Deletion / Duplication Mutation Detection Technology Evaluation And Diagnosis Strategies And A Congenital Nystagmus Family Gene Mutation Detection

Multiplex ligation-dependent probe amplification (MLPA) is a recently developed relative semi-quantitative technology. The procedure contains hybridization, ligation, PCR amplification and electrophoretic separation, by which copy number varations of up to 45 different nucleotide sequences can be detected in a single reaction simultaneously. It has been used successfully in clinical diagnosis such as Duchenne muscular dystrophy (DMD), mental retardation and kinds of syndromes caused by microdeletions or microduplications. In this study, MLPA was introducted to detect copy number variations in patients with DMD and Williams-Beuren syndrome (WBS). Based on the results and evaluation of the new method, more available and appropriate molecular diagnostic strategy for Chinese DMD/WBS patients was set up successfully, which will be popularized much easier among basic units.DMD is an X-linked recessive fatal disorder. It's the most common form of hereditary muscular dystrophy, with an incidence of about 1/3500 male births. Progressive proximal muscular dystrophy, powerless and characteristic pseudohypertrophy of the calves, caused by abnormal expression of dystrophin protein in the tissue of striped muscle is the most common clinical manifestations. The gene, DMD, was mapped to chromosome Xp21.1-21.3, which consists of 79 exons and spans more than 2.3 Mb. Exon deletions, duplications and other micro changes as point mutation are molecular defects underlying the disease. One hundred and sixteen DMD patients were detected by MLPA in the present study and the spectrum of deletion mutations in Chinese DMD patients was indicated by analysis of 380 deletion cases. A new multiplex PCR (mPCR) system'5×2+6'was established based on this analysis. In common units, especially grass-roots departments, it can be a preferred option for genetic analysis of DMD and MLPA as a supplement method. Co-application of mPCR and MLPA is to improve the molecular diagnostic strategy of Chinese DMD patients, making it more simple, rapid and efficient.Williams-Beuren syndrome is a rare genetic disorder (1/10,000-20,000 in live births) caused by a heterozygous deletion on chromosome 7q11.23. Common deletions in WBS patients span a genomic region of 1.55Mb. The disease is generally sporadic although rare familial cases of autosomal dominant transmission have been reported. In this study, five WBS patients were diagnosed by our homemade MLPA method. One was deletion of paternal, the rest four were deletions of maternal. When testing the MLPA results, we found that short tandem repeats (STR) within the critical deletion region of WBS were always of high informative. Then polymorphism analysis of these STRs was performed on normal controls for the first time interiorly, which provides a theoretical basis for optimizing DNA markers when detecting deletions in Chinese WBS patients. Therefore, genetic analysis can be performed firstly using STR sites picked up in this study, which will receive a much higher positive rate. Congenital nystagmus (CN) is an ocular hereditary disorder characterized by binocular spontaneous oscillations that is present at birth or develops within the first few months of life. So far, X-linked dominant and X-linked recessive (MIM 310700), autosomal dominant (MIM 164100, MIM 608345, MIM 193003), and autosomal recessive (MIM 257400) modes of inheritance have been reported, but X-linked inheritance with incomplete penetrance and variable expressivity is probably the most common. Three different genetic loci for X-linked congenital nystagmus (XLCN) have been mapped to chromosomes Xp11.3-11.4, Xp22 and Xq26-Xq27.The FERM domain-containing 7 (FRMD7, MIM 300628) gene located at Xq26-27 and the G protein-coupled receptor 143 (GPR143, MIM 300500) gene at Xp22, have been identified as disease-causing genes for XLCN, but the etiology or molecular pathogenic mechanism is largely unknown. To date, multiple mutations of FRMD7 have been reported. Another gene, GPR143, also known as the OA1 gene, causes ocular albinism type 1 (OA1). However, GPR143 gene mutations have been identified in two Chinese families with XLCN without any classical phenotype of OA1 but only with congenital nystagmus.In this study, we present a four generation Chinese family with XLCN. All the affected individuals suffer from nystagmus but without any typical sign of OA1. We mapped the disease-causing gene to Xp22.3 and characterised the underlying molecular defect as a novel 19-bp duplication in exon 1 of GPR143, causing a frame-shift in all affected males. Our results indicate that this novel GPR143 mutation might cause the XLCN in this Chinese family.