odour, failure to thrive, several acute metabolic decompensations with mild hyperammonemia during infections (fr)

                                      108

                                      m

                                      Asian

                                      2.5

                                      ++

                                      ++

                                      ++

                                      0.8

                                      456

                                      MCCC2

                                      c.518C>T c.518C>T

                                      p.S173L p.S173L

                                      recurrent infections, muscular hypertonia and hyperreflexia in infancy (fr)

                                      127

                                      m

                                      Arab

                                      2

                                      +

                                      na

                                      na

                                      92,9

                                      755

                                      MCCC2

                                      c.1423G>C

                                      p.G475R

                                      muscle weakness (fr)

                                                

                                      c.1423G>C

                                      p.G475R

                                       

                                      136

                                      f

                                      Caucasian

                                      8

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC2

                                      c.1149+1G>T c.1149+1G>T

                                      p.? p.?

                                      3 metabolic decompensations with acidosis, hypoglycaemia, vomiting, encephalopathy and coma (fr)

                                      31

                                      f

                                      Caucasian

                                      10

                                      na

                                      na

                                      na

                                      12.4

                                      518

                                      MCCC1

                                      c.1155A>C

                                      p.R385S

                                      ?

                                                

                                      c.400G>A

                                      p.E134K

                                       

                                      103

                                      m

                                      Caucasian

                                      3

                                      ++

                                      ++

                                      ++

                                      0

                                      545

                                      MCCC2

                                      (exon 7 to 14 skipping)

                                      (p.I209Pfs*43)

                                      ?

                                                

                                      (exon 7 to 14 skipping)

                                      (p.I209Pfs*43)

                                       

                                      111

                                      ?

                                      Caucasian

                                      1

                                      ++

                                      +

                                      +

                                      34.0

                                      1083

                                      MCCC2

                                      c.1015G>A

                                      p.V339M

                                      ?

                                                

                                      c.1309A>G

                                        
                                                

                                      (r.1309A>G+ r.1310_1373del64)

                                      (p.I437V+ p.I437Tfs*15)

                                       

                                      113

                                      ?

                                      Caucasian

                                      1

                                      +

                                      +

                                      n

                                      7.9

                                      407

                                      MCCC1

                                      c.193A>T

                                      p.M65L

                                      ?

                                                

                                      c.1193_1194delTG

                                      p.V398Gfs*19

                                       

                                      1 control values measured in 53 cell lines, expressed as median value and (range): MCC activity, 305 pmol/min/mg protein (134-671); PCC activity, 583 (208-1165); ratio of PCC/MCC activity, 1.93 (1.19 – 2.58).

                                      §information in brackets: fr followed regularly, ltf lost to follow-up, age of last follow-up, if known. + slightly elevated; ++ massively elevated; C5OH 3-hydroxyisovalerylcarnitine; DBS dried blood spots; 3-HIVA 3-hydroxyisovaleric acid; 3-MCG 3-methylcrotonylglycine; f female; fr followed regularly; ltf lost to follow-up; m male; MCC methylcrotonyl-CoA carboxylase; n normal; na not available; NBS newborn screening; PCC propionyl-CoA carboxylase; Pt # Patient number; RNA nd RNA not detectable; SMS selective metabolic screening; y years; ◊ mutation heterozygous on genomic PCR, homozygous in RT-PCR; # diagnosed following the positive NBS result of their baby; ? not known.

                                      Table 2

                                      Sociodemographic, biochemical, enzymatic, genetic and clinical information on 88 patients with MCC deficiency 18 individuals identified by selective metabolic screening due to clinical symptoms (n = 17, no clinical details in n = 1)

                                      Pt #

                                      Sex

                                      Ethnic origin

                                      age at diagnosis

                                      current age (y)

                                      Biochemical phenotype

                                      Carboxylase activities in fibroblasts (pmol/min/mg protein)1

                                      Genotype

                                      Clinical phenotype§

                                      DBS/ plasma

                                      urine

                                      affected gene

                                      Nucleotide change (at RNA level)

                                      Amino acid change (predicted from RNA)

                                      C5OH

                                      3-HIVA

                                      3MCG

                                      MCC

                                      PCC

                                      Allele 1 Allele 2

                                      30

                                      m

                                      Turkish

                                      newborn

                                      died at 33 days

                                      ++

                                      ++

                                      ++

                                      0

                                      637

                                      MCCC2

                                      c.1574+1G>A c.1574+1G>A

                                      (p.F497Gfs*4) (p.F497Gfs*4)

                                      acute decompensation on first day of life, acidosis, hypoglycaemia, hyperlactemia, hyperammonemia, encephalopathy, depressed neonatal reflexes, hypertonic episodes, prominent hypotonia, respiratory insufficiency requiring assisted ventilation, cardiac arrest, patient deceased on day 33

                                                   

                                      CT scan of the brain: multiple cysts, ventricular dilatation, cerebral atrophy

                                      32a

                                      m

                                      Arab

                                      4 years

                                      14

                                      ++

                                      ++

                                      ++

                                      5.0

                                      863

                                      MCCC2

                                      c.127C>T c.127C>T

                                      p.Q43* p.Q43*

                                      muscular hypotonia, weakness, mild motor delay (fr)

                                      35a

                                      m

                                      Caucasian

                                      9 months

                                      16

                                      na

                                      ++

                                      ++

                                      7.3

                                      976

                                      MCCC2

                                      (exon 7 to 14 skipping) (exon 7 to 14 skipping)

                                      (p.I209Pfs*43) (p.I209Pfs*43)

                                      developmental delay, familial nystagmus, hyperopia, significant hand tremor, mild learning disability, failure to thrive, unpleasant odour descibed as "smelling like cat`s urine", hypothermia, ketonuria, hypoglycemia and mild hyperammonemia prior to stabilisation on dietary therapy (ltf, 3y)

                                      36

                                      f

                                      Turkish

                                      3 years

                                      11

                                      ++

                                      ++

                                      ++

                                      0.4

                                      420

                                      MCCC1

                                      c.1527C>A c.1527C>A

                                      p.C509* p.C509*

                                      mental and speech retardation, spasticity, impaired physical performance (ltf)

                                      42

                                      f

                                      Caucasian

                                      ?

                                      24

                                      ++

                                      ++

                                      ++

                                      0

                                      664

                                      MCCC2

                                      c.929C>G c.929C>G

                                      p.P310R p.P310R

                                      severe muscular weakness, muscle pain (ltf, 16y)

                                      44

                                      m

                                      Caucasian

                                      1.5 years

                                      10

                                      na

                                      ++

                                      ++

                                      4.0

                                      425

                                      MCCC2

                                      c.463C>T c.463C>T

                                      p.R155W p.R155W

                                      psychomotor retardation, seizures, muscular hypotonia, metabolic stroke, failure to thrive, clinodactyly of the 5th fingers (fr)

                                      50

                                      f

                                      Arab

                                      13 years

                                      21

                                      na

                                      ++

                                      ++

                                      8.1

                                      761

                                      MCCC1

                                      c.1882G>T c.1114C>T

                                      p.E628* p.Q372*

                                      mild Reye-like episode and encephalitis during Influenza A infection at age 5 years, mild learning disability, severe attention-deficit hyperactivity disorder, multiple sclerosis (fr)

                                      54

                                      m

                                      Asian

                                      ?

                                      13

                                      ++

                                      ++

                                      ++

                                      1.3

                                      1162

                                      MCCC1

                                      c.980C>G c.639+2T>A

                                      p.S327* p.S164Rfs*3

                                      psychomotor retardation, attention deficit hyperactivity disorder, frequent skin picking behaviour (ltf)

                                      60

                                      f

                                      Turkish

                                      ?

                                      10

                                      ++

                                      ++

                                      ++

                                      6.4

                                      754

                                      MCCC1

                                      c.2079delA c.2079delA

                                      p.V694* p.V694*

                                      mild global psychomotor retardation, convulsions starting at the age of 18 months during febrile episode, continued as generalized tonic clonic seizures after the age of 3 years, nephrolithiasis, episodes of hematuria (ltf, 4 y)

                                      63

                                      m

                                      Turkish

                                      ?

                                      8

                                      ++

                                      ++

                                      na

                                      12.0

                                      729

                                      MCCC2

                                      c.464G>A c.464G>A

                                      p.R155Q p.R155Q

                                      3 metabolic decompensations with encephalopathy, seizures, acidosis, hypoglycemia, mild developmental retardation

                                      68

                                      m

                                      Turkish

                                      3 years

                                      9

                                      ++

                                      ++

                                      ++

                                      2.4

                                      335

                                      MCCC1

                                      c.1155A>C c.1155A>C

                                      R385S R385S

                                      severe metabolic decompensation with metabolic stroke, cerebral edema and hemiparesis, mild psychomotor retardation, seizures (fr)

                                      77

                                      m

                                      Arab

                                      8 months

                                      9

                                      na

                                      ++

                                      ++

                                      0

                                      777

                                      MCCC2

                                      c.463C>T c.463C>T

                                      p.R155W p.R155W

                                      psychomotor and speech retardation, kyphoscolisis, genu varum, hypogammaglobulinemia, chronic diarrhea, reversible cytopenia under TPN (ltf, 7y)

                                      80

                                      m

                                      Turkish

                                      1.5 years

                                      9

                                      ++

                                      ++

                                      n (6m)++ (1y)

                                      22.8

                                      1162

                                      MCCC2

                                      c.116C>T c.116C>T

                                      p.S39F p.S39F

                                      speech retardation, seizures, recurring attacks of status epilepticus (ltf, 3y)

                                      89

                                      f

                                      Caucasian

                                      7 months

                                      10

                                      na

                                      na

                                      na

                                      17.0

                                      986

                                      MCCC2

                                      (exon 8 to 10 skipping)

                                      (p.K248_V334del)

                                      failure to thrive, poor feeding (ltf, 5y)

                                                 

                                      (exon 8 to 10 skipping)

                                      (p.K248_V334del)

                                       

                                      92

                                      m

                                      Caucasian

                                      1 week

                                      5

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC2

                                      c.710G>A c.1149+5G>C

                                      p.G237D p.?

                                      acute metabolic crisis, mild retardation (fr)

                                      96a

                                      m

                                      Turkish

                                      1 year

                                      6

                                      ++

                                      ++

                                      ++

                                      7.3

                                      1212

                                      MCCC1

                                      c.873+ 4524_6787del2264

                                      large deletion

                                      acidosis at 1 year of age, atonic seizures starting at 1 year of age (fr)

                                                 

                                      c.873+ 4524_6787del2264

                                      large deletion

                                       

                                      99a

                                      f

                                      Turkish

                                      8 years

                                      died at 8 years

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC2

                                      c.392G>T c.392G>T

                                      p.C131F p.C131F

                                      catecholaminergic ventricular tachycardia (mutation in RyR2 gene) sudden cardiac death at age 8 years

                                      69

                                      ?

                                      Arab

                                      ?

                                      9

                                      na

                                      na

                                      na

                                      18.9

                                      1210

                                      MCCC2

                                      c.1567A>G

                                      p.S523G

                                      ?

                                                 

                                      c.1567A>G

                                      p.S523G

                                       

                                      1 control values measured in 53 cell lines, expressed as median value and (range): MCC activity, 305 pmol/min/mg protein (134-671); PCC activity, 583 (208-1165); ratio of PCC/MCC activity, 1.93 (1.19 – 2.58).

                                      § information in brackets: fr followed regularly, ltf lost to follow-up, age of last follow-up, if known. + slightly elevated; ++ massively elevated; C5OH 3-hydroxyisovalerylcarnitine; DBS dried blood spots; 3-HIVA 3-hydroxyisovaleric acid; 3-MCG 3-methylcrotonylglycine; f female; fr followed regularly; ltf lost to follow-up; m male; MCC methylcrotonyl-CoA carboxylase; n normal; na not available; NBS newborn screening; PCC propionyl-CoA carboxylase; Pt # Patient number; RNA nd RNA not detectable; SMS selective metabolic screening; y years; ◊ mutation heterozygous on genomic PCR, homozygous in RT-PCR; # diagnosed following the positive NBS result of their baby; ? not known.

                                      Table 3

                                      Sociodemographic, biochemical, enzymatic, genetic and clinical information on 88 patients with MCC deficiency 8 individuals identified by family screening (asymptomatic individuals (n = 3), symptomatic individuals (n = 3), no clinical data (n = 2))

                                      Pt #

                                      Sex

                                      Ethnic origin

                                      Age at diagnosis

                                      Current age (y)

                                      Biochemical phenotype

                                      Carboxylase activities in fibroblasts (pmol/min/ mg protein)1

                                      Genotype

                                      Clinical phenotype§

                                      DBS/ plasma

                                      urine

                                      affected gene

                                      Nucleotide change (at RNA level)

                                      Amino acid change (predicted from RNA)

                                      C5OH

                                      3-HIVA

                                      3MCG

                                      MCC

                                      PCC

                                      Allele 1 Allele 2

                                      32b

                                      m

                                      Arab

                                      17 years

                                      28

                                      ++

                                      ++

                                      ++

                                      5.3

                                      409

                                      na

                                      na

                                      na

                                      asymptomatic (fr)

                                      93a

                                      m

                                      Caucasian

                                      4 years

                                      12

                                      ++

                                      +

                                      +

                                      19.0

                                      402

                                      MCCC1

                                      c.558delA

                                      p.Q186Hfs*6

                                      asymptomatic (ltf)

                                                 

                                      c.558delA

                                      p.Q186Hfs*6

                                       

                                      99b

                                      f

                                      Turkish

                                      5.5 years

                                      8

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      na

                                      na

                                      na

                                      asymptomatic (fr)

                                      70b

                                      m

                                      Caucasian

                                      3.5 years

                                      10

                                      +

                                      +

                                      ++

                                      na

                                      na

                                      na

                                      na

                                      na

                                      speech retardation, muscle weakness, hyperactivity, refusal of meat (fr)

                                      96c

                                      m

                                      Turkish

                                      3 years

                                      8

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC1

                                      c.873+4524_ 6787del2264

                                      large deletion

                                      mild speech retardation, macrocephaly (ltf)

                                                 

                                      c.873+4524_ 6787del2264

                                      large deletion

                                       

                                      35b

                                      f

                                      Caucasian

                                      18 months

                                      18

                                      na

                                      ++

                                      ++

                                      na

                                      na

                                      na

                                      na

                                      na

                                      psychomotor retardation (by 2 years developmental age of 10 months), failure to thrive, hypothermia and ketonuria prior to stabilisation on dietary therapy (ltf, 1.75y)

                                      52b

                                      m

                                      Turkish

                                      ?

                                      ?

                                      na

                                      na

                                      na

                                      na

                                      na

                                      MCCC2

                                      c.803G>C

                                      p.R268T

                                      ?

                                                 

                                      (r.785_803del)

                                      (p.G262_ R268delfs*5)

                                       
                                                 

                                      c.803G>C

                                      p.R268T

                                       
                                                 

                                      (r.785_803del)

                                      (p.G262_ R268delfs*5)

                                       

                                      52c

                                      m

                                      Turkish

                                      ?

                                      ?

                                      na

                                      na

                                      na

                                      na

                                      na

                                      MCCC2

                                      c.803G>C

                                      p.R268T

                                      ?

                                                 

                                      (r.785_803del)

                                      (p.G262_ R268delfs*5)

                                       
                                                 

                                      c.803G>C

                                      p.R268T

                                       
                                                 

                                      (r.785_803del)

                                      (p.G262_ R268delfs*5)

                                       

                                      1 control values measured in 53 cell lines, expressed as median value and (range): MCC activity, 305 pmol/min/mg protein (134-671); PCC activity, 583 (208-1165); ratio of PCC/MCC activity, 1.93 (1.19 – 2.58).

                                      §information in brackets: fr followed regularly, ltf lost to follow-up, age of last follow-up, if known. + slightly elevated; ++ massively elevated; C5OH 3-hydroxyisovalerylcarnitine; DBS dried blood spots; 3-HIVA 3-hydroxyisovaleric acid; 3-MCG 3-methylcrotonylglycine; f female; fr followed regularly; ltf lost to follow-up; m male; MCC methylcrotonyl-CoA carboxylase; n normal; na not available; NBS newborn screening; PCC propionyl-CoA carboxylase; Pt # Patient number; RNA nd RNA not detectable; SMS selective metabolic screening; y years; ◊ mutation heterozygous on genomic PCR, homozygous in RT-PCR; # diagnosed following the positive NBS result of their baby; ? not known.

                                      Table 4

                                      Sociodemographic, biochemical, enzymatic, genetic and clinical information on 88 patients with MCC deficiency Mothers identified following the positive newborn screening result of their offspring (n = 9)

                                      Pt #

                                      Sex

                                      Ethnic origin

                                      Age at diagnosis

                                      Current age (y)

                                      Biochemical phenotype

                                      Carboxylase activities in fibroblasts (pmol/min/mg protein)1

                                      Genotype

                                      Clinical phenotype§

                                      DBS/ plasma

                                      urine

                                      affected gene

                                      Nucleotide change (at RNA level)

                                      Amino acid change (predicted from RNA)

                                      C5OH

                                      3-HIVA

                                      3MCG

                                      MCC

                                      PCC

                                      Allele 1 Allele 2

                                      37

                                      f

                                      Asian

                                      32 years

                                      40

                                      ++

                                      ++

                                      ++

                                      9.6

                                      1268

                                      MCCC2

                                      c.1367C>T

                                      p.A456V

                                      asymptomatic (ltf)

                                                 

                                      c.1367C>T

                                      p.A456V

                                       

                                      51

                                      f

                                      Asian

                                      24 years

                                      32

                                      ++

                                      na

                                      na

                                      0

                                      475

                                      MCCC2

                                      c.351_353delTGG◊ -

                                      p.G118del -

                                      asymptomatic (ltf)

                                      73c

                                      f

                                      Faroe Islands

                                      29 years

                                      37

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC1

                                      c.1526delG

                                      p.C509Sfs*14

                                      asymptomatic (fr)

                                                 

                                      c.1526delG

                                      p.C509Sfs*14

                                       

                                      83

                                      f

                                      Caucasian

                                      ?

                                      38

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC1

                                      c.539G>T

                                      p.G180V

                                      asymptomatic (fr)

                                                 

                                      c.558delA

                                      p.Q186Hfs*6

                                       

                                      85

                                      f

                                      Caucasian

                                      38 years

                                      49

                                      ++

                                      +

                                      n

                                      na

                                      na

                                      MCCC2

                                      c.517dupT

                                      p.S173Ffs*25

                                      asymptomatic (ltf)

                                                 

                                      c.599T>A

                                      p.I200N

                                       

                                      100

                                      f

                                      Caucasian

                                      29 years

                                      34

                                      ++

                                      ++

                                      ++

                                      na

                                      na

                                      MCCC2

                                      c.505T>G

                                      p.Y169D

                                      asymptomatic (fr)

                                                 

                                      c.1073-12C>G

                                        
                                                 

                                      (r.1073_1216del+ r.1073insr.1073- 48_r.1073-1)

                                      (p.G358Vfs*6+ p.G358Afs*12)

                                       

                                      66

                                      f

                                      Caucasian

                                      34 years

                                      41

                                      +

                                      +

                                      ++

                                      10.0

                                      807

                                      MCCC2

                                      c.436T>Ac.416_427del12ins16

                                      p.Y146Np.T139_G143> RWVPGEfs*35

                                      several metabolic crises with hypoglycemia during febrile illnesses, metabolic stroke, cardiomopathy, paraesthesias (ltf)

                                      87

                                      f

                                      Faroe Islands

                                      28 years

                                      33

                                      ++

                                      n

                                      n

                                      13.0

                                      826

                                      MCCC1

                                      c.1526delGc.1526delG

                                      p.C509Sfs*14p.C509Sfs*14

                                      chronic tiredness (fr), otherwise asymptomatic

                                      33

                                      f

                                      Turkish

                                      36 years

                                      45

                                      ++

                                      ++

                                      ++

                                      4.6

                                      520

                                      MCCC2

                                      c.282-1G>C

                                      p.S95_G128del

                                      ?

                                                 

                                      c.282-1G>C

                                      p.S95_G128del

                                       

                                      1 control values measured in 53 cell lines, expressed as median value and (range): MCC activity, 305 pmol/min/mg protein (134-671); PCC activity, 583 (208-1165); ratio of PCC/MCC activity, 1.93 (1.19 – 2.58).

                                      § information in brackets: fr followed regularly, ltf lost to follow-up, age of last follow-up, if known.

                                      + slightly elevated.

                                      ++ massively elevated; C5OH 3-hydroxyisovalerylcarnitine; DBS dried blood spots; 3-HIVA 3-hydroxyisovaleric acid; 3-MCG 3-methylcrotonylglycine; f female; fr followed regularly; ltf lost to follow-up; m male; MCC methylcrotonyl-CoA carboxylase

                                      n normal; na not available; NBS newborn screening; PCC propionyl-CoA carboxylase; Pt # Patient number; RNA nd RNA not detectable; SMS selective metabolic screening; y years

                                      mutation heterozygous on genomic PCR, homozygous in RT-PCR; # diagnosed following the positive NBS result of their baby; ? not known.

                                      Clinical, biochemical, enzymatic or mutation data of 32 individuals have been reported earlier [proband 20-32a, 33-34, 36-43a, 44 and 46 [11], 30 [23], 35a and 35b [30], 42 [37], 44 [38], 50 [28], 80 [9], 81 [39], 96a and 96c [10], and 136 [26]].

                                      Clinical data

                                      A questionnaire was designed and sent out to the treating physicians. This questionnaire specifically addressed the mode of diagnosis, clinical symptoms, the psychomotor development, biochemical markers and long-term treatment regimens. Additionally, in some cases medical reports that were sent in together with the diagnostic samples were available and used for clinical data collection.

                                      Cell lines and enzyme assays

                                      Fibroblasts were cultured in a culture medium containing 10% foetal calf serum, and the activities of PCC and MCC were assayed simultaneously in crude fibroblast homogenates by measuring the incorporation of 14C-bicarbonate into acid-non-volatile products as described earlier [40].

                                      MCCC1 and MCCC2 mutation analysis by RT-PCR and genomic PCR

                                      After obtaining informed consent mutation analysis was performed in 83 individuals. The 5 individuals in whom no mutation analysis was performed were siblings of index cases and presented with a metabolite profile typical for MCC deficiency. In probands from whom RNA and DNA were available RT-PCR amplification and sequencing of the entire MCCC2 ORF was first performed and if no clearly pathogenic coding alterations were detected in MCCC2, the entire MCCC1 ORF was also analyzed. Identified mutations were confirmed by PCR amplification of genomic DNA. In individuals from whom only DNA was available amplification of all MCCC1 and MCCC2 exons and flanking intronic sequences from genomic DNA followed by direct sequencing was performed.

                                      RNA and genomic DNA were extracted from cultured skin fibroblasts or peripheral blood leukocytes using the QIAamp® RNeasy or QIAamp® RNA Blood Mini Kit and the QIAamp® DNA Mini Kit (Qiagen AG, Basel, Switzerland), respectively. The RT-PCR reaction was performed using the One-Step RT-PCR kit (Qiagen AG, Basel, Switzerland) following the manufacturer’s instructions. First-strand MCCC1 and MCCC2 cDNA was amplified as described [2]. PCR products were sequenced in a thermocycler and analyzed with an ABI Prism 3100 Avant using the dye-terminator method (Applied Biosystem, Rotkreuz, Switzerland) according to the manufacturer’s instructions. To confirm mutations identified in RT-PCR products, a genomic fragment containing the corresponding exon was amplified using flanking intronic primers, and the PCR product was sequenced directly. In cases where only one of the two alleles could be identified in the standard RT-PCR product all exons and flanking intronic regions were sequenced. The sequences of all primers are available upon request.

                                      To exclude that the identified missense mutations are common polymorphisms we amplified the relevant exons from genomic DNA of 100 controls (200 alleles).

                                      Construction of wild type and mutant MCCC1 and MCCC2 expression vectors and transfections

                                      The following mutations were introduced by site-directed mutagenesis into the existing wildtype MCCC1 and MCCC2 pCR Blunt II TOPO vector (Invitrogen, Basel, Switzerland): MCCC1 p.E288G, p.G379D, p.I434M and MCCC2 p.S39F, p.G118del, p.Y146N, p.H282R, p.V434L, p.A456V, p.G475R, p.S523G. All constructs were then transferred into the mammalian expression vector pTracer-CMV2 (Invitrogen, Basel, Switzerland) and seq-uenced for validation. For expression studies the constructs were transiently transfected into transformed cultured fibroblasts deficient in either MCCC1 (homozygous for c.1264_1265insG/p.Q422Rfs*10 or compound heterozygous for c.1264_1265insG/p.Q422Rfs*10 and c.1682-3A > G/p.N561Kfs*10) or MCCC2 (homozygous for c.127 C > T/p.Q43*) by electroporation as described [2]. The cells were harvested 48 hours later and assayed for MCC and PCC activity.

                                      Western blot analysis of expressed proteins

                                      Western blot analysis of proteins extracted from cells harvested 48 h after transfection was performed as described earlier [41]. For immunostaining of MCCC2 a commercially available antibody was used (Abnova). Antiserum for MCCC1 was produced by inoculating rabbits with peptides corresponding to the hydrophilic strech of the last 19 C-terminal amino acids (RHTPLVEFEEEESDKRESE) of human MCCC1 conjugated to keyhole limpet hemocyanin (Covance, Denver, Colorado). β-Actin was stained as control.

                                      The biotin-containing MCC and PCC α-subunits and PC were also stained using streptavidin-alkaline phosphatase followed by colorimetric detection (Transcend™ Non-Radioactive Translation Detection Systems Kit, Promega, Dübendorf, Switzerland).

                                      In silico prediction of functional relevance of identified mutations

                                      The human MCCC1/2 enzyme has not been structurally characterized. Missense mutations were therefore interpreted structurally using the homologous structure of Pseudomonas aeruginosa MCC holoenzyme (PDB code 3U9T). Amino acid sequence alignment of MCCC1/2, ACC1/2, PCCα/β sequences was constructed using the ICM-Pro program (Molsoft, San Diego) with the implemented alignment algorithm. The protein sequences NG_008100.1 and NG_008882.1 [GenBank at the NCBI] were used as reference sequences for the alpha and beta subunit of MCC, respectively.

                                      Results

                                      A comprehensive summary of clinical, biochemical, enzymatic and molecular genetic information on each patient is given in Tables 1, 2, 3 and 4.

                                      Diagnosis of MCC deficiency was confirmed by assaying MCC and PCC activities in fibroblasts of 68 individuals. In 50 cell lines MCC activity was severely reduced to less than 5% of the median control value. In 16 further cell lines residual MCC activity varied between 5.1% and 20% and in 2 cell lines MCC activity was 31% and 34% of the control value. All cell lines had a highly increased PCC/MCC activity ratio of at least 7.1. From 20 subjects fibroblasts were not available and the diagnosis was confirmed by mutation analysis using genomic DNA.

                                      Clinical data

                                      Twenty-six individuals (30%) were diagnosed by selective metabolic screening (SMS) due to clinical symptoms (n = 18) or a positive family history (n = 8) while 53 individuals (60%) were identified by expanded NBS. Additionally, 9 mothers (10%) were diagnosed following a positive NBS result of their healthy offspring. In patients in whom a metabolic work-up was initiated due to clinical symptoms age at diagnosis ranged between one week and 13 years (median 1.5 years) (Table 2). Patients identified by family screening had a median age at diagnosis of 3.75 years (range 1.5-17 years) (Table 3), and mothers were diagnosed at a median age of 30.5 years (range 24-38 years) (Table 4).

                                      Clinical information was available from 80 individuals. Parental consanguinity was reported in 31 subjects with most parents being second-degree relatives. Fourty-four parents were non-consanguineous and of 13 individuals no information on consanguinity was available. Three children had deceased, two during an acute metabolic decompensation at the age of 33 days and 6 weeks, and the third, a 8-year-old girl with sudden cardiac death due to catecholaminergic polymorphic ventricular tachycardia (mutations in RyR2 gene).

                                      In 34 (43%) of 80 subjects clinical symptoms were reported ranging from acute metabolic decompensation with ketoacidosis, hypoglycemia and encephalopathy to neuromuscular symptoms, mental retardation or attention deficit hyperactivity disorders (Figure 1). Twelve patients, 5 of which were diagnosed by newborn screening, had at least one acute metabolic decompensation. The most common clinical symptoms of acute crises were vomiting and encephalopathy with impaired conciousness. Neurologic symptoms like seizures, metabolic stroke, hemiparesis and cerebral edema were less frequent. The most common laboratory findings were acidosis and hypoglycaemia. Among chronic symptoms mental retardation including speech retardation were the most common findings followed by seizures, muscular hypotonia, muscle weakness, muscle pain and failure to thrive. In 5 patients an attention deficit hyperactivity disorder was reported.
                                      http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-31/MediaObjects/13023_2012_408_Fig1_HTML.jpg
                                      Figure 1

                                      Clinical manifestation of 33 symptomatic individuals with MCC deficiency. One patient who died of sudden cardiac arrest at the age of 8 years was excluded from this figure as catecholaminergic ventricular tachycardia with mutations in the RyR2 gene was identified as a likely cause for the cardiac symptoms.

                                      Thirty-five out of 61 (57%) living individuals of whom recent follow-up information was available or who had been followed for at least until the age of three years have remained asymptomatic. Notably, 25 (69%) of the 36 subjects identified by NBS of whom either recent information or follow-up data until the age of at least three years were available, have remained without symptoms.

                                      Sixty-nine out of 75 individuals of whom information on treatment was available received dietary and/or medical therapy. Forty individuals were given a protein-restricted or leucine-restricted diet (which was stopped later in life in some cases) and in 23 a leucine-free amino acid mixture was administered at least temporarily. In 30 subjects oral biotin was given on a trial basis. 63 patients were supplemented with oral carnitine, and 10 patients received oral glycine.

                                      Biochemical phenotype at diagnosis

                                      Presence of C5OH in blood or dried blood spots and presence of elevated urinary excretion of 3-HIVA and 3-MCG at the time of diagnosis are shown in Tables 1, 2, 3 and 4. A biochemical phenotype characteristic for MCC deficiency with elevated excretion of 3-HIVA and/or 3-MCG, defined as more than twice the upper normal value, was found in 85% (68/80) of individuals. In 14% (11/80) only mildly elevated excretion was detected. In one patient (1%) no elevated excretion of 3-HIVA and 3-MCG was detected. Notably, 5 individuals including one patient reported earlier [39] did not excrete 3-MCG, the pathognomonic metabolite of MCC deficiency. However, one of these individuals showed massive excretion of 3-MCG when re-evaluated 6 months later.

                                      C5OH in dried blood spots was highly elevated in 85% (66/78) and slightly (less than twice the upper normal value) elevated in 15% (12/78) of individuals. Low C5OH concentrations were not always linked with low urinary excretion of metabolites and vice versa.

                                      In 43/68 individuals (63%) a secondary carnitine deficiency was present. Remarkably, 24 (60%) of those 40 children identified through NBS of whom information on free carnitine concentrations was available had decreased free carnitine levels already at the time of diagnosis or within the neonatal period. In 15 of them free carnitine concentration was below 5 μmol/l.

                                      Mutation analysis

                                      Of the 83 individuals in whom mutation analysis was performed, 31 had mutations in MCCC1 and 52 in MCCC2 (Tables 1, 2, 3 and 4). Forty-eight probands were found to be homozygous (12 for MCCC1 mutations and 36 for MCCC2 mutations) while 28 were compound heterozygous (15 for MCCC1 mutations and 13 for MCCC2 mutations). In 5 of these patients RT-PCR results showed exon skipping either for one or both alleles, but the underlying genomic mutation could not be identified. In the remaining 7 subjects (4 MCCC1 and 3 MCCC2) it was not possible to detect a second mutation in spite of sequencing all exons and flanking intronic sequences. However, the mutant allele identified appeared to be homozygous in RT-PCR, but was clearly heterozygous in genomic DNA. This suggests that the steady state level of mRNA from the second allele was not detectable as would be the case for a promoter mutation or an intragenic deletion or insertion missed by genomic PCR.

                                      We identified a total of 15 novel MCCC1 and 16 novel MCCC2 mutations (shown in bold in Tables 5 and 6). The 15 novel MCCC1 mutations comprise 7 missense, 2 nonsense, 1 splice site and 5 frameshift mutations (5 due to small deletions and one due to a small insertion). The 16 novel MCCC2 variants include 11 missense and 4 splice site mutations and 1 deletion of a single amino acid.
                                      Table 5

                                      Overview on 64 MCCC1 mutant alleles and their consequences

                                      Exon/Intron

                                      Nucleotide change at cDNA level

                                      Amino acid change (at RNA level)

                                      Consequence

                                      Patients, in whom this mutation was found in this study/ Reference of first description of the mutation

                                      exon 1

                                      c.43GC>T

                                      p.E15*

                                      nonsense

                                      Morscher et al. 2012

                                      intron 1

                                      c.89+2_89+34del

                                      p.?

                                      splice

                                      Morscher et al. 2012

                                      intron 2

                                      c.137-2A>G

                                      p.?

                                      splice

                                      Stadler et al. 2006

                                      exon 3

                                      c.137G>A

                                      p.G46E

                                      missense

                                      Nguyen et al. 2011

                                      exon 3

                                      c.168C>G

                                      p.N56K

                                      missense

                                      Morscher et al. 2012

                                      exon 3

                                      c.193A>T

                                      p.M65L

                                      missense

                                      #113/ This study

                                      exon 3

                                      c.227_228delTG

                                      p.V76Gfs*4

                                      frameshift

                                      unpublisheda

                                      exon 3

                                      c.251_252delGAb

                                      p.R84Kfs*10

                                      deletion/frameshift

                                      Stadler et al. 2006

                                      exon 4

                                      c.369G>C

                                      p.Q123H

                                      missense

                                      Stadler et al. 2006

                                      exon 5

                                      c.375C>G

                                      p.I125M

                                      missense

                                      Stadler et al. 2006

                                      exon 5

                                      c.400G>A

                                      p.E134K

                                      missense

                                      #32/ Dantas et al. 2005

                                      exon 5

                                      c.479T>G

                                      p.M160R

                                      missense

                                      Stadler et al. 2006

                                      exon 6

                                      c.539G>T

                                      p.G180V

                                      missense

                                      #83/ This study

                                      exon 6

                                      c.558delA

                                      p.Q186Hfs*6

                                      deletion/frameshift

                                      #83, 93a/ Morscher et al. 2012

                                      exon 6

                                      c.559T>C

                                      p.S187P

                                      missense

                                      #20, Dantas et al. 2005

                                      intron 6

                                      c.639+2T>A

                                      p.S164Rfs*3

                                      splice, exon 6 skipping

                                      #54/ This study

                                      exon 7

                                      c.640_641delGG

                                      p.G214Nfs*5c

                                      deletion/frameshift

                                      #43a, 43b/ Dantas et al. 2005

                                      exon 7

                                      c.658_662delTCAGA

                                      p.S220Tfs*15

                                      deletion/frameshift

                                      #112/ This study

                                      exon 7

                                      c.694C>T

                                      p.R232W

                                      missense

                                      #41/ Dantas et al. 2005

                                      intron 7

                                      c.762-1G>A

                                      p. ?

                                      splice

                                      Nguyen et al. 2011

                                      exon 8

                                      c.803C>A

                                      p.A268D

                                      missense

                                      #115/ This study

                                      exon 8

                                      c.841C>T

                                      p.R281*

                                      nonsense

                                      Morscher et al. 2012

                                      exon 8

                                      c.842G>A

                                      p.R281Q

                                      missense

                                      Morscher et al. 2012

                                      exon 8

                                      c.863A>G

                                      p.E288G

                                      missense

                                      #57/ This study

                                      exon 8

                                      c.866C>T

                                      p.A289V

                                      missense

                                      Baumgartner et al. 2001

                                      exon 8

                                      c.872C>T

                                      p.A291V

                                      missense

                                      #25/ Dantas et al. 2005

                                      intron 8 + exon 9

                                      c.873+4524_6787del2264

                                      2 transkripts: p.P292Gfs*18 p.P292_R361del

                                      large deletion, exon 9 and exon 9 and 10 skipping

                                      #62, 96a, 96c/ Eminoglu et al. 2009

                                      exon 9

                                      c.901_902delAA

                                      p.K301Afs*10

                                      deletion/ frameshift

                                      Uematsu et al. 2007

                                      exon 9

                                      c.945T>A

                                      p.Y315*

                                      nonsense

                                      Stadler et al. 2006

                                      exon 10

                                      c.974T>G

                                      p.M325R

                                      missense

                                      Gallardo et al. 2001

                                      exon 10

                                      c.980C>G

                                      p.S327*

                                      nonsense

                                      #54/ Morscher et al. 2012

                                      exon 10

                                      not published

                                      p.Q372P

                                      missense

                                      Desviat et al. 2003

                                      exon 11

                                      c.1114C>T

                                      p.Q372*

                                      nonsense

                                      #50/ This study

                                      exon 11

                                      c.1135G>A

                                      p.G379S

                                      missense

                                      Stadler et al. 2006

                                      exon 11

                                      c.1136G>A

                                      p.G379D

                                      missense

                                      #71/ This study

                                      exon 11

                                      c.1139A>C

                                      p.H380P

                                      missense

                                      Morscher et al. 2012

                                      exon 11

                                      c.1155A>C

                                      p.R385S

                                      missense

                                      #20, 27, 28, 31, 53, 68, 105, 115, 138/ Baumgartner et al. 2001, Gallardo et al. 2001

                                      exon 11

                                      c.1193_1194delTG

                                      p.V398Gfs*19

                                      deletion/frameshift

                                      #113/ This study

                                      exon 11

                                      c.1225C>T

                                      p.R409*

                                      nonsense

                                      Stadler et al. 2006

                                      exon 11

                                      c.1264_1265insGd

                                      p.Q422Rfs*10d

                                      insertion/frameshift

                                      Baumgartner et al. 2001

                                      intron 11

                                      c.1268-2A>G

                                      p.G423Efs*15

                                      splice, exon 12/13 skipping

                                      Stadler et al. 2006

                                      exon 12

                                      c.1302T>G

                                      p.I434M

                                      missense

                                      #74/ This study

                                      exon 12

                                      c.1310T>C

                                      p.L437P

                                      missense

                                      Baumgartner et al. 2001

                                      exon 12

                                      c.1315G>A

                                      p.V439M

                                      missense

                                      #53/ This study

                                      exon 12

                                      c.1333C>T

                                      p.Q445*

                                      nonsense

                                      Morscher et al. 2011

                                      exon13

                                      c.1380T>G

                                      p.I460M

                                      missense

                                      Uematsu et al. 2007

                                      exon 13

                                      c.1522_1544del

                                      p.L508Hfs*17

                                      deletion

                                      Morscher et al. 2012

                                      exon 13

                                      c.1526delGe

                                      p.C509Sfs*14

                                      deletion/frameshift

                                      #46, 59, 73c, 87/ Dantas et al. 2005

                                      exon 13

                                      c.1527C>A

                                      p.C509*

                                      nonsense

                                      #36/ Dantas et al. 2005

                                      exon 13

                                      c.1541dupG

                                      p.L515Sfs*18

                                      insertion/frameshift

                                      Morscher et al. 2012

                                      exon 13

                                      c.1594G>C

                                      p.D532H

                                      splice

                                      Baumgartner et al. 2001

                                      intron 13

                                      c.1594+3A>G

                                      p.V461Nfs*13

                                      splice, exon 13 skipping

                                      Morscher et al. 2012

                                      exon 14

                                      c.1604C>T

                                      p.S535F

                                      missense

                                      Holzinger et al. 2001

                                      intron 14

                                      c.1681+5G>A

                                      p.Q533_N561del

                                      splice, exon 14 skipping

                                      Stadler et al. 2006

                                      intron 14

                                      c.1682-3A>G

                                      p.N561Kfs*10

                                      splice/frameshift

                                      Dantas et al. 2005

                                      exon 15

                                      c.1695_1700del

                                      p.V566_T567del

                                      deletion

                                      Morscher et al. 2012

                                      exon 16

                                      c.1750C>T

                                      p.Q584*

                                      nonsense

                                      Uematsu et al. 2007

                                      exon 16

                                      c.1820delG

                                      p.S607Ifs*5

                                      deletion/frameshift

                                      #103/ This study

                                      exon 17

                                      c.1882G>T

                                      p.E628*

                                      nonsense

                                      #50/ This study

                                      exon 17

                                      c.1930G>T

                                      p.E644*

                                      nonsense

                                      #43a, 43b/ Dantas et al. 2005

                                      exon 18

                                      c.2009_2043del35

                                      p.A670Dfs*34

                                      deletion/frameshift

                                      #138/ This study

                                      exon 19

                                      c.2079delA

                                      p.V694*

                                      nonsense

                                      #60, 70a/ Holzinger et al. 2001

                                      exon 19

                                      c.2088dupA

                                      p.V697Sfs*19

                                      insertion/frameshift

                                      #46/ Dantas et al. 2005

                                      exon 19

                                      c.2123dupA

                                      p.H708Qfs*8

                                      insertion/frameshift

                                      #74/ This study

                                      a Found in our laboratory in a heterozygous individual, not yet published.b Published in the original paper as c.250_251delAG (p.R84Kfs*9), however AG is not found at this position in the reference sequence, but GA instead.c Published in the original paper as p.G214IfsX5, nomenclature has been adapted to new approved guidelines.d Published in the original paper as c.1264insG, Q421fs(+1), nomenclature has been adapted to new approved guidelines.e Mainly found in patients from the Faroe Islands.

                                      (NG_008100.1 [GenBank at the NCBI] was used as reference sequence. Consensus nomenclature according to approved guidelines ( http://​www.​hgvs.​org/​mutnomen/​))

                                      Table 6

                                      Overview on 68 MCCC2 mutant alleles and their consequences

                                      Exon/Intron

                                      Nucleotide change at cDNA level

                                      Amino acid change (at RNA level)

                                      Consequence

                                      Patients, in whom this mutation was found in this study/ Reference of first description of the mutation

                                      exon 1

                                      c.116C>T

                                      p.S39F

                                      missense

                                      #80/ Dirik et al. 2008

                                      exon 1

                                      c.127C>T

                                      p.Q43*

                                      nonsense

                                      #32/ Dantas et al. 2005

                                      exon 3

                                      c.214C>T

                                      p.R72*

                                      nonsense

                                      #40/ Dantas et al. 2005

                                      exon 3

                                      c.243dupT

                                      p.L81Ifs*7a

                                      insertion/frameshift

                                      Stadler et al. 2006

                                      intron 3

                                      c.281+5G>A

                                      p.?

                                      splice

                                      Stadler et al. 2006

                                      intron 3

                                      c.281+5G>T

                                      p.G67Lfs*35b

                                      splice/exon 3 skippingb

                                      Gallardo et al. 2001

                                      intron3

                                      c.282-1G>C

                                      p.S95_G128delc

                                      splice/exon 4 skipping

                                      #33/ Dantas et al. 2005

                                      exon 4

                                      c.295G>C

                                      p.E99Q

                                      missense

                                      #24, 29, 90, 91/ Baumgartner et al. 2001, Holzinger et al. 2001

                                      exon 4

                                      c.302C>T

                                      p.S101F

                                      missense

                                      Stadler et al. 2006

                                      exon 4

                                      c.351_353delTGG

                                      p.G118del

                                      deletion

                                      #51, 55/ This study

                                      intron 4

                                      c.383+1G>T

                                      p.?

                                      splice

                                      Stadler et al. 2006

                                      intron 4

                                      c.384-2A>G

                                      p.?

                                      splice

                                      Stadler et al. 2006

                                      exon 5

                                      c.392G>T

                                      p.C131F

                                      missense

                                      #99a/ This study

                                      exon 5

                                      c.416_427del12ins16

                                      p.T139_G143>RWVPGEfs*35

                                      deletion/insertion/frameshift

                                      #40, 66/ Dantas et al. 2005

                                      exon 5

                                      c.436T>A

                                      p.Y146N

                                      missense

                                      #66/ This study

                                      exon 5

                                      c.455A>C

                                      p.K152T

                                      missense

                                      #72/ This study

                                      exon 5

                                      c.463C>T

                                      p.R155W

                                      missense

                                      #44, 77/ Dantas et al. 2005

                                      exon 5

                                      c.464G>A

                                      p.R155Q

                                      missense

                                      #22, 63, 67/ Baumgartner et al. 2001

                                      exon 5

                                      c.469C>T

                                      p.Q157*

                                      nonsense

                                      #23/ Dantas et al. 2005

                                      exon 5

                                      c.499T>C

                                      p.C167R

                                      missense

                                      Gallardo et al. 2001

                                      exon 5

                                      c.505T>G

                                      p.Y169D

                                      missense

                                      #100/ This study

                                      intron 5

                                      c.512-1G>Ad

                                      p.?

                                      splice

                                      #82a, 82b/ Baumgartner et al. 2001

                                      exon 6

                                      c.517dupT

                                      p.S173Ffs*25

                                      insertion/frameshift

                                      #39, 85/ Baumgartner et al. 2001,Gallardo et al 2001

                                      exon 6

                                      c.518C>T

                                      p.S173L

                                      missense

                                      #108, 137/ Baumgartner et al. 2001

                                      exon 6

                                      c.538C>T

                                      p.R180*

                                      nonsense

                                      #58/ Stadler et al. 2006

                                      exon 6

                                      c.568C>T

                                      p.H190Y

                                      missense

                                      Dantas et al. 2005

                                      exon 6

                                      c.569A>G

                                      p.H190R

                                      missense

                                      Uematsu et al. 2007

                                      exon 6

                                      c.577C>T

                                      p.R193C

                                      missense

                                      Baumgartner et al. 2001

                                      exon 6

                                      c.578G>A

                                      p.R193H

                                      missense

                                      Stadler et al. 2006

                                      exon 6

                                      c.592C>T

                                      p.Q198*

                                      nonsense

                                      Uematsu et al. 2007

                                      exon 6

                                      c.599T>A

                                      p.I200N

                                      missense

                                      #85/ This study

                                      exon 7

                                      c.652G>A

                                      p.A218T

                                      missense

                                      Gallardo et al. 2001

                                      exon 7

                                      c.653C>T

                                      p.A218V

                                      missense

                                      Morscher et al. 2012

                                      exon 7

                                      c.653_654delCAinsTT

                                      p.A218V

                                      missense

                                      Uematsu et al. 2007

                                      exon 7

                                      c.659G>A

                                      p.G220E

                                      missense

                                      #55/ This study

                                      exon 7

                                      c.671C>T

                                      p.P224L

                                      missense

                                      #78/ This study

                                      exon 7

                                      c.710G>A

                                      p.G237D

                                      missense

                                      #92/ This study

                                      exon 8

                                      c.797A>Te

                                      p.H266Le

                                      missense

                                      Stadler et al. 2006

                                      exon 8

                                      c.803G>C (r.785_803del)

                                      p.R268T (p.G262_R268delfs*5)

                                      missense/splice

                                      #21, 52, 64/ Holzinger et al. 2001, Dantas et al. 2005

                                      exon 9

                                      c.838G>T

                                      p.D280Y

                                      missense

                                      Uematsu et al. 2007

                                      exon 9

                                      c.845A>G

                                      p.H282R

                                      missense

                                      #34/ Dantas et al. 2005

                                      intron 9

                                      c.903+6_903+9delTACG

                                      p.?

                                      splice/RNA nd

                                      #72/ This study

                                      exon 10

                                      c.929C>G

                                      p.P310R

                                      missense

                                      #42/ Baumgartner et al. 2001

                                      exon 10

                                      c.994C>T

                                      p.R332*

                                      nonsense

                                      Dantas et al. 2005

                                      exon 11

                                      c.1015G>A

                                      p.V339M

                                      missense

                                      #67, 81, 90, 111/ Baumgartner et al. 2001

                                      exon 11

                                      c.1019A>T

                                      p.D340V

                                      missense

                                      Stadler et al. 2006

                                      exon 11

                                      c.1054G>A

                                      (r.1054G>A + r.1000_1072delins r.999+858_r.999+922)

                                      p.G352R + (p.V334_G358delins KFFMKYFLRLDLNSYNSTWQH)

                                      missense/splice (skip exon 11, insert 64 bp from intron 10)

                                      Dantas et al. 2005

                                      exon 11

                                      c.1054_1055delGG

                                      p.G352Rfs*27f

                                      deletion/frameshift

                                      Uematsu et al. 2007

                                      exon 11

                                      c.1065A>T

                                      p.L355F

                                      missense

                                      Nguyen et al. 2011

                                      intron 11

                                      c.1073-12C>G (r.1073_1216del+ r.1073insr.1073-48_r.1073-1)

                                      2 transkripts: (p.G358Vfs*6+p. G358Afs*12)

                                      splice/2 transkripts: exon 12 and 13 skipping, insertion of 48 bp from intron 11

                                      #100/ This study

                                      exon 12

                                      c.1123G>T

                                      p.V375F

                                      missense

                                      #39/ Dantas et al. 2005

                                      intron 12

                                      c.1149+1G>T

                                      p.?

                                      splice

                                      #136 / This study

                                      intron12

                                      c.1149+5G>C

                                      p.?

                                      splice

                                      #92/ This study

                                      exon 13

                                      c.1208A>C

                                      p.N403T

                                      missense

                                      Stadler et al. 2006

                                      exon 14

                                      c.1300G>C

                                      p.V434L

                                      missense

                                      #126/ This study

                                      exon 14

                                      c.1309A>G(r.1309A>G+ r.1310_1373del64)

                                      2 transkripts: (p.I437V+p.I437Tfs*15)

                                      missense/splice (cryptic splice donor resulting in deletion of the last 64 bp of exon 14)

                                      #111/ Baumgartner et al. 2001

                                      exon 14

                                      c.1367C>T

                                      p.A456V

                                      missense

                                      #37/ Dantas et al. 2005

                                      exon 15

                                      c.1423G>A

                                      p.G475R

                                      missense

                                      #125/ This study

                                      exon 15

                                      c.1423G>C

                                      p.G475R

                                      missense

                                      #127/ This study

                                      exon 15

                                      c.1430A>G

                                      p.Q477R

                                      missense

                                      Nguyen et al. 2011

                                      exon 15

                                      c.1465C>T

                                      p.Q489*

                                      nonsense

                                      Stadler et al. 2006

                                      exon 16

                                      c.1549G>A

                                      p.G517R

                                      missense

                                      Nguyen et al. 2011

                                      exon 16

                                      c.1559A>C

                                      p.Y520S

                                      missense

                                      Nguyen et al. 2011

                                      exon 16

                                      c.1567A>G

                                      p.S523G

                                      missense

                                      #56, 69/ Morscher et al. 2011

                                      intron 16

                                      c.1574+1G>A

                                      p.F497Gfs*4g

                                      splice, exon 16 skipping

                                      #29, 30/ Dantas et al. 2005

                                      exon 17

                                      c.1624_1625dupGGh

                                      p.L543Vfs*11

                                      insertion/frameshift

                                      Uematsu et al. 2007

                                      exon 17

                                      c.1663A>G

                                      p.K555E

                                      missense

                                      Stadler et al. 2006

                                      exon 17

                                      c.1690T>C

                                      p.X564QLE

                                      add 3 aa at C-terminus

                                      #26/ Dantas et al. 2005

                                      (NG_008882.1 [GenBank at the NCBI] was used as reference sequence. Consensus nomenclature according to approved guidelines ( http://​www.​hgvs.​org/​mutnomen/​)).

                                      nd not detectable

                                      a This mutation has been published as p.L81Lfs*7 in the original paper, nomenclature has been adapted to new approved guidelines.

                                      b For this mutation "MCCB exon 3 skipping, frameshift after residue 66" has been published in the original paper, nomenclature has been adapted to new approved guidelines.

                                      c This mutation has been published as p.G94_S127del in the original paper, nomenclature has been adapted to new approved guidelines.

                                      d This mutation has been published as In5ac-1G→A in the original paper, nomenclature has been adapted to new approved guidelines.

                                      e This mutation has been published as c.979A>T, p.H266L in exon 8 in the original paper. However, c.979A>T would predict an Arginine to Tryptophan change in position 327 (p.R327W). The Histidine to Leucine change in position 266 (p.H266L) could be caused by c.797A>T, therefore, a typing error cannot be excluded.

                                      f This mutation has been published as p.G352RfsX26 in the original paper, nomenclature has been adapted to new approved guidelines.

                                      g This mutation has been published as p.F497_V526>GfsX4 in the original paper, nomenclature has been adapted to new approved guidelines.

                                      h This mutation has been published as c.1625_1626insGG in the original paper, nomenclature has been adapted to new approved guidelines.

                                      Expression studies and Western blot analysis

                                      The functional consequences of three MCCC1 (p.E288G, p.G379D, p.I434M) and 8 MCCC2 (p.S39F, p.G118del, p.Y146N, p.H282R, p.V434L, p.A456V, p.G475R and p.S523G) mutations were investigated by expression studies (Table 7). The MCCC1 p.E288G and p.G379D mutations showed no residual activity while the p.I434M mutant allele yielded on average 46% of MCC1 wildtype activity.
                                      Table 7

                                      Expression of MCCC1 and MCCC2 wildtype and mutant alleles

                                      Allele

                                      PCC and MCC activities (pmol/min/mg protein)*

                                      Experiment 1

                                      Experiment 2

                                      PCC

                                      MCC

                                      %**

                                      PCC

                                      MCC

                                      %**

                                      MCCC1-wildtype

                                      311

                                      193

                                      100

                                      331

                                      157

                                      100

                                      vector only

                                      335

                                      0

                                      0

                                      377

                                      0.3

                                      0.2

                                      MCCC1-p.E288G

                                      283

                                      0

                                      0

                                      352

                                      1.8

                                      1.1

                                      MCCC1-p.G379D

                                      293

                                      0

                                      0

                                      326

                                      0

                                      0

                                      MCCC1-p.I434M

                                      330

                                      87.6

                                      45.4

                                      324

                                      74.5

                                      47.4

                                      MCCC2-wildtype

                                      377

                                      75.4

                                      100

                                      341

                                      49.4

                                      100

                                      vector only

                                      364

                                      0.3

                                      0.4

                                      346

                                      0

                                      0

                                      MCCC2-p.S39F

                                      398

                                      29.6

                                      39.3

                                      368

                                      24.8

                                      50.2

                                      MCCC2-p.G118del

                                      366

                                      10.8

                                      14.3

                                      342

                                      3.2

                                      6.5

                                      MCCC2-p.Y146N

                                      341

                                      58.7

                                      77.9

                                      372

                                      44.1

                                      89.3

                                      MCCC2-p.H282R

                                      301

                                      9.6

                                      12.7

                                      339

                                      2.3

                                      4.7

                                      MCCC2-p.A456V

                                      265

                                      2.0

                                      2.7

                                      340

                                      0.1

                                      0.2

                                      MCCC2-p.S523G

                                      313

                                      56.1

                                      74.4

                                      344

                                      30.9

                                      62.6

                                      MCCC2-wildtype

                                      335

                                      76.9

                                      100

                                      446

                                      43.5

                                      100

                                      vector only

                                      371

                                      0

                                      0

                                      540

                                      0.5

                                      1.1

                                      MCCC2-p.V434L

                                      295

                                      56.6

                                      73.7

                                      449

                                      33.5

                                      77.0

                                      MCCC2-p.G475R

                                      290

                                      33.7

                                      43.8

                                      414

                                      21.1

                                      48.5

                                      Transient expression was performed in transformed MCCC1 and MCCC2 deficient fibroblasts followed by the assay of propionyl-CoA carboxylase (PCC) and 3-methylcrotonyl-CoA carboxylase (MCC) activities. Transfection with an empty vector (vector only) was used as negative control.

                                      *activities are the mean of duplicate determination.

                                      ** % of MCC activity of simultaneously expressed wildtype allele.

                                      Only one of the MCCC2 mutations, p.A456V, showed virtually no enzyme activity whereas seven mutations were found to have residual activity ranging on average from 9 to 84% of MCC2 wildtype activity. Western blot analysis of the expressed proteins revealed virtually wildtype levels for the MCCC1 p.I434M protein while the levels of MCCC1 p.E288G and MCCC1 p.G379D proteins were severely reduced or not detectable (Figure 2). Five of the 8 expressed MCCC2 proteins (p.Y146N, p.H282R, p.A456V, p.G475R and p.S523G) were detected at normal or only slightly reduced levels, while the p.S39F and p.V434L protein levels were severely reduced and no protein was detectable after transfection with the p.G118del construct (Figure 3).
                                      http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-31/MediaObjects/13023_2012_408_Fig2_HTML.jpg
                                      Figure 2

                                      Western blot analysis of expressed MCCC1 wildtype and mutant proteins. Constructs with MCCC1 wildtype and 3 mutant cDNAs in pTracer vector were transfected into MCCC1 deficient reference cell lines by electroporation and harvested 48 hours later for Western blot analysis. 50 μg of protein were used per lane. The MCCC1 subunit was visualized by a) immunostaining using β-actin (4 μg) as control, or by b) colorimetric reaction after coupling with avidin-alkaline phosphatase. Transfection with an empty vector (vector only) was used as a negative control. For further details see “Methods”.

                                      http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-31/MediaObjects/13023_2012_408_Fig3_HTML.jpg
                                      Figure 3

                                      Western blot analysis of expressed MCCC2 wildtype and mutant proteins. Constructs with MCCC2 wildtype and 8 mutant cDNAs in pTracer vector were transfected into MCCC2 deficient reference cell lines by electroporation and harvested 48 hours later for Western blot analysis. 50 μg of protein were used per lane. The MCCC2 subunit was visualized by immunostaining using β-actin (6 μg) as control. Transfection with an empty vector (vector only) was used as a negative control. For further details see “Methods”.

                                      Discussion

                                      This study summarizes clinical, biochemical, enzymatic and mutation data on 88 MCC deficient individuals and summarizes all MCCC1 and MCCC2 mutations described so far.

                                      Clinical phenotype

                                      MCC deficiency has been described as a genetic condition with low clinical penetrance [6]. Results of a comparative analysis of case reports with NBS data reported by Stadler and co-workers [6] suggest that less than 10% of affected individuals ever develop minor symptoms and only less than 1 to 2% might have a risk for severe adverse outcome. In their study all 14 individuals diagnosed by NBS remained asymptomatic during a follow-up period of 1.75 to 6.5 years. In contrast, in our study only 69% of the 36 subjects identified by NBS with a follow-up of at least 3 years have stayed completely asymptomatic while the remainder (31% =11) developed clinical symptoms including various neurologic symptoms as well as at least one acute metabolic decompensation in 5 children. This indicates that early diagnosis with concomitant early initiation of therapy and counselling of parents cannot prevent a clinical manifestation in all cases. However, our data have to be interpreted with caution, since the source of patients being those sent to a diagnostic referral laboratory may lead to a selection bias in favour of symptomatic individuals since it can be assumed that samples for confirmatory diagnosis are more likely to be obtained from symptomatic subjects than from asymptomatic individuals. Additionally, the clinical phenotype of MCC deficiency is still not well-defined.

                                      In our study population the most common clinical features were acute episodes of metabolic acidosis with vomiting, hypoglycemia and acidosis, muscular symptoms such as muscular hypotonia, muscle weakness and muscle pain and neurological abnormalities including developmental delay and seizures as well as attention deficit hyperactivity disorders (Tables 1, 2, 3 and 4). The acute metabolic crises reported in 12 patients are likely to be caused by the underlying metabolic defect. However, the causative attribution of all other clinical signs, especially of unspecific chronic neurologic symptoms such as mental retardation, attention deficit disorders and fatigue to MCC deficiency remains questionable. It could be speculated that these symptoms might as well be caused by undiagnosed genetic defects other than MCC deficiency. Such an additional genetic disorder –though unprobable- would be more likely in individuals that are the product of a consanguineous union; thus a higher share of symptomatic individuals would be expected in this subgroup when compared to subjects whose parents are reported not to be consanguineous. However, no significant difference in the clinical manifestation rate between the two subgroups could be shown (41% symptomatic patients in both subgroups, 12 of 29 individuals with reported parental consanguinity versus 18 of 44 subjects with parents reported not to be consanguineous).

                                      Of the three lethal cases in our study cohort, two could be attributed to a severe metabolic decompensation due to MCC deficiency. The sudden cardiac death of a 9 year old girl was shown to be caused by catecholaminergic polymorphic ventricular tachycardia with mutations in the RyR2 gene. Thus, in the cohort of 80 individuals of whom clinical information was available, lethality that may be associated with MCC deficiency was 2.5% (2/80).

                                      Altogether, though the share of individuals with clinical symptoms was higher in our study population, our data underline the observation of Stadler and co-workers [6] that compared to other organic acidemias, individuals with MCC deficiency appear to have a significantly higher tolerance toward metabolic stress; even complete absence of MCC activity seems to cause clinical manifestations only in association with environmental triggering factors in a rather small subgroup of individuals. Dietetic treatment is usually not required. However, considering the frequency of carnitine deficiency in our study population, regular monitoring of free carnitine concentrations and – if necessary - oral carnitine substitution seems to be warranted. Also, an emergency regimen during intercurrent illness may be advisable.

                                      Biochemical phenotype/MCC activity

                                      As shown in Tables 1, 2, 3 and 4 the vast majority of individuals displayed a typical biochemical phenotype with accumulation of metabolites characteristic for MCC deficiency in both blood and urine at the time of diagnosis. Mild elevations of one or more metabolites were the exception and were not more common in asymptomatic individuals. A completely unremarkable urine organic acid pattern was detected in only one woman. However, the concentration of C5OH in her blood was clearly elevated at the same time.

                                      The phenomenon that mutations may cause a clear biochemical phenotype in otherwise asymptomatic individuals is well known from other inborn errors of metabolism implemented in expanded NBS such as isovaleric acidemia [42] or medium-chain acyl-CoA dehydrogenase deficiency [43]. From the data available in this study we were able to confirm earlier observations that in MCC deficiency there is no apparent association between the biochemical and the clinical phenotype and that a mild biochemical phenotype does not seem to be a predictor of a mild clinical expression [2, 6, 11]. Furthermore, we have recently shown that also individuals who are carriers of a single mutation at the MCCC1 locus and have residual MCC activity greater than 20% of control may present with a mild biochemical phenotype characteristic for MCC deficiency [12].

                                      In all but two individuals MCC activity in fibroblasts was severely reduced combined with normal activity of PCC. As for the biochemical phenotype, we were not able to demonstrate a correlation between the residual enzyme activity and the clinical phenotype. When individuals with a less severe deficiency of MCC as indicated by a PCC/MCC ratio of < 50 were compared to a subgroup with a more severe deficiency and a ratio of > 50, the manifestation of clinical symptoms was not significantly more common in the latter group (40% (6/15) versus 47% (22/47), respectively).

                                      Molecular heterogeneity

                                      Molecular genetic analysis of 83 subjects enrolled in the current study revealed a total of 31 new MCCC1 (n = 15) and MCCC2 (n = 16) mutations considered to be causative of MCC deficiency. This brings up the total of mutations published to date to 64 for MCCC1 and 68 for MCCC2 (Tables 5 and 6) [212].

                                      In our study cohort MCCC2 mutations were 1.7 times more common than MCCC1 mutations (63% versus 37%, respectively).

                                      Tables 5 and 6 illustrate a broad genetic heterogeneity at both the MCCC1 and MCCC2 locus. Mutations are distributed along almost the entire coding regions of both genes with the exception of exon 2 of both the MCCC1 and MCCC2 gene which do not host any mutations.

                                      The majority of mutations are private. Notably, in our cohort of 78 families only 5 MCCC1 and 10 MCCC2 mutations have been found in more than one family. The most common mutation was the p.R385S mutation in MCCC1 which was found in 8 individuals and 9 alleles. This missense mutation has been shown to have a dominant negative effect in the presence of the wild type allele and may lead to biochemical and clinical abnormalities in heterozygous individuals [44]. However, in agreement with earlier reports p.R385S appears not to be a predictor of a particular phenotype and has been found in severely affected patients as well as in asymptomatic individuals (this study, [2, 3, 44].

                                      Another recurring mutation was c.1526delG (p.C509Sfs*14) which was the only mutation found in all 3 individuals from the Faroe Islands suggesting that this is a founder mutation.

                                      Novel mutations

                                      Among the novel mutations identified in this study (shown in bold in Tables 5 and 6) the most common were missense mutations (n = 18). Frameshift (n = 5) and splice site mutations (n = 5) were more frequent than nonsense mutations (n = 2) and small deletions (of a single amino acid) (n = 1).

                                      We assume deleterious functional consequences for the frameshift mutations MCCC1 c.658_662delTCAGA (p.S220Tfs*15), c.1193_1194delTG (p.V398Gfs*19), c.1820delG (p.S607Ifs*5), c.2009_2043del35 (p.A670Dfs*34), c.2123dupA (p.H708Qfs*8)], the splice site mutations MCCC1 c.639 + 2 T > A (p.S164Rfs*3); MCCC2 c.903 + 6_903 + 9delTACG (p.?), c.1073-12 C > G (p.G358Vfs*6 + p.G358Afs*12), c.1149 + 1 G > T (p.?), c.1149 + 5 G > C (p.?)] and the nonsense mutations MCCC1 c.1114 C > T (p.Q372*), c.1882 G > T (p.E628*)] because they result in truncated proteins lacking functionally important domains such as the BC (in MCCC1) or CT (in MCCC2) domains [2, 45].

                                      The MCCC2 splice site mutation c.1073-12 C > G is interesting since sequence analysis of cDNA of patient #107 revealed two overlapping sequences. One transcript showed exon 12 and 13 skipping, while the other transcript contained an inframe insertion of a 48 bp sequence from intron 11. No wildtype transcript could be detected. It is conceivable that the c.1073-12 C > G mutation creates a cryptic splice site resulting in partial skipping of exons 12 and 13 and in the partial insertion of an additional exon.

                                      Among the novel MCCC1 missense mutations all variants [p.M65L, p.G180V, p.A268D, p.E288G, p.G379D, p.I434M, p.V439M] change residues within the BC domain, while all novel MCCC2 missense mutations [p.C131F, p.Y146N, p.K152T, p.Y169D, p.I200N, p.G220E, p.P224L, p.G237D, p.V434L, p.G475R, p.G475R] affect the CT domain of the MCCC2 protein. During the final stage of this manuscript preparation, the crystal structure of P. aeruginosa MCC holoenzyme, with >50% sequence identity to the human counterpart (Additional file 1: Figure S1 Additional file 2: Figure S2), was reported [46]. The structure reveals a markedly different holoenzyme architecture compared to other biotin-dependent enzymes, hence providing an unprecedented opportunity to understand MCCC1/2 missense mutations in the protein context. We rationalize that the reported variants are very likely to affect protein function for the following reasons: 1) No other alterations in the MCCC1 and MCCC2 gene have been found despite sequencing of the complete coding regions of both genes in all individuals carrying one of those mutations; 2) Amino acid sequence alignments revealed that all but one MCCC2 c.599 T > A (p.I200N)] of the nucleotide changes affect highly conserved residues across species (PolyPhen2, http://​/​/​genetics.​bwh.​harvard.​edu/​pph/​), and many are also conserved among other biotin-dependent enzymes (Additional file 1: Figure S1, Additional file 2: Figure S2); 3) A majority of the mutations are mapped onto the catalytic core of the MCCC1 BC domain and the substrate binding region of the MCCC2 CT domain (yellow spheres in Figure 4A), suggesting their functional importance; 4) Most of the missense mutations alter the physicochemical properties of the amino acid position (e.g. replacing small with bulky, unipolar with polar, or uncharged with charged residues) and hence are likely to affect the local molecular environment; 5) None of these 19 variants is present in the 1000 Genomes Project dataset ( http://​/​/​www.​1000genomes.​org/​home); 6) It is of note that some MCCC1/2 missense mutations, affecting residues at the inter-subunit interface (Figure 4B and C), may affect not only the corresponding domain fold but also disrupt the assembly of the α and β subunits into the functional hetero-dodecamer.
                                      http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-31/MediaObjects/13023_2012_408_Fig4_HTML.jpg
                                      Figure 4

                                      Mapping of missense mutations onto the P. aeruginosa MCC holoenzyme structure. (A) Mapping of the novel missense mutations (yellow spheres) onto the P. aeruginosa MCC holoenzyme structure (PDB code 3U9T), showing that they are clustered in the BC domain of MCCC1 (pink) or the CT domain of MCCC2 (green). (B) and (C) Mutation sites of Asn200, Gly220, Pro224 and Gly475 are located at the interface between two MCCC2 subunits (coloured green and purple). (D) Structural environment of the MCCC1 Glu288 and Gly379 mutation sites. All the residues shown in sticks are conserved between the human and P. aeruginosa enzymes. The Glu288-Arg444 ionic interaction is indicated by dashed lines.

                                      Expression data

                                      So far, functional consequences of only 4 MCCC1 and 8 MCCC2 mutant alleles have been proven by expression in a mammalian expression system [2, 11]. In this study we expressed 3 further MCCC1 and 8 MCCC2 mutations (Table 7) including those expected to show residual enzyme activity based on studies in fibroblasts. Expression of two of the 3 MCCC1 [p.E288G and p.G379D] and one of the 8 MCCC2 [p.G118del] mutants resulted in severely reduced MCC activity and protein levels on Western blot analysis confirming deleterious consequences of these mutations on enzyme function and protein stability. Glu288 and Gly379 in MCCC1 are highly conserved residues in the BC domains among biotin-dependent enzymes (Additional file 1: Figure S1). In the P. aeruginosa MCC holoenzyme structure, Glu288 forms an ionic pair interaction (Glu288-Arg444) to hold two secondary structure elements together, as observed in the homologous structures of ACC1 (Glu427-Arg604; PDB code 2YL2), ACC2 (Glu533-Arg710; 3GLK) and PCCα (Glu302-Arg459; 3N6R). Gly379 is expected to be at an invariant position at the beginning of a nearby β-strand, directly facing the aforementioned arginine residue. Mutations at Glu288 and Gly379 therefore may disrupt the packing of these secondary structure elements (Figure 4D).

                                      Deleterious effects of two further MCCC2 mutant alleles [p.H282R and p.A456V] were shown by severely reduced MCC activities of less than 13% of simultaneously expressed wildtype activity. However, in both cases protein levels were normal, and the two affected residues His282 and Ala456 are located at the monomeric surface, indicating that these mutations do not affect the stability of the protein. Expression of all other mutant alleles [MCCC1 p.I434M, MCCC2 p.S39F, p.Y146N, p.V434L, p.G475R and p.S523G] yielded considerable levels of residual MCC activity (Table 7). The equivalent of MCCC1 Ile434 in other biotin-dependent carboxylases can be Phe or Leu (Additional file 2: Figure S2), suggesting that substitution to a Met (similar size to Phe and Leu) in the p.I434M mutation may well be tolerated. All mutated residues from the 7 MCCC2 missense constructs (p.S39F, p.Y146N, p.282R, p.V434L, p.A456V, p.G475R, p.S523G) are at least partially exposed to the surface of the monomer, and not buried within the enzymatic core. Fibroblasts of 4 individuals (No 69, 80, 126 and 127) each homozygous for one of these mutations also showed residual enzyme activity, which was, however, much lower (6.2 – 31% of the median control value) than the activities of the expressed mutant alleles (39% - 77% of simultaneously expressed wildtype activity). Inspite of the high MCC activities after expression Western blot analysis revealed severely reduced protein levels in 2 cases (MCCC2 p.S39F, p.V434L). Normal levels of protein were expressed by two other mutant alleles (MCCC2 p.S523G, p.G475R). Even more drastic differences were found between MCC activity of the expressed protein (45%-89%) and that of fibroblasts (3.2% - 7.6%) that are compound heterozygous for the MCCC1 mutation p.I434M and MCCC2 mutations p.Y146N and p.S523G (individuals No 56, 66 and 74). Thus, expression studies may not demonstrate/identify the specific functional abnormalities for at least some of the mutant alleles with residual enzyme activity. However, no other mutations were found despite sequencing of the complete coding region of both MCC genes.

                                      Genotype-phenotype correlations

                                      Our data confirm previous studies reporting no clear genotype-phenotype correlation in MCC deficiency [3, 6, 11] suggesting that factors other than the genotype at the MCC loci have a major influence on the clinical phenotype [11]. In line with this we identified clinically asymptomatic female adults carrying null mutations in homozygosity and siblings of which one was asymptomatic and the other showed neurologic symptoms compatible with influence of environmental factors on the clinical outcome of affected individuals.

                                      None of the mutations found in asymptomatic individuals with MCC deficiency detected by NBS was prevalent in this group, which is in contrast to other inborn errors of metabolism such as isovaleric acidemia [42] or medium-chain acyl-CoA dehydrogenase deficiency [43]. Consequently, genotyping still appears to be of no help in predicting the clinical outcome of individuals with MCC deficiency.

                                      Conclusions

                                      Our data confirm that MCC deficiency despite its low penetrance can lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting which affected individual is at risk of developing clinical symptoms.

                                      Declarations

                                      Acknowledgements

                                      We thank the following doctors and physicians for providing clinical information: Dr. M. Du Moulin (Münster, Germany), Prof. Dr. A. Das (Hannover, Germany), Dr. Konstantinos Tsiakas (Hamburg, Germany), Prof. Dr. F. Trefz (Reutlingen, Germany), Prof. Dr. J. Kreuder (Gießen, Germany), Dr. P. Hofstetter (Wiesbaden, Germany), Dr. B. Müksch (Köln, Germany), Dr. S. Beblo (Leipzig, Germany), Dr. C. Prasad (London, Canada), Dr. M. Schiff (Paris, France), Prof. Dr. A. Burlina (Padova, Italy), Dr. N. Darin (Göteborg, Sweden), Dr. Hanna Mandel (Haifa, Israel), Dr. F.S. Ezgü (Ankara, Turkey), Dr. D. Lianou-Trapezanoglou (Athens, Greece), Dr. N. Al Sannaà (Dhahran, Saudi Arabia), Dr. Y.H. Chien (Taipei, Taiwan), Dr. L. Lapagesse (Porto Alegre, Brazil), Dr. S. Cederbaum (Los Angeles, USA) and Dr. G. Horvath (Vancouver, Canada). We are also grateful to all physicians who referred patient samples to our laboratory.

                                      Sarah C. Grünert was supported by the Deutsche Forschungsgemeinschaft, Grant GR 3918/1-1. Martin Stucki was supported by the Swiss National Science Foundation Grant 32003AO-109219. Additional financial support of this work was provided by personal research grants dedicated to K.O. Schwab.

                                      Authors’ Affiliations

                                      (1)
                                      Division of Metabolism and Children’s Research Center (CRC), University Children’s Hospital Zurich
                                      (2)
                                      Center for Pediatrics and Adolescent Medicine, University Hospital Freiburg
                                      (3)
                                      Zürich Center for Integrative Human Physiology (ZHIP), University of Zürich
                                      (4)
                                      Metabolic Unit, University Children’s Hospital
                                      (5)
                                      Department of Clinical Genetics, Rigshospitalet
                                      (6)
                                      Perelman School of Medicine, Section of Biochemical Genetics, Children’s Hospital of Philadelphia, University of Pennsylvania
                                      (7)
                                      University Children’s Hospital Frankfurt
                                      (8)
                                      Division of Inherited Metabolic Diseases, University Children’s Hospital
                                      (9)
                                      Department of Pediatric and Adolescent Medicine, University Hospital Vienna
                                      (10)
                                      Metabolic and Muscular Unit, Clinic of Pediatric Neurology, Meyer Children’s Hospital
                                      (11)
                                      Department of Pediatrics, University Medical Center Hamburg-Eppendorf
                                      (12)
                                      Department of Biochemical Genetics, The Children’s Hospital at Westmead
                                      (13)
                                      Structural Genomics Consortium, University of Oxford

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