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Abnormally increased carotid intima media-thickness and elasticity in patients with Morquio A disease
Orphanet Journal of Rare Diseases volumeĀ 15, ArticleĀ number:Ā 73 (2020)
Abstract
Background
Cardiovascular disease frequently causes morbidity and mortality in mucopolysaccharidoses (MPS); however, cardiovascular anatomy and dysfunction in MPS IVA (Morquio A disease) is not well described. Consequently, the study aimed to compare carotid artery structure and elasticity of MPS IVA patients with other MPS patients and healthy control subjects, and quantitate frequency of MPS IVA cardiac structural and functional abnormalities.
Methods
Prospective, multi-center echocardiogram and carotid ultrasound evaluations of 12 Morquio A patients were compared with other MPS and healthy control subjects. Average differences between groups were adjusted for age, sex, and height with robust variance estimation for confidence intervals and P-values.
Results
Morquio A patients demonstrated significantly higher (Pā<ā0.001) adjusted carotid intima-media thickness (cIMT), mean (SD) of 0.56āmm (0.03) compared to control subjects, 0.44āmm (0.04). The Morquio A cohort had significantly greater adjusted carotid elasticity (carotid cross-sectional compliance +ā43%, Pā<ā0.001; carotid incremental elastic modulus āā33%, Pā=ā0.003) than control subjects and other MPS patients. Aortic root dilatation was noted in 56% of the Morquio A cohort, which also had highly prevalent mitral (73%) and aortic (82%) valve thickening, though hemodynamically significant valve dysfunction was less frequent (9%).
Conclusions
Increased carotid elasticity in Morquio A patients is an unexpected contrast to the reduced elasticity observed in other MPS. These Morquio A cIMT findings corroborate MPS IVA arterial post-mortem reports and are consistent with cIMT of other MPS. Aortic root dilatation in Morquio A indicates arterial elastin dysfunction, but their carotid hyperelasticity indicates other vascular intima/media components, such as proteoglycans, may also influence artery function. Studying MPS I and IVA model systems may uniquely illuminate the function of glycosaminoglycan-bearing proteoglycans in arterial health.
Background
Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease, MIM#253000) is an autosomal recessive lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). This deficiency results in excessive systemic storage of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) in tissues and organs [1]. Morquio A patients have coarse facial features, hearing and vision loss, systemic skeletal dysplasia, disproportionate short-trunked short stature, joint abnormalities, hepatomegaly, pulmonary compromise, and valvular heart disease [2]. Though the focus is often on skeletal deformities, cardiac anomalies have been reported as the first sign of Morquio A as early as 3.6āmonths of age [3]. Cardiac anomalies observed in Morquio A include mitral and/or aortic insufficiency, aortic stenosis, tricuspid regurgitation, thickened interventricular septum, and hypertrophic cardiomyopathy [3,4,5,6]. Echocardiography indicates that cardiac lesions worsen in patients [3]. An observational study that included 54 Morquio A patients showed enlarged aortic roots, thickened left ventricles, smaller end-diastolic dimensions of the heart, reduced stroke volumes and impaired diastolic filling patterns [7]. Effect of enzyme replacement therapy (ERT) seemed negligible after a median follow up of 2.6ā3.0āyears [5, 7]. In addition, histopathology in a 20-year-old patient revealed foam cells and macrophages in aorta, aortic, mitral and tricuspid valves [8]. Cells in the aortic intima and media were vacuolated with GAGs, and elastin fibers were attenuated and fragmented. The intima was thickened by GAG accumulation, atherosclerotic plaques were found, and the aortic valve was hypertrophic [8].
Studies show carotid intima-media thickness (cIMT) could be a marker of atherosclerosis and cardiovascular risk in patients [9]. cIMT and carotid stiffness have been consistently increased in MPS patients when compared to age-matched controls [10, 11]. To date, no study explores cIMT in Morquio A. This study aimed to compare cIMT and carotid stiffness of Morquio A patients with other MPS patients and healthy controls.
Results
Morquio A carotid intima media-thickness
Details regarding the control and non-Morquio MPS cohorts have been published [11, 12]. The Morquio A cohort (nā=ā12) was 58% male and 42% female (TableĀ 1). Patient 2 had a follow-up study visit with carotid ultrasound 16āmonths after initiating enzyme replacement. Data from this subsequent visit are included in reporting and statistical analysis, resulting in 13 data points (TableĀ 2). Although the age at visit is slightly older (meanā±āSD), (14.0ā±ā10āyears) due to the inclusion of four adults, mean height of the Morquio A cohort (108ā±ā21.89ācm) was much less than the control (155ā±ā18.30ācm) and non-Morquio MPS (132ā±ā16.55ācm) cohorts. The mean Morquio A cohort cIMT was 0.56ā±ā0.03āmm, while the non-Morquio MPS cohort cIMT was 0.56ā±ā0.06āmm, and the control cohort cIMT was 0.44ā±ā0.04āmm. The mean Morquio A cIMT was 0.12āmm greater than the mean control cohort cIMT, an increase of 25% (See TableĀ 3 for detailed comparisons).
Comparison of mean carotid measures in all cohorts were adjusted for sex, age, and height, known covariates of cIMT and carotid stiffness (Table 3). The covariate-adjusted mean cIMT of the Morquio A patients was significantly greater than that of the control cohort (Pā<ā0.001) and comparable to the non-Morquio MPS patient cohort (Pā=ā0.866).
Carotid artery compliance and distensibility assessment
In contrast to the non-Morquio MPS cohort, which had significantly increased carotid stiffness compared to controls, the Morquio A cohort demonstrated significantly increased carotid elasticity compared to controls. Following adjustment for age, sex, and height, the Morquio A cohort had significantly higher cCSC1 (+ā0.07āmm2 Ā· mm Hgāā1; 95% confidence interval [+ā0.03 to +ā0.11] mm2 Ā· mm Hgāā1; Pā=ā0.001) and lower cIEM (āā294āmmHg; [āā503 to āā85.7] mm Hg; Pā=ā0.006) than the control patient cohort. This reflects a 43% increase [+ā15% to 75%] in cross-sectional compliance, and a 33% [āā49% to āā13%] reduction in incremental elastic modulus, of the Morquio A patients when compared to the control cohort. Carotid cross-sectional distensibility did not differ significantly between Morquio A and control cohorts (āā7.5%; [āā30% to +ā12%]; Pā=ā0.52).
The Morquio A cohort also had significantly higher cCSC (+ā0.09āmm2 Ā· mm Hgāā1; [+ā0.05 to +ā0.14] mm2 Ā· mm Hgāā1; Pā<ā0.001) and lower cIEM (āā681āmmHg; [āā984 to āā379] mm Hg; Pā<ā0.001) than the non-Morquio MPS cohort, indicating increased Morquio A carotid elasticity compared to MPS patients who did not have Morquio A syndrome. The mean Morquio A cross-sectional compliance was 64% greater, and the incremental elastic modulus 54% reduced, compared to the non-Morquio MPS patients. Overall, age was the strongest and most consistent significant co-variate aside from MPS diagnosis for increasing carotid stiffness, with reduced cCSD: 29% decline per 10āyears, Pā<ā0.001; cCSC1: 17% decline per 10āyears, Pā<ā0.001; and increased cIEM: +ā48% increase per 10āyears, Pā<ā0.001).
Treatment effects upon carotid measurements
No clear differences in cIMT or other measurements were observed in the two studies obtained from patient 2 (first measurement pre-ERT and second measurement 14āmonths post-ERT). To further examine effects of treatment upon carotid structure and function, a comparison was made between the patients who were receiving ERT at the time of study, and those who were not receiving ERT. There were no significant differences in cIMT (0.00āmm, 95% confidence intervalāāā0.03 to +ā0.03āmm; Pā=ā0.92), cCSD (āā2.20%, [āā8.91% to +ā4.51%]; Pā=ā0.52), or cCSC (āā0.03āmm2 Ā· mm Hgāā1, [āā0.08 to +ā0.02] mm2 Ā· mm Hgāā1; Pā=ā0.28) between the Morquio A patients currently treated with ERT and those not treated with ERT adjusting for sex, age, and height (TableĀ 4). Morquio A patients treated with ERT had significantly increased cIEM compared to those not treated (+ā137, [+ā2.44 to +ā271.29] mm Hg; Pā=ā0.046). For this comparison, height was a significant co-variate for carotid stiffness, with reduced cCSD: āā2.98% / 10ācm, Pā<ā0.001; cCSC1: āā0.02āmm2 Ā· mm Hgāā1/ 10ācm, Pā<ā0.001; and increased cIEM: +ā131āmmHg / 10ācm, Pā<ā0.001).
Cardiac structure and function
The Morquio A cohort had high occurrence of mitral (72%; 8 of 11, 95% confidence interval 39 to 93%) and aortic valve (82%; 9 of 11, [48 to 97%]) thickening (TableĀ 5). Though abnormal valve anatomy was frequent, dysfunction was not severe. Trivial mitral valve regurgitation was noted in 18% (2 of 11), [3 to 52%] of Morquio patients. There did not appear to be an age predisposition for mitral valve dysfunction, as the two individuals were 3.9 and 8.5āyears of age and were noted to have āthickenedā and āmildly dysplasticā mitral valves, respectively. Aortic valve regurgitation was present in 27% of (3 of 11), [7 to 61%] Morquio A patients. Two patients had trivial aortic regurgitation; they were aged 8.5 and 16.1āyears. One patient had combined regurgitation of both mitral and aortic valves. One patient, aged 22.3āyears, had more prominent aortic regurgitation that had resulted in mild left ventricular dilatation. No patients demonstrated abnormal cardiac contractility or function. Aortic root dilatation, defined as a measurement at the level of the Sinuses of Valsava exceeding 2 standard deviations above the mean, was also frequent in the Morquio A population (56%; 5 of 9, [23 to 85%]).
Patient brief summaries
Patient 1
Iraqi Caucasian girl, diagnosed with Morquio A at 10.6āyears of age. Sequencing of GALNS showed that she was homozygous for the pathogenic variant c.230Cā>āG (p.P77R). Study echocardiography and carotid ultrasounds were performed at 11.2 and 12.7āyears of age. Her first Morquio A-related symptom was kyphosis at 4āmonths of age; she suffered from frequent upper respiratory infections and was unable to walk by 5āyears of age. Physical examination identified coarse facial features, corneal clouding, macrocephaly, short nose, poor dentition with wide spaced teeth, and mandibular prominence. Skeletal dysplasia was evident, with short stature, short trunk, kyphosis, lordosis, pectus carinatum, lower rib flaring, short neck with decreased rotation and flexion, genu valgum, platyspondyly, and marked laxity in wrists and fingers leading to difficulties with fine and gross motor tasks. She had contractures at knees, hips and ankles, canal stenosis, cervical cord compression, myelopathy and cervical instability. There was progressive weakness and difficulty breathing at rest. Patient had obstructive sleep apnea. The echocardiogram showed mild thickening of the mitral valve, no significant stenosis, no significant ventricular hypertrophy and normal biventricular systolic function. Patient had her first recombinant human GALNS (rhGALNS) enzyme infusion at 11.6āyears old without any complications. At 11.7āyears, she underwent tracheostomy, cervical fusion and decompression. At 12.5āyears, bilateral sensorineural hearing loss was diagnosed.
Patient 2
Iraqi Caucasian boy; he is the younger sibling of patient 1 and was diagnosed with Morquio A at 6.2āyears of age with homozygosity of the c.230Cā>āG (p.P77R) family variant. Study echocardiography and carotid ultrasounds were performed at 7.9 and 9.2āyears of age. Physical examination identified macrocephaly, coarse facial features, corneal clouding, poor dentition, gingival hypertrophy, and mandibular prominence. He had skeletal dysplasia with short stature, short and stiff neck, platyspondyly, kyphosis at the thoracolumbar junction, anterior beaking in vertebral bodies, oar-shaped deformity of ribs, deformed femoral heads, flat acetabular roofs, bilateral coxa valga, genu valgum, pectus carinatum, wrist deformity, progressive joint laxity, difficulty with fine and gross motor tasks, and diminished motion of shoulders. He had difficulty walking, a gait problem and general weakness. At 7āyears, atlantoaxial subluxation with hypoplastic C2 odontoid process was confirmed, with subsequent occipital-cervical instability, myelopathy, and decline of ambulation. Echocardiogram showed very mild thickening of aortic and mitral valves, no significant valvar stenosis, regurgitation or ventricular hypertrophy. He had normal biventricular systolic function. Pulmonary studies suggested a narrowed oropharynx, impaired cough clearance, severe respiratory muscle weakness, neuromuscular weakness, daytime hypersomnolence, upper airway obstruction, restrictive lung disease with shortness of breath and obstructive sleep apnea, and multiple upper respiratory infections. First rhGALNS enzyme replacement therapy (ERT) occurred at 7.2āyears without complications. He reported increased stamina after ERT, but subsequently developed a rash after enzyme infusions.
Patient 3
American Caucasian man of Middle Eastern ancestry. He was diagnosed at 12āyears of age. Molecular studies identified the GALNS mutations c.1353Gā>āT / c.1353Gā>āT (p.R451S/R451S). Study echocardiography and carotid ultrasound were performed at 38.4āyears of age. His skeletal dysplasia includes decreased bone density, generalized platyspondyly, bullet-shaped vertebra at the thoracolumbar junction, and generalized central canal stenosis of the cervical spine. He also had anterior atlantoaxial instability with a hypoplastic or absent odontoid process. Other notable medical problems include mild corneal clouding, joint contractures, and diffuse joint, muscle, and bone pain. Multiple surgeries have included bilateral hip replacement, gynecomastia reduction, gastric bypass, and cholecystectomy. There have been no reports of hearing problems, pulmonary or cardiac symptoms. At 37, he started enzyme replacement, which has resulted in improvement of restricted joint mobility.
Patient 4
Southern Chinese girl diagnosed at age 2āyears (leukocyte GALNS 0.58ānmol/17āh/mg protein; reference range 42.3ā441.96; GALNS variants c.106_111delCTGCTC / c.953āTā>āG). Study assessments were performed at 6.4āyears of age. Physical examination at assessment demonstrated disproportionate short stature, with a height Z-score of āā6.0; mild coarse facial features, depressed nasal bridge, pectus carinatum, kyphosis, ulnar deviation of forearms, genu valgum, and severe joint hyperlaxity. Spinal magnetic resonance imaging (MRI) identified platyspondyly with cervicomedullary and T10 spinal canal stenosis, and absence of spinal instability despite incomplete ossification of the odontoid. Her echocardiogram identified normal left ventricular size and function with mild thickening of the mitral and aortic valve leaflets without regurgitation or stenosis. The aortic root absolute measurement was 18āmm (Z scoreā+ā1.20). She received rhGALNS from age 6.4 until age 8.2 with moderate reactions (hypotension, tachycardia, nausea, and emesis). ERT was discontinued at parental request due to arrest of linear growth and progression of genu valgum.
Patient 5
Hispanic male with GALNS pathogenic variants c.1156Cā>āT and 32ākb deletion. At the time of study assessment, he was 18.3āyears old and demonstrated disproportionate short stature, with a height Z-score of āā8.11; coarse facial features and depressed nasal bridge, and ulnar deviation of his forearms with wrist and ankle hyperlaxity. He required tracheostomy due to upper airway obstruction and tracheomalacia. He had been receiving rhGALNS ERT beginning at age 17.3 without any infusion-associated reactions.
Patient 6
Hispanic male diagnosed at 2.8āyears of age (GALNS c.901Gā>āT / c.1520Gā>āT pathogenic variants) after presenting at 3āmonths of age with lumbar gibbus and subsequently developing disproportionate short stature. At study assessment, he was 3.87āyears old and his height Z score was āā3.73. Physical examination identified frontal bossing, depressed nasal bridge, pectus carinatum, thoracolumbar kyphosis, global joint hyperlaxity, and genu valgum. Spinal MRI identified platyspondyly with moderate cervical canal stenosis, and hypoplastic dens without craniocervical instability. Echocardiogram identified thickening of mitral and aortic valve leaflets with trivial mitral valve regurgitation and normal aortic valve function. His aortic root was moderately dilated with a measurement of 21.6āmm (Z scoreā+ā3.28). He began receiving rhGALNS at age 3.2 ERT without reactions. Subsequently, he has required acetabuloplasty and femoral/tibial epiphyseodesis due to hip dysplasia and genu valgum.
Patient 7
Caucasian female who presented at 18āmonths of age with genu valgum and spinal kyphosis, and subsequently diagnosed with Morquio A at age 3 (deficient leukocyte GALNS enzyme; one pathogenic GALNS c.239Cā>āT variant identified). Her echocardiogram, carotid ultrasound, and other study assessments were performed at age 8.5āyears. Physical examination at assessment demonstrated a height Z score of āā6.77, disproportionate short stature, depressed nasal bridge, prominent pectus carinatum, severe wrist and ankle joint hyperlaxity, and genu valgum. Spinal MRI showed platyspondyly, craniocervical stenosis without instability, thoracic kyphosis, and lumbar hyperlordosis. Cardiac imaging identified trivial mitral and aortic valve regurgitation, and an aortic root measurement of 20.6āmm (Z scoreā+ā1.97). She has required bilateral hip acetabuloplasty and femoral/tibial epiphyseodesis. She had received investigational rhGALNS and declined to continue enzyme therapy.
Patient 8
She is the younger sister of patient 7 and was diagnosed at birth due to family history. She was 4.6āyears old at time of study assessment, which identified a height Z score was āā2.17 with physical examination significant for disproportionate short stature, depressed nasal bridge, pectus carinatum, mild wrist / ankle joint hyperlaxity, thoracolumbar gibbus deformity, and genu valgum. Spinal MRI showed platyspondyly, and mild cervical spinal canal stenosis without instability. Echocardiogram identified thickening of the mitral and aortic valves without valve dysfunction. The aortic root measured 18āmm (Z scoreā+ā1.06). She has also undergone bilateral hip acetabuloplasty and femoral/tibial epiphyseodesis. She received investigational rhGALNS as an infant and declined to continue with enzyme therapy. At most recent clinical assessment, her short stature has worsened, and her current height Z score is āā6.34 at age 8.25āyears, demonstrating a severe Morquio A phenotype like her older sister.
Patient 9
He is a Hispanic male initially misdiagnosed as spondyloepiphyseal dysplasia, subsequently identified to have Morquio A at 15āyears of age after biochemical evaluation discovered elevated urinary KS and deficient fibroblast GALNS enzymatic activity. His study echocardiogram, carotid ultrasound, and other evaluations were performed at 25.9āyears of age. Study evaluation was significant for disproportionate short stature (adult height 134.4ācm; approximate height Z scoreāāā6.09), minimally coarse facial features, ulnar deviation of his forearms, mild joint hyperlaxity, and minimal genu valgum status post femoral/tibial epiphyseodesis. He had also undergone T10 ā L3 spinal fusion due to kyphosis. Echocardiogram identified mild thickening of the mitral valve with neither mitral nor aortic valve dysfunction. His aortic arch could not be well-visualized due to habitus. He declined rhGALNS therapy.
Patient 10
He is a Hispanic male who was diagnosed at 3āyears of age following identification of dysostosis multiplex and subsequent demonstration of urinary keratan sulfaturia with deficient fibroblast GALNS enzyme (7000āunits; reference rangeā>ā40,000) and eventual demonstration of GALNS pathogenic variants (c.1156Cā>āT / c.924āTā>āGā+āc.930Gā>āC). Study assessments were performed at 22.26āyears of age. Physical examination identified macrocephaly (56.5ācm), short stature (adult height 109.2ācm; Z scoreāāā9.1), mild corneal clouding, coarse facial features, marked wrist and ankle joint laxity, genu valgum, brachydactyly, pectus carinatum, and thoracolumbar kyphosis. His echocardiogram was significant for normal mitral valve anatomy and function, but moderate aortic valve regurgitation, mild left ventricular dilatation (Left ventricular / LV mass index 170.1āg/m2), and aortic root dilatation (32.9āmm, Z-scoreā+ā4.95). He had undergone occipitocervical decompression and fusion, as well as bilateral hip osteotomies prior to study assessments. He has been treated with rhGALNS without reactions.
Patient 11
She is a Cuban/Caucasian female, first noted by parents at 2āyears of age to have lumbar kyphosis. Subsequent spinal MRI suggested MPS, and biochemical evaluations identified elevated urinary KS, deficient fibroblast GALNS (0.05āunits, reference rangeā>ā0.7), and eventual molecular confirmation of GALNS pathogenic mutations (c.776Gā>āA / c.1520Gā>āT). Study echocardiography, ultrasound, and examination occurred at 10.4āyears of age. Study physical examination identified mild disproportionate short stature (height Z-scoreāāā2.23), minimally coarse facial features, normal corneas, absence of pectus carinatum, and mild joint hyperlaxity. S-shaped curvature of the cervical-thoracic junction manifested with cord compression at age 11 and required decompression with spinal fusion. Her echocardiogram showed minimally redundant mitral and aortic valve leaflets without regurgitation or stenosis. Her aortic root measured 21āmm (Z-scoreā+ā0.43). She has been treated with intravenous rhGALNS without complications.
Patient 12
Hispanic male whose first notable symptom of Morquio A was pectus carinatum at age 2āyears. After developing abnormal gait at 5āyears of age, he was referred for clinical genetics evaluation and formally diagnosed with Morquio A at age 8. Urinary GAG excretion was elevated; his leukocyte GALNS activity was undetectable, and sequencing of GALNS identified c.281Gā>āA / c.1156Cā>āT pathogenic variants. Study assessments were performed at 16.1āyears of age. Study physical exam demonstrated disproportionate short stature (height Z-scoreāāā4.27), mild facial coarseness, pectus carinatum, thoracolumbar gibbus, forearm ulnar deviation, and hyperlaxity of joints. Mitral valve anatomy and function were normal, while he demonstrated trivial aortic valve regurgitation. Aortic root was mildly dilated, measuring 28.2āmm (Z-scoreā+ā2.23). He has been treated with intravenous rhGALNS without reactions.
Correlation between genotype and phenotype
Patients 1 and 2 are homozygous for the mutation p.P77R (c.230Cā>āG) in exon 2, which is associated with a severe phenotype due to a non-conservative change of proline to arginine in a conserved amino acid region of GALNS affecting its hydrophobic core [13, 14]. These patients show mildly thickened mitral valve and/or mildly thickened aortic valve with normal function. Patients 3 and 11 exhibit an attenuated phenotype based on their growth charts. Despite the difference in their age and gender, these patients have normal or minimally thickened mitral and aortic valves with normal function and normal aortic root Z score. Patient 3 is homozygous for the novel mutation p.R451S (c.1353āGā>āT) in exon 12 that is likely to be pathogenic with an in-silico score of 0.918 (sensitivity: 0.81; specificity: 0.94) [15]. Patient 11 has the allele c.776āGā>āA (p.R259Q, in exon 8), which is located on the surface of GALNS enzyme and gives an attenuated phenotype [13]. The other allele was c.1520āGā>āT (p.C507F) in exon 14 which has been classified as ālikely disease-associatedā mutation and has not been characterized in detail [16].
Patients 10 and 12 (22.3 and 16.1āyears old, respectively) have severe phenotype of the disease with aortic valve pathology including cusp asymmetry or slightly thickened aortic valves with moderate regurgitation. In addition, both patients show a dilated aortic root. Their genotypes share the allele c.1156Cā>āT (p.R386C) which is the most frequently reported GALNS mutation, located in exon 11 and associated with severe phenotype [14, 17, 18].
Patients 4 and 6, who were 6.4 and 3.9āyears of age, have severe phenotype with severe heart disease evidenced by mitral and aortic valve pathology. Both have exon 9 mutations (p.M318R and p.G301C) known to impact protein folding and cause severe phenotype [13, 19,20,21].
Patients 7 and 8 (8.5 and 4.6āyears old, respectively) are siblings with severe phenotype with mitral and valve pathology but with normal aortic root Z-score. They share the mutation p.S80L which affects the active site of GALNS enzyme [21,22,23]. The other allele has not been elucidated, but the Morquio A diagnosis is secure given patient 7ās low GALNS enzymatic activity.
Discussion
This study of twelve patients with MPS IVA, or Morquio A disease, demonstrates a high prevalence of structural and functional abnormalities in their carotid arteries and left-sided cardiac valves. The small MPS IVA cohort and the cross-sectional nature of sonographic assessments represent limitations to this study; however, we have utilized the same approach for statistical analyses with other, similarly-sized MPS cohorts [11, 12].
Carotid intima-media thickness
Mean cIMT of Morquio A patients was 0.56āmm, comparable to the mean cIMT of 0.56āmm found in 33 patients with other types of MPS (17 MPS I, 9 MPS II, 4 MPS IIIA, and 3 MPS VI), and 0.12āmm (26%) thicker than the mean cIMT of a large, healthy control cohort. ERT did not affect cIMT measurements in the Morquio A cohort. The mechanism of cIMT thickening in Morquio A is very similar to other MPS types: a combination of mechanical expansion caused by GAG storage, extracellular matrix proliferation, and recruitment of myofibroblasts and activated macrophages throughout arterial vasculature [12]. The in vivo cIMT findings corroborate the post-mortem examination of aortic intima and media from a 20-year old male with Morquio A disease, which identified infiltration of foamy GAG-laden macrophages and abundant accumulation of C6S within the intima-media matrix [8].
Mitral and aortic valve disease
Hypertrophy of the decedentās mitral and aortic valves, which contained large numbers of foam cells and inflammatory T-cell and macrophages has been reported [8]. Such findings correlate with the in vivo findings of our Morquio A cohort, which demonstrated a high proportion of patients with thickening of the mitral and aortic valves, and a smaller percentage who had valvular regurgitation. Age did not clearly correlate with severity of valve thickening or dysfunction. Valve thickening was observed in young and adult patients alike, and the three patients who did not have mitral valve thickening were 16.1, 22.3, and 38.4āyears old. Young patientsā valve thickening may reflect greater Morquio A severity. Likewise, absence of valve thickening in the oldest patient reflects a more attenuated disorder. Despite this observation, we note that the 22.3-year-old who had a normal mitral valve also had the most dysplastic and regurgitant aortic valve. Thus, valve disease in Morquio A may also be simultaneously discordant and progressive (this patientās aortic regurgitation worsened with age).
Carotid hyper-elasticity
An unexpected finding of this study was markedly increased carotid elasticity in Morquio A patients. The Morquio A cohort had cCSC1 that was 43% and 64% greater than the control and non-Morquio A MPS cohorts, and a cIEM that was 33% and 54% reduced compared to the control and non-Morquio A MPS cohorts. This is counter to the significantly reduced carotid elasticity in patients with non-Morquio A MPS previously identified by our group [10,11,12]. Compliance or distensibility did not differ between Morquio A patients on or off ERT, but cIEM was higher in the ERT-treated group after adjustment for sex, age, and height. Whether this difference is an effect of treatment will require additional, longitudinal assessments. Elastin, a highly abundant protein within the arterial media, is arranged in tightly coiled laminae and allows arteries to withstand blood pressure oscillations throughout the cardiac cycle. Attenuated, fragmented arterial elastin laminae have been observed in MPS types I, VI, and VII patients and animal models [12, 24], a result of proteolysis by cathepsins and matrix metalloproteinases secreted by GAG-activated macrophages [25]. We hypothesized that reduced carotid elasticity in MPS I, II, IIIA, and VI patients was a consequence of the destruction in arterial elastin. Precedence for this hypothesis comes from Marfan syndrome patients, where disarrayed and fragmented arterial elastin fibrils are a cardinal feature, giving rise to reduced carotid compliance and distensibility and ultimately life-threatening aortic root dissection [26]. Since elastin attenuation and fragmentation was observed in Yasudaās Morquio A patient [8], we expected the Morquio A cohort to demonstrate abnormally low carotid elasticity compared to our other MPS cohorts.
Despite the unexpectedly increased carotid elasticity in the Morquio A cohort, the cohortās high prevalence of aortic root dilatation (56%), comparable to the 69% (11 of 16) prevalence in an independently ascertained Morquio A cohort [27] and a high prevalence in older Morquio A patients [7] comprise evidence that pathology of arterial elastin in Morquio A has clinical significance. While no Morquio A patient has yet been reported to experience aortic dissection, improved life spans of MPS IVA patients necessitate education for families and physician providers to recognize and promptly seek medical intervention if dissection symptoms develop. An additional potential step, mirroring aortic root management in Marfan syndrome, would be to determine what magnitude of aortic root dilatation, if any, would warrant elective surgical correction. The existence of multiple co-morbidities in Morquio A, notably airway access challenges [28], redundancy and malacia of the tracheobronchial tree [29], and risk of watershed spinal ischemia [30], result in an extremely high-risk surgery. Thus, the authors are reluctant to recommend any aortic root surgery until further natural history studies document the occurrence of aortic root dissection in Morquio A.
Carotid hyper-elasticity in the context of arterial elastin fragmentation in Morquio A seems incongruous, but may uniquely illuminate the function GAG-bearing proteoglycans (PGs) in arterial health. Differences in GAG species accumulated in Morquio A (KS and C6S), compared to MPS types I / II / VI (heparan and dermatan sulfates; HS and DS, respectively), may be the key to understanding the stark differences in carotid function between Morquio A and other types of MPS. GAGs represent 1ā2% of arterial dry weight in healthy individuals [31, 32]. Attached to PGs and secreted into the extracellular matrix, they form complexes with elastin, collagen, and other fibrillar proteins that generate viscoelastic properties required to maintain arterial integrity through a lifetime of cardiac cycles. Alterations in balance between KS, C6S, HS, and DS-bearing PGs may alter arterial elastic properties independent of elastin content. Indeed, versican, a C6S-PG, is known to accumulate in early intimal thickening, large atherosclerotic lesions, and areas of vascular injury and serves as a ligand for inflammatory macrophage adhesion and chemokines [33]. Short of atherosclerosis, many of these findings are observed in Morquio A patients and model systems, making such versican and other KS/C6S-bearing PGs the focal point of MPS vascular pathology investigation. Perhaps these investigations will also explain joint hyperlaxity only observed in Morquio A patients, a contrast to the joint stiffness and contractures in all other MPS types. Additional studies are in progress to better document the cardiovascular natural history in Morquio A patients, and to compare with great detail the alterations and downstream effects of arterial KS/C6S storage in the Morquio A model system, versus arterial HS/DS storage in MPS I model system.
Conclusions
Carotid hyper elasticity, increased thickening of left-sided heart valves, and increased cIMT constitute most of the structural and functional abnormalities observed in this cohort of 12 Morquio A patients. Since cardiac lesions worsen with time, patients should be evaluated periodically by their cardiologist. Adding carotid ultrasonography with evaluation of cIMT and vessel elasticity to the diagnostic toolbox will aid in understanding Morquio A progression and evaluating current and novel treatment strategies.
Methods
Study aim, design, and settings
The study aimed to compare echocardiographic findings and carotid intima-media thickness and stiffness in individuals with Morquio A disease, other MPS types, and unaffected controls utilizing a prospective, case-control design. The study took place at Childrenās Hospital of Orange County, Saint Louis University, and the University of Minnesota.
Human subjects
Studies were conducted following informed consent / assent (CHOC IRB#131107, SLU IRB#24454). Morquio A was diagnosed based on reduced GALNS enzyme activity of ā¤5% the normal level in plasma, fibroblasts or leukocytes, and/or via GALNS molecular testing demonstrating two or more pathogenic mutations. All Morquio A patients followed clinically at CHOC and Saint Louis University were eligible for the study, and all enrolled; control patients were ascertained at the University of Minnesota. We classified disease severity by patient height, with attenuated disease classified as ā„90th centile height isopleth, and severe disease as <90th centile height isopleth for Morquio A patients [34]. Patient urine was collected after obtaining informed patient consent or guardian assent. Enzyme activity in patient samples was compared to activity in urine samples from five healthy controls to confirm Morquio A diagnosis. Chart review was conducted for the 12 Morquio A patients, and relevant summaries of clinical, biochemical, radiographic, and molecular diagnostics evaluations are summarized in Table 1.
Carotid ultrasound
Ultrasonographers with at least 7āyears of experience examined patients, assisted by a senior technologist. All exams were performed with a General Electric Logiq E9 machine (GE Healthcare, Waukesha, WI) and linear 9 mHz transducer. A standard automated blood pressure monitor was used with pediatric cuff. Subjects were placed supine without a pillow and with legs uncrossed. Subjects were then asked to extend their neck and tilt their head back and to the right to expose the left common carotid artery. Subjects were asked to close their mouths and to not speak or swallow during the exam. An automatic blood pressure cuff was applied to the right upper arm. The ultrasound transducer was placed approximately 10āmm proximal to the bifurcation of the internal and external carotid arteries in the longitudinal plane. The transducer was manipulated to optimize visualization of the far wall intima-media thickness. Presets were adjusted to carotid mode; parameters such as focal zone placement, gain, and time gain compensation were optimized by the examiner on a case by case basis. The automatic blood pressure machine was engaged. The technologist then took a 10-s cine series while blood pressure was obtained. Exams were saved in the Picture Archiving and Communication System (PACS) and analyzed at a central facility (University of Minnesota). Of note, Patient 2 underwent two carotid ultrasounds at 7.0 and 8.4āyears of age. Data from both time points are reported and incorporated in statistical analyses.
Carotid structure and function definitions
Carotid intima-media thickness (cIMT), cross-sectional distensibility (cCSD), cross-sectional compliance (cCSC1), and incremental elastic modulus (cIEM) [35] were calculated utilizing Vascular Research Tools 5, Medical Imaging Application, LLC, Coralville, IA, USA. Although multiple technicians were involved in collection of carotid ultrasounds, this software suite automates the quantification of the parameters and is operator-independent, minimizing any variability between technicians [35].
cCSD is defined by the percentage change in area of the carotid lumen from diastole to systole and is defined by the equation [35]:
where sD indicates the maximum carotid lumen diameter during systole, and dD is the minimum carotid lumen diameter during diastole. A higher vessel cCSD indicates increased elasticity / reduced stiffness; a lower vessel cCSD corresponds to reduced elasticity / increased stiffness.
cCSC1, which has a unit of mm2 / mm Hg, is defined by the relative change in carotid lumen area from diastole to systole per unit change in blood pressure and is defined by the equation:
where SBP and DBP refer to systolic and diastolic blood pressure, respectively. Another way to conceptualize cCSC1 is the increase in carotid lumen area for each increase in blood pressure of 1āmmHg. Similar to cCSD, increasing cCSC1 represents increased elasticity / reduced stiffness (more change in carotid area for a given change in blood pressure), and vice versa.
Finally, cIEM represents the carotid elasticity constant. Increased cIEM indicates more difficulty to distend the vessel and therefore reduced elasticity / increased stiffness, and vice versa. The equation for cIEM, which has the unit of mm Hg, is:
Apparatus and sample preparation
The chromatographic system consists of an HP1100 system (Agilent Technologies, USA) and a Hypercarb column (2.0āmm i.d. 150āmm, 5āμm, Thermo Electron, USA). An API-4000 mass spectrometer (Applied Biosystems) equipped with a turbo ionspray ion source was used. Stock solution of GalĆ1,4GlcNAc(6S) (500āμg/ml), ĪDiHS-0S (100āμg/ml), ĪDiHS-NS (10āμg/ml), ĪDi-4S (100āμg/ml), ĪDi-6S (100āμg/ml) (Seikagaku, Tokyo, Japan), and internal standard (IS-chondrosine) (50āμg/ml) (Glycosyn, New Zealand) were prepared in water. Urine was centrifuged and supernatants digested overnight with 1āmU of chondroitinase b, 1āmU heparitinase, and 1āmU keratanase II (Seikagaku, Tokyo, Japan) in buffer solution (1% BSA, 50āmM Tris-HCl, 5āmg/ml Chondrosine). Recovered samples were analyzed by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and normalized by creatinine concentration in urine. Disaccharide GAG concentrations were calculated by Analyst 1.5.1 software (AB SCIEX, Foster City, CA).
Statistical analysis
Descriptive summaries were tabulated separately by disease group. These included the mean and standard deviation for continuous variables and frequency for categorical variables. One MPS IVA patient was measured at two time points, 1.35āyears apart: both measurements were included in analysis. Confidence intervals for proportions were computed by inverting the score test. Linear regression was used to evaluate differences in mean values between groups adjusting for age, sex, and height with robust variance estimation used for confidence intervals and P-values. Regression to evaluate ratios of means were evaluated similarly, but with a log-link. Statistical analyses were performed using R v3.5.1 [36].
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- C6S:
-
Chondroitin-6-sulfate
- cCSC:
-
Carotid Cross-Sectional Compliance
- cCSD:
-
Carotid Cross-Sectional Distensibility
- cIEM:
-
Carotid Incremental Elastic Modulus
- cIMT:
-
Carotid Intima-Media Thickness
- DBP:
-
Diastolic blood pressure
- DS:
-
Dermatan sulfate
- ERT:
-
Enzyme Replacement Therapy
- GAGs:
-
Glycosaminoglycans
- GALNS:
-
N-acetylgalactosamine-6-sulfate sulfatase
- HS:
-
Heparan sulfate
- KS:
-
Keratan sulfate
- LC-MS/MS:
-
Liquid chromatography ā tandem mass spectrometry
- LV:
-
Left ventricular
- mm Hg:
-
Millimeters of mercury
- MPS:
-
Mucopolysaccharidoses
- MRI:
-
Magnetic resonance imaging
- rhGALNS:
-
Recombinant Human N-acetylgalactosamine-6-sulfate sulfatase
- SBP:
-
Systolic blood pressure
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Acknowledgements
We are grateful to M. Marcinkowski for editorial assistance.
Funding
This research was supported by the National MPS Society (AMM and RYW), the Liferay Foundation (RYW), the Campbell Foundation of Caring (RYW), and Saint Louis University, Department of Pediatrics in association with the Cardinal Glennon Foundation (AMM). Additional support was provided to the University of Minnesota Clinical and Translational Science Institute by National Center for Advancing Translational Sciences, award #UL1TR002494 (KR), R01DK072124-01A3 (JS), and R01CA113930-01A1 (JS). The funding agencies played no role in the design of the study and collection, analysis, and interpretation of data and in writing of the manuscript.
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RYW and AMM conceptualized and designed the study, acquired and interpreted data, drafted the initial manuscript, and reviewed and revised the manuscript. They take full responsibility for the content of the manuscript. KDR analyzed and interpreted data, drafted the initial manuscript, and reviewed and revised the manuscript. DRD and NE acquired, analyzed, and interpreted data, and reviewed and revised the manuscript. QG analyzed and interpreted data, and reviewed and revised the manuscript. NM, JS, RG, KC, DB, SRB, and MS acquired data and reviewed and revised the manuscript. All authors read and approved the final manuscript.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the authorsā institutional research committees. This study was approved by the Institutional Review Board of CHOC Childrenās Specialists (IRB # 131107) and Saint Louis University (IRB # 24454), and complied with the standards of the 1964 Declaration of Helsinki and current ethical guidelines.
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All patients or their parents consented to publication of clinical, biochemical, and molecular details.
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Wang, R.Y., Rudser, K.D., Dengel, D.R. et al. Abnormally increased carotid intima media-thickness and elasticity in patients with Morquio A disease. Orphanet J Rare Dis 15, 73 (2020). https://doi.org/10.1186/s13023-020-1331-y
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DOI: https://doi.org/10.1186/s13023-020-1331-y