Application Of Combining Direct Sequencing And CNV Detection Technology In Waardenburg Syndrome-related Genes Screening

Background:Waardenburg syndrome (WS) is the most common autosomal dominant inherited disorder in syndromic hearing loss and is characterized by the association of pigmentation abnormalities, including depigmented patches of the skin and hair, vivid blue eyes or heterochromia irides, and sensorineural hearing loss. Depending on other clinical features such as dystopia canthorum, musculoskeletal abnormalities of the limbs, Hirschsprung disease, WS can be classified into four types (WS Ⅰ-Ⅳ). Six genes are involved in this syndrome:PAX3(encoding the paired box3transcription factor), MITF (microphthalmia-associated transcription factor), EDN3(endothelin3), EDNRB (endothelin receptor type B), SOX10(encoding the Sry bOX10transcription factor), and SNAI2(snail homolog2).As to March2014, the LOVD-Leiden Open Variation Database (http://grenada.lumc.nl/LOVD2/WS/) had reported113PAX3,78SOX10,41MITF,15EDN3,31EDNRB and2SNAI2gene mutations.Most of these reported mutations were delected by direct gene sequencing. Even though direct gene sequencing enables the discovery of point mutations and small alterations in the genes, it cannot reliably detect whole exon or whole gene copy number changes, which have been reported for many genes resulting in a specific genetic disorder.The exact description of the mutations responsible for the WS type is of significant importance in genetic counseling of WS patients and their families. In recent years, a number of PCR-based methods of CNV detecting have been widely used in molecular genetic diagnostics to detect or exclude copy number changes in targeted genes.Foreign research groups have also used Multiplex ligation-dependent probe amplification (MLPA) in WS to detect copy number changes in WS related genes.In this study we applied both direct gene sequencing and CNVplex analysis to detect point mutations and copy number variations in the six WS related genes.Objective:Our study intended to determine the frequency of mutations and deletions in these genes, to assess the clinical phenotype in detail and to identify rational priorities for molecular genetic diagnostics procedures.Methods:We analyzed the clinical features and identified the WS type of29WS Patients. Both direct gene sequencing and CNVplex analysis were applied to detect point mutations and CNVs in the six WS related genes.Then, we used quantitative PCR (QPCR) for the CNV verification and preliminary to identify their range and location. Finally, we summarized the results of both test to assess the clinical phenotype and analysis of mutation frequency.Results:Results of this study:1.In four new WS cases, two WS Ⅰ and two WS Ⅱ, all of these cases had detected mutation by direct gene sequencing.Two WS Ⅰ cases’mutation located on PAX3(c.232G> A, c.792+1G>A), two WS Ⅱ cases’located on MITF c.649-651delAGA.2.There are4WS Ⅰ,23WS Ⅱ and2WSⅣ in the29WS patients.Identified by direct sequencing, mutations found in all WS Ⅱ/WS Ⅳ and65%(15/23) of WS Ⅱ.All WS Ⅰ patients with PAX3mutation;39%(9/23) WSⅡ patients with MITF mutation;two WSⅣ patients with SOX10mutation. 3. The CNVplex result showed9abnormal cases,7suggestive of point mutations, which locations consistent with previous direct gene sequencing results. Further, we also found two CNVs involved MITF/SOXIO, CNV involved MITF was repetition type, the other involved SOXIO was deficiency type.Both of them were verification and preliminary identified their range and location by QPCR.One of them located on upstream to the MITF first exon, size4-5Kbp, the other involved whole SOX10, size more than160Kbp.Conclusions:This is the frist report about WS CNV study of Chinese population. We draw conclusions from the results in these studies:1.WS in Chinese population is mainly WS Ⅰ and WS Ⅱ subtypes, WSIV and WSⅢ are rare. PAX3gene mutations were most likely involved in WS Ⅰ cases; MITF and SOX10are the major causative gene of WSⅡ (65%);WSⅣ mainly caused by SOXIO mutations.2. CNVs were found in Chinese WS patients, CNV detecting of targeted genes can increase the detection rate of abnormal change in WS-related genes, and complement with direct gene sequencing.3. CNVplex could suggest gene mutations either, and verified by direct gene sequncing lately.4. On the basis of these funding we suggest that combining direct gene sequencing with CNV detecting should be recommended in the routine molecular diagnostic setting for WS patients.