The present study is the first to report the clinical and genetic profiles of patients with Morquio-A syndrome in Indian Gujarati patients. Our study cohort consisted of 25 patients with consanguinity in 28% and endogamy in the remaining. A previous study by Bidchol et al (15) was also carried out in Indian patients but mainly comprising northern and southern region of the country and none from Gujarat.
The mean age at the first symptom was 4.32 years. The mean age reported in various studies differ across populations with Bidchol et al (15) reporting a higher mean age of 6.7 years at presentation, whereas, Montano et al (23) and Lin et al (24) have reported a lower mean age of 2.1 years and 2.0 years respectively. This depends on awareness among clinicians, patients and availability of a diagnostic facility. In the present study, majority of the referrals were from pediatric orthopedic specialists. Use of urine GAG electrophoresis with high excretion of KS with CS seems to be the reason for early diagnosis.
The clinical phenotype of the patients consisted of marked features like short stature, short fingers to kyphosis, scoliosis, genu valgum and skeletal dysplasia. Leong et al (12) reported such severe phenotypes in all the Malaysian patients with Morquio A syndrome. A similar phenotypic spectrum was reported in Indian Morquio-A syndrome patients (15) where short stature, genu valgum and pectus carinatum were the most commonly observed. The International Morquio-A syndrome registry has given the 5 most common initial symptoms in Morquio-A syndrome to be short stature (49.9%), genu valgum (45.1%), kyphosis (44.4%), pectus carinatum (43.6%), and abnormal gait (37.8%). In this study we observed short stature (100%), genu valgum (32%) and kyphosis (40%). We found, mild kyphosis and scoliosis were common at an early age, as is expected. However, serious skeletal abnormality and dysostosis multiplex was observed in patients at an advanced age. These findings support the fact that the phenotypic severity increases with advancement of age as reviewed (25).
All the patients had persistent increased urine excretion of GAGs predominantly KS with or without HS (heparan sulphate). The similar observation has been reported in Morquio-A syndrome patients (3). This clearly indicates the use of urinary GAG electrophoresis as the first line screening test and as a differential diagnosis of Morquio-A syndrome. The GALNS enzyme activity in the KS positive patients ranged from undetectable to less than 10% of the total enzyme activity. Our results are consistent with that observed by Leong et al (12) in the Malaysian population and other reported studies (15, 26, 27).
On mutational analysis of the GALNS gene in the biochemically diagnosed Morquio-A syndrome patients, we found that out of the 14 exons, exons 1, 2, 5 and 11 harbor most mutations. The study by Bidchol et al (15) showed exon 8 to harbor most of the mutations followed by exon 1 and 7. This is in contrast to our observation of exon 2 (p.P77R) being the hot spot site for mutation. This is likely to be due to absence of Gujarati patients in the earlier study from India.
Our study has identified a total of eleven mutations, out of which eight are missense mutations. This finding is similar to that reported by Bidchol et al (15) as well as matches with the trend observed in HGMD, where for Morquio A, maximum missense mutations have been reported followed by other types. Five out of eight missense variants in our study have been previously reported. None of the ten most common mutations as per the earlier report (3) were found in our study. This suggests that there is heterogeneity at the molecular level. Thus, we can infer that mutation spectrum is influenced by ethnicity. One of the missense variants (p.P77R) more frequently seen in our study has been reported earlier to be present in Indian patient (20) with unknown ethnicity. This mutation was also reported being linked to severe phenotype (28), indicating the association of the variant with severe phenotype in Indian patients in the present study. Bidchol et al (15) has also reported a mutation at the same position (p.P77S), suggesting that the proline at position 77 is important for the enzyme activity and any change can lead to clinical manifestations of Morquio-A syndrome. As reported by Sukegawa (29), proline at 77 has a key role in preventing the internalization in hydrophobic patch of the protein. The positively charged side chain of arginine instead of proline is expected to have a destabilizing effect in this process. The missense variant p.L36R was also found in two patients. This variant has been previously reported by Tomatsu et al (30) and Morrone et al (14) indicating its association with attenuated phenotype. Another missense variant p.P151L reported in this study was also reported previously by Tomatsu et al (20). Proline is conserved at 151 position, thereby any substitution at this site will lead to alteration in packing of protein (29). The missense variant p.C79R was first reported by Bidchol et al (15) in an Indian patient. Our study also reports this variant in one patient in homozygous condition. The missense variant p.P125L was found in a patient in compound heterozygous condition. This variant has been reported earlier by Tomatsu et al (21) in Japanese population showing severe phenotype for Morquio-A.
Our study has also identified 6 novel mutations that include 3 missense mutations, 1 splice site, 1 small insertion and 1 small deletion. The novel missense mutation p.D39G is an active site mutation which was found in one patient (P7). The noticeable phenotype was short stature, kyphosis and hydrocephalus. Morrone et al (14) and Tuyuz et al (28) have reported p.D40N to be associated with severe phenotype in Morquio-A syndrome patient. The other two novel missense variants p.G255A and p.L350P were found in compound heterozygous and homozygous conditions respectively. The splice site variant c.121-7 C > G identified in the present study has not been reported till date. The patient with this mutation presented with X-ray showing spine lumbar lordosis and wrist broadening. Generally, mutations in the splice site region leads to the formation of abnormal protein due to splicing defect. In our study, we have also identified a small insertion p.I416HfsTer2 in one patient which is a novel mutation. The prominent clinical features of skeletal abnormality, kyphoscoliosis, frontal bossing and platyspondyly were observed in this patient. A small deletion in exon 8 was found in a patient in compound heterozygous condition. As per the data by Tomatsu et al (30), 20 small deletions have been reported in the GALNS gene. The deletion in the present study was not previously reported in Indian population.
A set of bioinformatics tools have predicted the pathogenicity of the novel mutations by in silico analysis as described earlier. And all of them were at the conserved regions and mutations at these positions are highly likely to be pathogenic and causative of Morquio-A syndrome.
Interestingly, p.P77R was predominantly found in the Patel community of Gujarat. In order to confirm the founder effect, haplotype study was carried out in unrelated individuals of the same ethnicity using dinucleotide microsatellite markers. All the tested individuals were negative for this mutation except two individuals showing a carrier status. This confirms that p.P77R is a founder mutation in the Gujarati Patel population. Our study further indicates the reason of significant burden of rare diseases in India as has been observed recently (31). A greater attention needs to be paid to the different subpopulations and the effect of rare disease on community health needs to be elucidated.