The Study Of Pathogenic Gene In 10 Congenital Nystagmus Families

Objective Congenital Nystagmus is a genetically heterogeneous condition with autosomal dominant, autosomal recessive, and x-linked modes of inheritance reported. Up to date, the FRMD7 gene residing at Xq26.2 is the only identified gene that causes the happening of Congenital Idiopathic Nystagmus. This study did FRMD7 gene sequencing in 10 CN families and 5 sporadic cases directly, aimed at screening pathogenic mutations within it, analysing the effect of mutations on the structure and function of FRMD7 protein, building immortalized cell lines combined with FRMD7 pathogenic mutations to lay the foundation of later FRMD7 function research.Methods 1. Collecting 10 CN families and 5 sporadic patients in Department of Pediatric Ophthalmology and Strabismus at Tianjin Eye Hospital from June 2013 to June 2014, analysing of the clinical phenotype of the CIN patients and their direct relatives. 2. Human genomic DNA was extracted from every patient’s peripheral blood.12 exon pieces of FRMD7 gene were amplified by PCR reaction. After purification and recycling the purpose fragments, using ABI 3130-avant genetic analyzer to do direct sequencing of the FRMD7 gene and DNAstar software to analyze data. 3. Analysing the effects of novel mutations within FRMD7 gene on its protein: using Human Splicing Finder-version 2.4.1software to forecast the influence on FRMD7 protein made by splicing mutations. 4. Establishing cell lines combined with FRMD7 gene pathogenic mutations: using the way of lymphocyte conversion to establish immortalized B lymphocyte cell lines; using method of dispase â…¡enzyme digestion to establish skin fibroblast cell lines.Results 1. In this study, we found 6 mutations of FRMD7 gene totoally, they are c.57+2T>C、c.58-3T>A、 c.685C>T、c.910C>T、c.980₉83del ATTA&c.986C>A and c.1860₁861del AG, which had not been found in 100 normal individuals in control. 2. c.1860₁861del AG, c.58-3T >A and c.57+2T >C were three novel mutations of FRMD7 gene: c.1860₁861del AG mutation might produce a truncated protein containing 647 amino acids; Human Splicing Finder software showed c.57+2T>C mutation would make the wild type splicing site broken,â–³CV was-30.71 and 58-3T>A mutation palyed little influence on splicing,â–³CV was-2.17. 3. In this study, the probands of CN families having FRMD7 gene mutations displayed horizontal impulse type nystagmus, 3/6 patients accompanied by compensatory head posture,but not associated with strabismus; the probands of CN families having no FRMD7 gene mutations displayed horizontal nystagmus combined with vertical and rotational components, either jerk type or pendular type,some patients can show a nodding spasm, 3/9 with abnormal eye, 4/9 accompanied by compensatory head posture, 2/9 eye and head position were normal. 4. Whether or not having FRMD7 gene mutation, CN patients had different degrees of decreased visual acuity and ametropia; the clinical phenotype of patients with the same FRMD7 gene mutation were different. 5. Successfully established immortalized B lymphocyte cell lines and skin fibroblasts cell lines containing FRMD7 pathogenic mutations, which can unlimitly be used to extract genomic DNA and do FRMD7 gene function research.Conclusion 1. There were 6 FRMD7 gene mutations totally found in 5 CN families and 1 sporadic patient,including 3 novel mutations and 3 known mutations,which expanded the mutation sprectrum of FRMD7 in association with CN and had great importance in exploring the pathogenesis of X linked-CIN caused by FRMD7 gene mutations. 2. Clinical phenotypes of CN were directly associated with the FRMD7 gene mutations. No mater if the ancestral source was same, the clinical phenotype of CN patients were different. It still need large sample of clinical data to statistically analyze the relationship between genotype and clinical phenotype. 3. There were 5 CN families and 4 sporadic patients with no FRMD7 gene mutation and the clinical phenotype of those patients were more complicatied than patients containing FRMD7 gene mutations, it still need futher research to explore the pathogenic genes of these patients. 4. Successfully establishing immortalized cell lines for the patients accompanied with novel mutations of FRMD7 gene or not, which could lay foundation for FRMD7 gene functional experiment and finding new pathogenic genes of CN in further research.