Identication of Missense Mutation Pro77Arg As A Founder Mutation in Morquio-A Syndrome in Indian Gujarati Patel Ethnicity

Background: Morquio A syndrome (MPS IVA) is a mucopolysaccharide group storage disorder caused due to the decient activity of the lysosomal enzyme N-acetylgalactoseamine-6-sulfatase encoded by GALNS. The present study represents the mutation spectrum of GALNS in 25 Gujarati Patel patients of India clinically and biochemically conrmed with Morquio-A disorder. Methods: Urinary GAG quantitation and leucocyte enzyme assay was carried out in all 25 patients. This was followed by molecular characterization by amplication and sequencing of the exons and adjacent intronic regions of GALNS gene. Haplotype analysis was performed in patients showing p.P77R variant, using microsatellite markers D16S3121, D16S3026 and D16S3023 and SNPs. Results: We identied 11 mutations that include eight missense mutations: (p.L36R, p.D39G, p.P77R, p.C79R, pP125L, p.P151L, p.G255A and p.L350P), one splice site mutation: (c.121-7C>G), one small insertion: (c.1241_1242insA, p.I416HfsTer2) and one small deletion: (c.839_41delACA). Of these, three missense mutations (p.D39G, p.G255A and p.L350P), one splice site and the two indels mentioned above are novel. In the present study, we found maximum number of mutant alleles in exon 2, and of note, the variant p.P77R was seen in fourteen patients. Conclusion: p.P77R variant was predominantly found in Gujarati Patel community and the results of haplotype analysis indicated it to be the founder mutation in this community. Further, a study of 200 unrelated healthy control participants from Gujarat has identied this mutation in the heterozygous status in two individuals. Overall, our study suggests that p.P77R is likely to be a founder mutation for Morquio-A syndrome in Gujarati Patel ethnicity. 94ºC for 30 seconds, annealing (normal primer - 50ºC, mutant primer - 61ºC) for 45 seconds, elongation at 72ºC for 1 minute and the nal elongation was at 72ºC for 5 minutes of 1 cycle. Amplied products were separated on 2% agarose gel at 100V for 30 minutes and viewed on UV image analyzer. packing of protein The missense variant p.C79R rst by in an Our study 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 identied 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 identied 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 identied 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.


Background
Morquio-A syndrome, (MIM# 253000) is an autosomal recessive glycosaminoglycan storage disorder with progressive skeletal dysplasia, among other symptoms. It is caused due to de cient activity of the lysosomal enzyme N-acetylgalactoseamine-6-sulfatase (GALNS, E.C.3.1.6. 4) resulting from mutations in GALNS gene (OMIM* 612222) (1,2). The de ciency of GALNS in Morquio-A syndrome patients results in the accumulation of glycosaminoglycans (GAG) such as keratan sulfate (KS) and chondroitin-6-sulfate (C6S) (3). The clinical features are characterized by a noticeable short stature, protrusion of the chest, kyphoscoliosis, genu valgum, laxity of joints and abnormal gait (4)(5)(6). Spine is predominantly involved in these patients (7), without affecting neuronal function. This is a rare lysosomal storage disorder, with an estimated incidence ranging from one in 76,000 in Northern Ireland to one in 640,000 births in Western Australia (4,8,9) and one in 450,000 in the Netherlands, and Portugal (8, 10). Though study in Asian population is scarce, the available estimates of birth prevalence include 1 in 500,000 live births in Japan, 1 in 304,000 in Taiwan (11), and 1 in 701,000 live births in Malaysia (12).
Among these, the mutations that have been commonly observed in particular ethnicities are: c.120 + 1G > A (Tunisian-91%), c.757C > T (Pakistani-89%), c.953T > G (Chinese-58%) c.1171A > G (64%) and c.337A > T (52%) in the Irish population. Thus, the prevalence of certain alleles in particular ethnicities is likely to be due to founder effects. Bidchol et al. (15) were the rst to report the mutation spectrum of the GALNS gene in Indian Morquio-A syndrome patients. In that study, the patients studied were primarily from the Northern and Southern regions of the country. Nonetheless, considering the diverse population of India with different ethnicities, the scope for better diagnosis and management of Morquio-A syndrome would be enhanced if the mutation spectrum is known speci cally for the individual ethnicities.
In present study we aimed to identify the mutation spectrum in Gujarati Indian patients with Morquio-A syndrome.

Study subjects and ethics statement
The study was carried out in 25 children from Gujarat-India, affected with dysostosis multiplex as per the clinical diagnosis. The age range of the subjects was 11 months to 21 years. Clinical details were noted in a case record form and an informed consent was obtained from each family. The study protocol was approved by Institutional Ethics committee of FRIGE, Ahmedabad (Reg No-E/13237) as per the Helsinki declaration. 5ml peripheral blood was collected from the patients for leucocyte enzyme assay and isolation of DNA.
Urinary GAG Testing and Leucocyte Enzyme Assay Urinary GAG quantitative study was performed by dimethyl methylene blue (DMB) dye method (16). The concentration of urine creatinine of individual patients was measured by a biochemistry analyzer using a commercial kit and expressed in mg/mmol of creatinine. The pretreatment of urine prior to qualitative analysis of GAG by electrophoresis, was performed by a method described by Hopwood and Harrison, 1982 (17). Lysosomal enzyme β-galactose-6-sulfate-sulfatase activity was carried out from leucocytes using uorogenic synthetic substrate 4methylumbelliferyl-β-galactose-6-sulfate-triethyl ammonium (18). The uorescence of free 4-methylumbelliferone (4 MU) was measured by a spectro uorometer (LS55-Perkin Elmer) to determine the β-galactose-6-sulfate-sulfatase activity. The enzyme activity was expressed as the amount of substrate in nmol cleaved per hour per mg of protein in the cell lysates.

Molecular characterization
For all enzymatically con rmed cases of Morquio-A syndrome, genomic DNA was isolated from peripheral blood by the standard salting out method (19). The exonic regions and the anking intronic sequences of the GALNS gene were ampli ed by PCR in 14 fragments using prescribed primers (See Additional le 1). A 10μl reaction mixture for the DNA ampli cation of each fragment was made up of 100ng genomic DNA, 1mM dNTPs, and 10XPCR buffer. 30 cycles of ampli cation were performed, each consisting denaturation at 94 C for 1 minute, annealing at 60-65 C suitable for each exon for 45 seconds and extension at 72 C for 45 seconds in a thermal cycler. The nal extension time was of 10 minutes. PCR products along with the appropriate negative controls and a 100 base-pair DNA ladder were then subjected to electrophoresis in 2% agarose gel and puri ed using Exo-SAP-IT™ (USB Corporation, OH, USA). The puri ed products were sequenced using BigDye Terminator v3.1 and capillary electrophoresis was performed using an automated sequencer ABI-3500 (Applied Biosystems, CA, USA) for mutation analysis of GALNS. The sequences through the bidirectional sequencing was analyzed by comparing with the reference sequence of GALNS (RefSeq cDNA NM_000512.5). The mutations identi ed were annotated with Human Gene Mutation Database, dbSNP and McGill University database
Haplotype analysis was performed in all Morquio-A syndrome patients using microsatellite markers D16S3121, D16S3026 and D16S3023 ( Figure 1) and SNPs. To con rm the results obtained from haplotype analysis, 200 normal unrelated individuals were screened using ARMS-PCR for P77R variant (See Additional le 1). A reaction mixture of 13µl was prepared by adding the components in their required quantity that included Emerald master mix from Takara (10µl/ tube); distilled water (2µl/ tube) and 5pmol common reverse primer (1µl/ tube). Normal forward and mutant forward primers (1µl/ tube) and 100ng of DNA was added along with the reaction mixture to the respective PCR tubes. The PCR was conducted as per the required condition such as, initial denaturation at 94ºC for 5 minutes of 1 cycle followed by 30 cycles of denaturation at 94ºC for 30 seconds, annealing (normal primer -50ºC, mutant primer -61ºC) for 45 seconds, elongation at 72ºC for 1 minute and the nal elongation was at 72ºC for 5 minutes of 1 cycle. Ampli ed products were separated on 2% agarose gel at 100V for 30 minutes and viewed on UV image analyzer.

Results
The present study included 25 Gujarati Indian patients clinically and enzymatically diagnosed as Morquio-A. Seven of the 25 patients had consanguineous parents and remaining 18 patients were endogamous. The age of the patients at diagnosis ranged from 11 months to 21 years with a mean age of 4.32 years. The detailed information about the clinical features of all the patients is provided in Table 1. The clinical features present in all the patients included short stature, short ngers and short neck-trunk, and frontal bossing. Kyphosis was seen in 10/25 patients and platyspondyly in 5/25 patients. The other prominent symptoms associated with Morquio-A were observed with a low frequency in the present study such as corneal clouding was seen only in 1/20 (5.00%), knock knee in 6/25 (24.00%) patients. On radiological assessment most of them showed anterior beaking of vertebral bodies, acetabular dysplasia, platyspondyly and multiple dysplastic epiphyses. The urinary GAG analysis and the GALNS enzyme activity were determined for all the patients. Elevated levels of urinary GAG were observed in all the patients with excess of KS and C6S by 1D electrophoresis. The enzyme activity of N-acetylgalactosamine-6sulfate sulfatase in all the patients was in the range of 0.02-11.1 nmol/hour/mg of protein and the mean enzyme activity was 2.30 nmol/hour/mg of protein which was less than 10% of the mean normal enzyme activity ( Table 1).
The molecular testing of the GALNS gene has identi ed 11 mutations in the GALNS gene from the 24 patients (Fig. 2, Table 1 Three missense mutations, one splice site mutation and two indels were novel (p.D39G, p.G255A, p.L350P, c.121-7C > G, c.839_41delACA and c.1241_1242insA/p.I416HfsTer2) (Fig. 2). All the mutations were present in homozygous state except two mutations in compound heterozygous state (p.P77R/p.G255A and c.839_41delACA /p.P125L) and one in heterozygous (p.P77R) state with another variant remaining unidenti ed.
All the novel mutations were tested for their pathogenicity using pathogenicity prediction tools MutationTaster, MutPred, PolyPhen2, SIFT and PROVEAN. All the three missense mutations were predicted as pathogenic by aforementioned bioinformatics predictive tools. The respective scores and likely basis of effect on the protein for the non-synonymous single nucleotide substitutions found are presented in Table 2. The pathogenicity of the novel splice site mutation and small indels was con rmed by MutationTaster. The sequence alignment and ortholog of GALNS are highly conserved among various species for the residues D39, P77, C79, P125, P151, G255, L350, in which missense mutations were found in this study. Additional le 2 gives the details of conservation of GALNS in orthologs (Additional le 2). Effects of the missense variants on the structure and function of GALNS, as predicted by use of bioinformatics tools. Novel variants are in red. All the variants are likely to be pathogenic, based on predictions by all the tools used. In MutPred, probability > 0.75 is considered a "very con dent hypothesis" for a variant to be causative of disease. In PolyPhen-2, the scores for neutral to damaging range from 0-1, with 1 being "probably damaging". According to SIFT, a variant is "damaging" if the score is ≤ 0.05 and tolerated if the score is > 0.05. In PROVEAN, an amino acid substitution with a score <-2.5 is deleterious.  We found ve previously known missense mutations (p.L36R, p.P77R, p.C79R, p.P125L, p.P151L) (14,15,(20)(21)(22). Out of these, the two missense variants p.P77R and p.C79R have been reported in Indian patients (15), whereas the remaining variants were reported from Mexican, Japanese and Italian population. The proportion of p.L36R in our cohort was 8% and that of p.P77R was 60%. This indicates the higher prevalence of p.P77R in our study population, speci cally in the Patel community. This high frequency of the variant in the population is likely to be due to the founder effect. In order to con rm this, we carried out haplotype analysis in patients using dinucleotide microsatellite markers. The results for the same is depicted in Table 3A. Also, all the patients with p.P77R showed a unique identical genotype for the 28 SNP alleles investigated in the present study (Table 3B). A further study comprising 200 unrelated normal Gujarati individuals was carried out to check for the presence of this variant in the population using ARMS-PCR. None of the individuals showed the presence of the variant except for two individuals who were found to be in carrier state. This data also provides evidence that the p.P77R variant is a founder mutation for Morquio-A syndrome especially in Patel community of Gujarati Indians.

Discussion
The present study is the rst to report the clinical and genetic pro les 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 rst 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 ngers 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 ndings 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 rst 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 identi ed a total of eleven mutations, out of which eight are missense mutations. This nding 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 in uenced 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 rst 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 identi ed 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 identi ed 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 identi ed 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 con rm 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 con rms that p.P77R is a founder mutation in the Gujarati Patel population.
Our study further indicates the reason of signi cant 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.

Conclusion
The mutation spectrum for Morquio-A syndrome patients of Gujarati Indians is unique, wherein P77R is likely to be the founder mutation in Gujarati Patel ethnicity. This can be further utilized as a molecular marker and for carrier screening of the aforementioned population. An informed consent for publication was also obtained from the individuals included in the submission [This was in accordance with the requirement of the institutional ethics committee].

Consent for publication
Informed written consent was obtained from all the participants for publication of their clinical details and/or clinical images. A copy of the written consent is available for review by the editor of this journal.

Availability of data and materials
The dataset generated and/or analyzed during the current study is submitted in the ClinVar repository. Additional File 3 gives information on the ClinVar accession IDs of all the variants of GALNS gene generated or analyzed in the present study.

Competing interests
All authors declare that they have no con ict of interests ( nancial or non-nancial).

Funding
This work is partially supported by Indian Council of Medical Research (ICMR) Project No.: BMS:54/2010. The funding agency was not directly or indirectly involved in the study design, specimen collection, analysis, interpretation and preparation of the manuscript.

Details of the contributions of individual authors
Designing the study, carrying out the protocol for enzyme assays and molecular study -JS