The genetic basis of classical galactosaemia in Polish patients

Classic galactosemia (OMIM #230400) is an autosomal recessive disorder caused by homozygous or compound heterozygous pathogenic variants in the galactose-1-phosphate uridylyltransferase gene (GALT; 606999) on chromosome 9p13. Its diagnosis is established by detecting elevated erythrocyte galactose-1-phosphate concentration, reduced erythrocyte galactose-1-phosphate uridylyltransferase (GALT) enzyme activity. Biallelic pathogenic variants in the GALT gene is confirmed by DNA analysis. Our paper presents molecular characteristics of 195 Polish patients diagnosed with galactosemia I, intending to expand the current knowledge of this rare disease's molecular etiology. To the best of our knowledge, the described cohort of galactosemia patients is the largest single-center cohort presented so far. Supplementary Information The online version contains supplementary material available at 10.1186/s13023-021-01869-3.

GALT defects lead to two types of galactosemia: the classic and biochemical variant ones (often referred to as Duarte galactosemia). The classic form usually manifests in the neonatal period, after galactose ingestion, with jaundice, hypoglycemia, liver insufficiency, vomiting and diarrhoea, renal tubular dysfunction, muscle hypotonia, cataract and urosepsis. Long-term complications, may include intellectual disability, verbal dyspraxia, motor abnormalities, and hypergonadotropic hypogonadism [2]. Early diagnosis, followed by treatment with a lactose-restricted (dairy-free) diet, is undeniably essential to avoid health issues, but some complications may still occur [3]. On the contrary, in Duarte galactosemia, where enzyme activity is typically reduced by 75%, the symptoms are much milder and dietary intervention is usually unnecessary [4]. On the molecular level, the classic galactosemia is caused by biallelic pathogenic variants, while in Duarte galactosemia, the so-called Duarte 2 variant (c.[-119_-116delGTCA;940A>G]) is either homozygous or heterozygous with pathogenic GALT variant [5]. Within the symptomatic spectrum of galactosemia, clinical variant galactosemia is also known. It occurs in Open Access *Correspondence: jezela@gmail.com † Jezela-Stanek Aleksandra and Bauer Anna have contributed equally to this work 1 Department of Genetics and Clinical Immunology, National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland Full list of author information is available at the end of the article African Americans, and native Africans in South Africa thus is not covered within our article [Berry 2000].
The prevalence of classic galactosemia depends on ancestry. This disease is more common among individuals of European ancestry than in the Asia population. Its prevalence in the UK ranges from 1:23,500 to 1:44,000, while it differs from 1:50,000 in the US to 1:400,000 in Taiwan and 1:700,000 in Japan [6,7]. In Poland, the prevalence of "classical galactosemia" is estimated at 1:35,000 [8,9].
In many countries, due to treatment options, hereditary galactosemia is a part of the Newborn Screening (NBS) programs [10]. However, GALAC1 is not included in Poland's newborn NBS program; therefore, most of the patients are diagnosed due to severe life-threatening symptoms, followed by an enzymatic test and molecular studies and/or positive family history. Only in the West Pomeranian region of the country, NBS for GALAC1 has been performed within a cross-border cooperation program.
The study aimed to summarize the results of genotyping of a Polish cohort of patients from a single centre-Institute of Mother and Child (who serves as an unofficial national reference centre for the patients with galactosemia), to get further insight into molecular defects in the GALT gene and to present the phenotypic variability on the examples of two families with novel GALT variants.

General characteristics of reported patients
One hundred ninety-five patients from 175 families (hence 175 probands and 20 siblings) born between 1972 and 2020 were included in the study based on clinical/ biochemical galactosemia suspicion and/or positive family history. As many as 190/195 (97%) patients from this group are treated in a single reference centre (Department of Inborn Errors of Metabolism and Paediatrics, Institute of Mother and Child in Warsaw).
In the neonatal period, the probands often presented cachexia, hyperbilirubinemia, coagulation disorders, tubulopathy, generalized oedema or cataract. Sepsis, urinary tract infections, or meningitis also occurred in many cases. The younger siblings were tested on day 1 of their lives and received a lactose-free formula from the first feeding. As a result, if confirmed, they were diagnosed in the asymptomatic period. Besides, one patient born in Ireland was diagnosed with NBS there; in four families, the proband was the younger child, and the diagnosis of older siblings was made with a delay.
Despite dietary treatment, many patients develop long-term complications. We often observed cognitive problems, intellectual disability, defects in speech and language development. Some patients complain of hand tremors. Primary ovarian failure is seen in many women with galactosemia. A significant problem in these patients is infertility, although five women from our group gave birth to healthy offspring. Diminished bone mineral density is not common thanks to the supplementation of calcium and vitamin D3.

History of individuals in whom novel GALT variants have been identified Family A
The first child with atypical classic galactosemia was diagnosed at the age of 5 months. Currently, a 5-year-old girl is the first child of unrelated, healthy parents. She was born after the uncomplicated pregnancy and delivery. She was breastfed, but due to her weight loss of up to 10% on the third day of life, urine analysis was ordered and revealed slight transient proteinuria from 1 month (0.4-1.85 g/l; normal values < 0.12 g/l). Urinary tract infection was excluded, and then her weight gain was adequate. The ultrasound examination of the abdominal organs demonstrated no abnormalities. Initially, the liver enzymes were in the normal range, but at 2 months of age, slight hypertransaminemia was observed (ALT 105 U/L, AST 165 U/L; normal values 7-40, 20-72, respectively). No coagulopathy was found. Two months later, her parents noticed nystagmus. An ophthalmological examination showed bilateral cataracts.
Further investigations revealed a significantly reduced GALT activity (5%; 0.12 mmol UDPgal/h/RBC; normal values 1.81-2.57) which confirmed the diagnosis of classic galactosemia. A lactose-free diet was introduced at the age of 5 months, and then proteinuria and hypertransaminasemia resolved. The girl underwent cataract surgery and intraocular lens implantation.
The proband's younger sister had an enzyme analysis on the first day of her life. A significant decrease in GALT activity was found (1%; 0.02 mmol UDPgal/h/RBC; normal values 1.46-2.21). The child was fed a lactose-free formula from the beginning and showed no clinical symptoms.
The girls are currently 5 and 3 years old. Their intellectual and speech development is as expected. Due to their still young age, the occurrence of long-term galactosemia complications cannot be assessed yet.

Family B
A boy was born at 40 weeks of gestation after an uneventful delivery. After birth, when breastfed, he developed severe jaundice from the second day of life (with a total bilirubin of 23.4 mg/dl), cholestasis, hypertransaminasemia and coagulation disorders. Moreover, urinary tract infection and E. coli sepsis were found. On day 5 of life, enzymatic research revealed undetectable GALT activity confirming a diagnosis of galactosemia. The diet was changed to lactose-free, with gradual clinical improvement and symptom relief.
Despite a properly followed diet, the boy had longterm galactosemia complications such as delayed speech development, impaired sensory integration, learning difficulties, and disharmonious intellectual development.
The boy had severe symptoms in newborn age and then long-term complications of galactosemia, and his enzyme activity was undetectable.

Molecular methods
DNA was isolated from peripheral blood leukocytes. The molecular test was perform using Sanger sequencing. The analysis procedure involved a two-step approachfirst, analysis of exons 6-9, and, if the genotype was determined, the remaining ones in the second step. The PCR and sequencing primers were self-designed (Additional file 1: Table 1). Fluorograms were analyzed using Mutation Surveyor Software (Softgenetics) and evaluate using HGMD (Human Gene Mutation Database, http:// www. hgmd. cf. ac. uk), GALT database [11], and ACMG (American College of Medical Genetics and Genomics) classification [12] using tools available at the VAR-SOME website (https:// varso me. com). The sequence NM_000155.4 was used as a reference.

Molecular characteristics of diagnosed individuals Novel variants
The p.Pro87Leu is not registered in any database, while the p.Glu352Gln has only been reported once in Gno-mAD (per 251,424 tested alleles) and is not present in any clinical resource. Both variants are assigned as likely pathogenic using ACMG criteria, and bioinformatic evaluation on the VARSOME platform showed that, in both cases, 19 out of 21 pathogenicity ranging algorithms classifies them as damaging. The p.Pro87Leu was detected in trans with a known pathogenic variants p.Tyr209Ser in patients from Family A, while p.Glu352Gln was in trans with p.Gln188Arg in patients from Family B.

Characteristics of the studied cohort
Among 175 probands, the full genotype was determined in 173 cases (99%). In 2 remaining patients with a mutation in one allele of GALT, only checking exons 6-9 was performed. In total, we identified 29 different mutations in the GALT gene on 348 of 350 alleles, including two novel variants: p.Pro87Leu and p.Glu352Gln (Tables 1,  2).

Discussion
Among the commonest GALT variants described in HGMD, c.563A>G (p.Gln188Arg) and c.855G>T (p.Lys285Asn) are the most abundant in our Polish group, identified with the frequency of 49.4% and 32.2%, respectively. Other recurrent variants like c.329-2A>C (3.2%), c.997C>T (p.Arg333Trp; 1.7%), and c.626A>C (p.Tyr209Ser; 1.7%) are far less frequent, while the majority of remaining variants are rare and were detected in less than 1.5% of the alleles. In terms of p.Gln188Arg and p.Lys285Asn, the observed frequencies are similar to those published by our group in 2001 [13], where those mutations were identified in 54% and 28% of mutated alleles, respectively. According to other research, the p.Gln188Arg was reported in approximately 70% of the alleles affected with GALT deficiency from the population of northern Europe [14], which is much higher than in our cohort. Simultaneously, the p.Lys285Asn was more prevalent in central European countries, suggesting its Slavic origin [14]. Indeed, according to international multicenter data gathered from December 2014 to July 2018 among 404 genotyped patients, 233/404 (57.7%) were homozygous for p.Gln188Arg, 7/404 (1.7%) were homozygous for p.Lys285Asn, and 29/404 (7.2%) were heterozygous p.Gln188Arg/p.Lys285Asn [15]. On the contrary, in our group, 53/175 (30.3%) and 26/175 (14.8%) probands were homozygotes for p.Gln188Arg and p.Lys285Asn, respectively. The 39/175 (22.3%) were identified as harboring p.Gln188Arg in trans with p.Lys285Asn. The limitation of our study is that the DNA of the parents was not accessible, therefore, we cannot rule out that certain homozygous cases were in fact heterozygous for identified mutation and 5-kb deletion (precisely: NM_000155. Regarding the rare variants, accounting for over 18% of GALT alleles in our study, we noticed that 20 out of 29 detected variants were identified in one or two cases, including two novels: p.Pro87Leu and p.Glu352Gln. This observation shows that the heterogeneity of these variants in Polish patients is much more significant than expected. Although most classical cases manifest clinical symptoms soon after birth, in the proband from Family A (genotype p.Pro87Leu/p.Tyr209Ser), a rare asymptomatic course of GALAC1 in the neonatal period was observed. The first symptoms were observed after 4 weeks of breastfeeding, but galactosemia was finally diagnosed at the age of 5 months, and GALT activity was about 5% measured at that time. Interestingly, in the patient's younger sister, the GALT activity was about 1% on the first day of life.
In the literature, galactosemia with GALT activity between 1 and 10% is sometimes referred to as clinic variant galactosemia. The most typical variant causing this phenotype is p.Ser135Leu. Unfortunately, we do not have biochemical data on the mentioned variants among our patients. Most probands did not have a galactose-1-phosphate assay performed prior to starting the diet because, as a rule, the diet should be started as soon as galactosemia is suspected, before the diagnosis is confirmed. The gal-1-P assay is only performed at the Institute of Mother and Child, and most patients had it completed at the first visit to our department, being already on a diet. In the case of another novel variant-p.Glu352Gln, detected in trans with p.Gln188Arg, the clinical picture was typical for classic galactosemia with undetectable GALT activity.
The majority of probands in our cohort manifests symptomatic course of galactosemia (data not shown), which could be anticipated, considering the high percentage of patients harboring p.Gln188Arg and p.Lys285Asn variants, associated with very low (0.2% compared to wild type) or non-detectable erythrocyte GALT enzyme activity [16], and the fact that they were diagnosed based on clinical symptoms.
Except for classic galactosemia discussed mainly herein, only three individuals have been diagnosed with Duarte galactosemia (biochemical variant   [17]. This clearly shows that the molecular spectrum of GALT gene variants is not yet fully discovered.

Summary
To the best of our knowledge, the described cohort of 195 patients with galactosemia is the largest singlecenter cohort presented so far. Our results support the previous observations that the p.Lys285Asn variant originates from Central Europe. However, over 18% of GALT alleles are sporadic variants, including two novel ones, p.Pro87Leu and p.Glu352Gln. Our results broaden the knowledge on the molecular pathology and epidemiology of GALT deficiency.