www.symbiosisonline.org Symbiosis www.symbiosisonlinepublishing.com Research Article International Journal of Genetic Science Open Access Genetic Analysis of CHST6 Gene in Indian Families with Macular Corneal Dystrophy Durga Murugan1, Namperumalsamy Venkatesh Prajna2, Lumbini Devi2, Periasamy Sundaresan1* 1Department of Molecular Genetics, Aravind Medical Research Foundation, Aravind Eye Hospital, Madurai, Tamilnadu, India 2Cornea Clinic, Aravind Eye Hospital, Madurai, Tamilnadu, India Received: November 22, 2017; Accepted: November 30, 2017; Published: December 06, 2017 *Corresponding author: Periasamy Sundaresan, Senior Scientist-V, Department of Genetics, Aravind Medical Research Foundation, Aravind Eye Hospital, Tamil Nadu, India, Tel: 91 452 4356100; Fax: 91 452 2530984; E-mail: [email protected] Introduction Abstract Macular corneal dystrophy is an autosomal recessive disorder, Background:Unlike the western world, Macular Corneal Dystrophy (MCD) is the most common corneal stromal dystrophy in caused by CHST6 India. It is caused by mutations in the carbohydrate sulfotransferase 6 in 1890. Due to high degree of consanguinity in some ethnic (CHST6) gene. So far, there are limited numbers of reports on CHST6 population, this disorder gene [1]. is highlyIt was prevalentfirst described in Saudi by ArabiaGroenouw and screening. Therefore, our screening of CHST6 gene in Indian families with MCD will be useful for genetic diagnosis, carrier detection and usually occurs by irregular, focal haze formation that leads to grey- genetic counseling to families included in this study and other families whiteSouth Indianopacities population eventually [2,3]. leading The Clinical to decreased manifestations visual acuity of MCD by with similar disease condition. Purpose: The main purpose of this study was to perform the MCD showing diffusely distributed, rounded stromal opacities genetic analysis of 55 Indian MCD families. (Figurethe fifth 1). decade In early of life stages, [4]. Slit Photo lamp Therapeutic view of cornea Keratectomy of patient (PTK) with Methods: We have recruited 55 affected, 11 unaffected members can be done, whereas in advanced disease stage, it requires from 55 MCD families along with 100 controls. All the study subjects full thickness or deep anterior lamellar keratoplasty [5,6]. The underwent ocular examination before collecting the blood for the screening of CHST6 gene. Polymerase chain reaction was performed corneal grafts even after 10 years of Penetrating Keratoplasty followed by bi-directional sequencing. The novel mutations were (PKP)recurrence [7]. rate of corneal opacities is more than 40% in macular predicted by Polyphene-2, SIFT, Mutation taster and SOPMA tool. Results: We identified 14 different mutations, 3 known SNP’s mutationsin 44 MCD inpatients the coding amongst region 6 were of CHST6 novel. geneAlso 2in hotspot 11 MCD mutations patients (20%).were identified amongst the 14 mutations. We could not identify any Conclusion: ethnic populations. Our Our study study identified also increases six novel the mutationalmutations whichlandscape will ofadd CHST6 up to gene. the list We ofconcluded already knowndue to genetic mutations heterogeneity identified therein different might be some other gene involved in Indian MCD patients who are negative for CHST6 mutations. Keywords: CHST6 gene; Cornea; Heterogeneity; Mutations; Abbreviations MCD: Macular Corneal Dystrophy; CHST6: Carbohydrate Slit lamp view of cornea with macular dystrophy demonstrat- Sulfotransferase 6; SIFT: Sorting Intolerant From Tolerant; Figure 1: ing multiple irregular grey-white opacities with intervening central SOPMA: Self-Optimized Prediction Method with Alignment; stromal haze. Symbiosis Group *Corresponding author email: [email protected] Genetic Analysis of CHST6 Gene in Indian Families with Macular Corneal Dystrophy Copyright: © 2017 Sundaresan P, et al. CHST6 gene is the only candidate gene so far known in MCD Analyser; Applied Biosystems) and the results were compared and has been further screened in different ethnic populations with the reference sequences of CHST6 Chromas lite (2.1) software. gene using BLAST and across the world [2,3,8-17]. It is located on chromosome 16q22 Bioinformatics Analysis of[18,19]. keratan It encodes sulfate a(KS) golgi essential resident forenzyme corneal N-acetyl transparency glucosamine- [20- The Self-Optimized Prediction Method with Alignment 22].6-O-sulfotransferase Defect in CHST6 (C-GlcNac-6-ST) gene results in thatunsulfated catalyses keratan the sulfation sulfate html) was used for the prediction of secondary structure of CHST6 protein.(SOPMA) In tool addition, (https://npsa Polyphene prabi.ibcp.fr/NPSA/npsa_sopma. 2, SIFT and Mutation taster deposition eventually leading to MCD phenotype [23]. were used for predicting the pathogenicity of novel mutations CHST6 gene and also the conservation of the novel characterizedBased on bythe absence histochemical or low levelfeatures, of sulfated MCD KSis (AgKS)classified in corneainto three as wellimmunophenotypes as serum. Type III, isII characterizedand IA [24-26]. by normalType I oris multipleidentified sequence in alignment software (http://www.clustal.org/ marginally reduced level of AgKS in cornea as well as serum. Type omega).mutations identified was being analysed by CLUSTALW (1.2.2) IA is characterized by low level of antigenic AgKS in serum and detectable level in keratocytes [26]. Results The main purpose of this study was to screen the coding We have recruited 55 families with MCD at the cornea unit region of CHST6 gene in 55 Indian families with MCD. were carrying history of consanguineous marriages. All the study Materials and Methods subjectsof Aravind were eye screened care system. for CHST6 Out of gene the mutations.55 families, Interestingly, 30 families Patients The subjects for this study (cases and controls) were recruited we identified 6 novel mutations (Ser53X, Ser81X, Val172Met, from the outpatient services of a tertiary eye care hospital in Arg202His, Ser248Asp, Glu274Gln) in 7 different MCD families South India (Aravind Eye Care System, Madurai, India). The study and 8 known mutations (H42Y, S53L, R93H, R127C, Q58X, adhered to the tenets of the Declaration of Helsinki, and ethics V66VfsX3, Q182RfsX198, N194_196delinsRC) in 29 different committee approval was obtained from the Institutional Ethics TableMCD families. 1. Rest of Also the we unaffected have identified 11 family 3 known members single was nucleotide negative Committee of the Aravind Eye Care System. All subjects read and forpolymorphisms these mutations. (R50C, InR205Q, addition, R205W) 100 in age 8 families matched described controls in signed informed consent except for illiterate subjects, who had were also negative for these mutations. We considered 6 novel sequence variants as mutations based on the following criteria: for participation consent. the information leaflet read out and provided a thumb impression 1. The six novel changes were segregating in the family in an Genetic Analysis autosomal recessive fashion. DNA extraction and PCR amplification 2. All the six novel mutations were absent in the following With informed consent from the study subjects, 5 ml of blood extracted by salting out method [27]. There are four coding databases (Ensemble, HGVS, 1000 genome project) regionswas collected in from all the study subjects and genomic DNA was CHST6 mutation3. Three of wasthe novelnot missenseconserved mutations (Arg202His) were highlyacross conserveddifferent as already reported in several independent studies. Based on (Val172Met, Ser248Asn, Glu274Gln) and one novel missense gene [18,19]. Out of these, exon 3 is unique orthologous species (Figure 2) using predesigned primers [16]. Each PCR was carried out in a the uniqueness of exon 3, we have also targeted the exon 3 by causing and the polyphen2, SIFT programme predicted all are damaging.4. Mutation taster predicted all the novel mutations to be disease potassium50 µl reaction chloride; mixture 1.5 containing mM magnesium 100 ng chloride of genomic and DNA,0.001% 1X buffer (PCR buffer (10 mM TRIS hydrochloride, pH 8.3; 50 mM A novel homozygous missense mutation Val172Met was gelatin)), 0.5 pmol of each primer 200 μM of deoxynucleotide identified in a patient from family 5 with consanguinity, Arg202His triphosphate and 1 U of Taq DNA polymerase (Sigma Aldrich). Biosystems-Invitrogen). The thermal cycling program started was identified in a patient from family 42 with consanguinity, withAmplification an initial wasdenaturation performed of 10in aminutes DNA Thermal at 96o cycler (Applied 9Ser248Asn with consanguinity was present (Table in 1). a Additionally, patient from a novel family homozygous 25 with cycles of 96o o consanguinity, Glu274Gln was identified in a patient from family 72o oC,C forfollowed 5 minutes by 37 C for 30 seconds, 60 C for 30 seconds of annealing, nonsense mutation Ser53X was identified in 2 patients (patient & SangerC for 45Sequencing seconds with a final extension at 72 patient’s younger brother) from family 8 without consanguinity but with MCD history (Figure 3a). One more novel homozygous nonsense mutation Ser81X was identified in a patient from family The amplified DNA products were purified by QIA quick 36 without consanguinity and MCD history. PCR purification kit method (Bio Basic Inc.,) followed by cyclic PCR. Bi-directional sequencing was performed (3130 Genetic Citation: CHST6 Page 2 of 10 Sundaresan P, Durga M, Namperumalsamy VP, Devi L (2017) Genetic Analysis