Molecular Genetic Study Of Four Chinese Families With Infantile Nystagmus Syndrome

Infantile nystagmus syndrome(INS), formerly known as congenital nystagmus(CN), is characterized by an involuntary back and forth eye movement and usually occurs at birth or in the first few months of life. INS can be classified into idiopathic infantile nystagmus(IIN) and sensory defect nystagmus(SDN) based on distinct clinical manifestations. Patients with IIN usually exhibit isolated nystagmus, while dysfunctions of visual afferent system(e.g. aniridia and congenital cataract) are frequently observed in patients with SDN. The inheritance modes for both IIN and SDN comprise autosomal dominant, autosomal recessive, and X-linked. Up to now, 7 genetic loci of IIN(NYS1~NYS7) have been mapped through linkage analysis, and FRMD7 and GPR143 genes have been identified on NYS1 and NYS6 loci, respectively. Recently, an IIN pathogenic gene MANBA has been identified by whole exome sequencing. Meanwhile, many investigations on the relationships between gene mutations and SDN have been reported, for instance, mutation in PAX6 result in autosomal dominant SDN coupled with iris hypoplasia and/or cataract. In this study, 2 IIN families and 2 SDN families were recruited and identified. Microsatellite markers based positional cloning and high throughput whole exome sequencing techniques were applied to explore the genetic mechanisms of INS in the 4 families. In summary, a new genetic locus of IIN has been mapped to chromosome 9q33.1–q34.2, and two novel pathogenic mutations underlying INS, c.973A>G(p.R325G) in FRMD7 and c.1183+1G>A in PAX6, have been identified, as well as distinct clinical manifestations resulted from a known INS causing mutation c.622C>T(p.R208W) in PAX6 has been described in this research. The detailed research results of this study were presented as follows: 1. Identification of a novel genetic locus for idiopathic infantile nystagmus and mutational analysis of candidate genesIn this study, a Chinese Han family(NX-NYS) with autosomal dominant IIN was collected and identified in Ningxia Hui Autonomous Region. There are 5 generations and 47 individuals in the family, and 11 of whom are affected by IIN. Among these individuals, 15 normal individuals and 8 affected individuals were enrolled in this research. The proband exhibited horizontal pendular nystagmus combined with abnormal electroretinogram and visual evoked potential, but no other ocular or neurological anomaly was presented. All the other affected individuals showed nystagmus in the first 6 months, and the waveform of which comprise pendular, jerky, rotatory and mixed waveform. Microsatellite markers in the four genetic loci for autosomal dominant IIN NYS2(6p12), NYS3(7p11.2), NYS4(13q31-q33) and NYS7(1q31.3-q32.1), as well as microsatellite markers flanking the autosomal dominant pathogenic gene MANBA, were selected for linkage analysis. However, all the genetic loci and genes were excluded for the negative two-point LOD scores of all the microsatellite markers. Subsequently, the pathogenic gene of NX-NYS family has been mapped to chromosome 9q33.1–34.2 between D9S1776 and D9S164 through whole genome linkage analysis of 382 microsatellite markers, and the two-point LOD scores for four microsatellite markers was graeter than three. This is a novel genetic locus for autosomal dominant IIN, and was named as NYS9. The locus spans a genomic region of 18.3 Mb, and contains 202 protein-coding genes. Direct Sanger sequencing was performed on the proband, but there was no pathogenic mutation was detected in LHX2, LHX6 and NCS1 genes, which were considered as the most likely genes involved in nystagmus. Subsequently, whole exome sequencing and bioinformatic analysis were carried out on the proband, the coverage of target region was 98.84% and the average sequencing depth of target region was 64.21 X. Sanger sequencing was applied to verify the 11 variations detected in NYS9 locus, and c.412C>T(p.R138C) in CCBL1 was found co-segregated with the nystagmus phenotype in the family. R138 C is not conserved through phylogenetic analysis, whether CCBL1 is responsible to IIN need further investigations. 2. Identification of a novel FRMD7 mutation in a family with idiopathic infantile nystagmus and functional characterization of the mutantA five-generation Chinese family(HB-NYS) with IIN was collected and identified. Among the 38 family members, 10 of whom suffered from nystagmus since early infancy. The proband showed nystagmus combined with strabismus, amblyopia and myopia, without any other ocular or neurological anomalies. Affected individuals were observed in each generation but no male-to-male transmission has been observed, and incomplete penetrance was noted in the family, which meet the criteria of X-linked IIN result from FRMD7 mutation. Direct sequencing found that the proband carried a c.973A>G(p.R325G) variation in exon 10 of FRMD7. Further Sanger sequencing showed this variation co-segregated with the nystagmus phenotype in HB-NYS family, except for one asymptomatic female carrier. The R325 residue of FRMD7 is conserved from zebrafish to human, suggesting an important role of this residue in FRMD7 function. However, in vitro experiments showed that the R325 mutant had normal subcellular localization, and its CASK protein binding ability was comparable to the wild-type FRMD7 protein, suggesting that the R325 mutation might change the function of FRMD7 in an unknown way. 3. Precise genetic diagnosis of two families with sensory defect nystagmusIn this study, two families with sensory defect nystagmus were collected and identified, which were named as HNXY-NYS and HNJZ-NYS, respectively. In HNXY-NYS family, the proband exhibited horizontal nystagmus accompanied with iris hypoplasia since early infancy, and the other affected family members showed similar clinical symptoms and age of onset. In HNJZ-NYS family, two individuals suffered from early-onset horizontal jerky nystagmus in the first six months of life and one patient manifested by cataract in addition to nystagmus. PAX6 is crucial in the morphogenesis and development of the eyes, and heterozygous mutation of this gene is known to be a cause of dysplasia of cornea, iris, lens, fovea and optic nerve combined with infantile nystagmus. These two SDN families can’t be excluded from PAX6 mutation, so precise genetic diagnosis was performed on the two SDN families.In HNXY-NYS family, a c.1183+1G>A variation was detected in intron-12, which destroyed the splicing donor site of intron-12. Mini-gene based in vitro splicing assay demonstrated that exon-12 was skipped in c.1183+1G>A mutant mini-gene, resulting in a reading-frame shift after codon 344. Similarly, a c.622C>T(p.R208W) substitution of PAX6 has been identified in exon-8 in HNJZ-NYS family, and the same mutation was detected in the other patient. R208 W was located in the link domain of PAX6 protein and highly conserved from jellyfish to human in evolution. The R208 W mutation had been reported twice and was linked to hereditary ocular disease prominently presented as aniridia/iris hypoplasia, while distinct clinical manifestations were noted as nystagmus and cataract in HNJZ-NYS family. This result suggested the R208 W is a hotspot of PAX6 mutation, and further confirmed the clinical heterogeneity of PAX6 mutation.In summary, two families with IIN and two families with SDN were enrolled and identified in this study. Linkage analysis, whole exome sequencing as well as mutational analysis were performed on these families, and one novel genetic locus of IIN and two new mutations of INS pathogenic genes have been identified as well as distinct clinical phenotypes result from a known INS mutation has been observed. This study further confirmed the genetic heterogeneity and clinical heterogeneity of INS, and enriched the understanding of the relationships between genotype and phenotype of INS. Meanwhile, this research is of great significance to understand the molecular pathogenesis of INS, and provide valuable information for genetic counseling and precise genetic diagnosis of INS.