Mutation Screening of ENAM, KLK4, MMP20 and FAM83H Genes among the Members of Five Iranian Families Affected with Autosomal Recessive Hypoplastic Amelogenesis Imperfecta

Amelogenesis Imperfectas (AIs) are clinically and genetically heterogeneous conditions characterized by a wide range of clinical features. These abnormalities of enamel formation are categorized into three main groups, hypoplastic, hypomaturation and hypocalcified with different modes of inheritance such as autosomal recessive (AR), autosomal dominant (AD) and X-lined recessive (XLR). In spite of the fact that frequent studies have explained the histological features of AIs, our knowledge regarding the molecular etiology of the affected enamel is not adequate. Up to now, different loci have been suggested to associate with the causation of AIs. Several genetic mutations including enamelin (ENAM), amelogenin (AMELX), ameloblastin (AMBN), tuftelin (TUFT1), kallikrein 4 (KLK4), matrix metalloproteinase 20 (MMP20) and family with sequence similarity 83, member H (FAM83H) have been suggested to play critical roles in the pathogenesis of these disorders. Therefore, the aim of this investigation was to study of mutation screening in ENAM, KLK4, MMP20 and FAM83H genes, responsible for AIs development in five Iranian families in which the probands were diagnosed with autosomal recessive hypoplastic amelogenesis imperfecta (ARHAI). Genomic DNA was extracted from probands and exon/intron boundaries of afore-mentioned genes were amplified by PCR and subjected to direct sequencing. We could not detect any mutation in the studied genes however; two different novel polymorphisms including T18515C and G18504A were identified in the intron 4 of MMP20 in the probands of two families. Our findings support the notion that different genes may be involved in the development of amelogenesis imperfectas

Two novel missense substitutions in the VSX1 gene: clinical and genetic analysis of families with Keratoconus from India

BACKGROUND: Visual system homeobox gene (VSX1) plays a major role in the early development of craniofacial and ocular tissues including cone opsin gene in the human retina. To date, few disease-causing mutations of VSX1 have been linked to familial and sporadic keratoconus (KC) in humans. In this study, we describe the clinical features and screening for VSX1 gene in families with KC from India. METHODS: Clinical data and genomic DNA were collected from patients with clinically diagnosed KC and their family members. The study was conducted on 20 subjects of eight families from India. The coding exons of VSX1 gene were amplified using PCR and amplicons were analyzed by direct sequencing. Predictive effect of the mutations was performed using Polyphen-2, SIFT and mutation assessor algorithms. Additionally, haplotypes of VSX1 gene were constructed for affected and unaffected individuals using SNPs. RESULTS: In the coding region of VSX1, one novel missense heterozygous change (p.Leu268His) was identified in five KC patients from two unrelated families. Another family of three members had a novel heterozygous change (p.Ser251Thr). These variants co-segregated with the disease phenotype in all affected individuals but not in the unaffected family members and 105 normal controls. In silico analysis suggested that p.Leu268His could have a deleterious effect on the protein coded by VSX1, while p.Ser251Thr has a neutral effect on the functional properties of VSX1. Haplotype examination revealed common SNPs around the missense change (p.Leu268His) in two unrelated KC families. CONCLUSIONS: In this study, we add p.Leu268His, a novel missense variation in the coding region of VSX1 to the existing repertoire of VSX1 coding variations observed in Indian patients with the characteristic phenotype of KC. The variant p.Ser251Thr might be a benign polymorphism, but further biophysical studies are necessary to evaluate its molecular mechanism. The shared haplotype by two families with the same variant suggests the possibility of a founder effect, which requires further elucidation. We suggest that p.Leu268His might be involved in the pathogenesis of KC, which may help in the genetic counselling of patients and their family

Mutation analysis of VSX1 and SOD1 in Iranian patients with keratoconus

Purpose: To evaluate mutations in the visual system homeobox gene 1 (VSX1) and superoxide dismutase 1 (SOD1) genes with keratoconus (KTCN), direct sequencing was performed in an Iranian population. Methods: One hundred and twelve autosomal dominant KTCN patients and fifty-two unaffected individuals from twentysix Iranian families, as well as one hundred healthy people as controls were enrolled. Genomic DNA was extracted from whole blood sample. Then to study the possible linkage between KTCN and six known loci linkage analysis was performed using 12 short tandem repeat (STR) markers. Also, the entire coding region and intron-exon boundaries of VSX1 and SOD1 were amplified by the PCR technique in each proband. Subsequently, PCR products were subjected to direct sequencing. Co-segregation analysis of the identified mutation was conducted in the family members. An Amplification Refractory Mutation System PCR (ARMS-PCR) was additionally employed for detection of the identified mutation in healthy controls. Results: Linkage analysis of aforementioned loci did not detect evidence for linkage to KTCN. Direct PCR sequencing revealed two single nucleotide polymorphisms (SNPs; g.1502T>G and g.9683C>T), as well as two missense mutations that have been previously reported (R166W and H244R) in VSX1. We also found three undescribed SNPs (g.4886G>A, g.4990C>G, and g.9061T>A) in SOD1. The R166W and H244R mutations were co-segregated in affected family members but not in those that were unaffected. Moreover, the ARMS-PCR strategy did not detect the identified mutations in controls. Conclusions: Our data suggest a significant association between KTCN patients and VSX1 genetic alterations (p.R166W and p.H244R). Although our findings support VSX1 as a plausible candidate gene responsible for keratoconus, other chromosomal loci and genes could be involved in KTCN development. Taken together, our results suggest that p.R166W and p.H244R could have possible pathogenic influences on KTCN