Genetic Analysis Of Bartter Syndrome And Development Of Deafness Microarray

Part1:Genetic analysis of type IV Bartter syndromeBackground:Bartter syndrome is an autosomal recessive disease, with a group of manifestations such as renal loss of salt, hypokalemia, metabolic alkalosis, enhanced renin-angiotensin-aldosterone system activity. Bartter syndrome has traditionally been classified into3main clinical variants:neonatal （or antenatal） Bartter syndrome, classic Bartter syndrome, and neonatal （or antenatal） Bartter syndrome with sensorineural deafness. Advances in molecular diagnostics have revealed that Bartter syndrome results from mutations in numerous genes:NKCC2, ROMK, CLCNKB, BSND, CLCNKA and NCCT。According to the genetic features, BS can be divided into six types:type Ⅰ, type Ⅱ, type Ⅲ, type Ⅳ, type Ⅴ and typeⅥ. Type Ⅳ BS and type Ⅴ BS are neonatal （or antenatal） Bartter syndrome with sensorineural deafness. Type Ⅵ BS is Gitelman syndrome.Objective:A sporadic case with Bartter syndrome and was collected. We intended to analyze clinical features and carry out gene mutation detection to find out the etiology for this patient.Methods:The patient and her parents’history, physical examination, and laboratory examinations were carried out.100normal people were selected as the control group. And then, the exons of GJB2, BSND, CLCNKA and CLCNKB were detected.Results:The patient was neonatal-onset, and the clinical features were increased renin-angiotensin-aldosterone system activity, hypokalemia, hypochloremia, growth retardation, deafness polyhydramnios, preterm birth and normal kidney imaging. This was in line with the typical symptoms of type IV Bartter syndrome. At the same time, the patient had homozygous mutations c.22C>T and c.127G>A in BSND, and also compound heterozygous mutations c.235delC and c.109G>A in GJB2. Both parents had heterozygous mutations c.22C>T and c.127G>A in BSND. Father’s had a heterozygous mutation c.109G>A in GJB2, and mother had heterozygous mutation c.235delC in GJB2. Otherwise, CLCNKA and CLCNKB mutations were not found out for the patient and parents.37persons of control group had heterozygous c.127G>A in BSND, and2persons had homozygous c.127G>A.Conclusions:1. It was first reported around the world that the patient with type IV Bartter syndrome carried both BSND and GJB2mutations;2. The patient with type IV BS was caused by homozygous mutation c.22C>T in BSND, and compound heterozygous mutations in GJB2might take a role in the process of deafness;3. The mutation BSND c.127G>A carried by the patient was single nucleotide polymorphism. Part2:Optimization of Goldengate deafness microarrayBackground:Hearing loss is the most common sensory nerve disease, which not only causes hearing disability, but also affects mental health. At the same time, the deafness bring the society a heavy burden. It is reported that250million people worldwide suffer from moderate to severe hearing loss. Genetics studies have shown that22of23pairs of chromosomes and mitochondrial genes are distributed by deafness loci. About half of congenital deafness associated with genetic factors. Gene diagnostic tools are derived with the development of genetics. However, these methods are not characterized by high-throughput, wide coverage, low-cost and high-accuracy. Previously, we designed GoldenGate deafness gene chip with384sites, including240deafness mutation detection sites（77dominant mutation sites and163recessive mutation sites） and144SNPs.465DNA templates were screened by this chip. And then SNP allele frequency and call rate of the chip were analysized. The results showed that the chip total call rate was96.32%; false positive rate of GJB2₂35delC was3.1%, and false negative rate was0; linkage analysis of a dominant hereditary non-syndromic deafness pedigree had similar result with traditional micro satellite positioning scanning method. However, several items could not be explained:First, some mutation sites had high positive detection rates, such as57.36%for PJVK₉88delG,7.14%for SLC26A4_Gly497Ser; Second, some sites had low call rate; Third, some patients carried a variety of homozygous gene mutations; Fourth, some normal persons carried one or more mutations, but were not deafness. For these reasons, our research team intended to verify the chip and optimize it in order to obtain a more reasonable chip with higher accuracy.Objective:To verify the accuracy of the first edition GoldenGate deafness microarray and optimize it on the basis of the clinical features, making its application in line with the genetic features of Chinese deafness crowd.Methods:There were384sites in the Goldengate gene chip,19of which were randomly selected to verify sensitivity and specificity by Sanger sequencing method. And then, optimization of the first version chip sites was based on verification. The records of deafness related mutation sites were made by retrieving literature, which was followed by selecting the sites according to several rules. The paramount principle was that the mutation sites was reported more than2times, giving privacy to the sites reported in Chinese population. The secondary principles were following:（1） adjacent sites were separated by over60bp;（2） probe scoring was over0.6（some hotspot mutations were exceptional）;（4） elimating long-missing and insertional variants;（5） excluding the sites whose call rate were less than70%in the first version chip. SNP exchanging principles were as follows:（1） SNP-related genes have been cloned in the Asian population;（2） adjacent sites were separated by over60bp;（3） probe scoring was over0.6;（4） elimating long-missing and insertional variants;（5） excluding the SNP whose call rate were below0.6in the first version chip.Result:1. Sensitivity of the first edition GoldenGate deafness gene chip ranged from0%to100%and specificity was100%;2. Optimized chip covered200deafness nonsyndromic deafness detection sites,40syndromic deafness detection sites and144SNP;3. Optimized chip covered eight most common hot spot mutations in China, as follows: GJB2_1BP_DEL₃5delG, GJB2_1BP_DEL₂35C, GJB2_2BP_DEL₂99₃00at, GJB3_Arg180Term[C/T], SLC26A4_Asn392Tyr[A/T], the SLC26A4_IVS7₂[A/G], SLC26A4_His723Arg [A/G], mitochondrial12rsRNA1555[A/G];4. Optimized chip included46deafness related nucleus genes and mitochondria gene;5. Probe design rating of two editions chip were compared by t test, P<0.05, which meant statistically significant difference;6. Optimized chip sites were named to interpret and determined the results easily.Conclusions:1. The first version chip had high specificity, but the sensitivity varies, owing to process of design and the quality of templates. Hence, the chip was needed for further optimization.2. Optimized GoldenGate deafness gene chip covered common non-syndromic deafness mutation hot spots, and several common syndromic deafness gene mutation sites, which was closely integrated with clinical features.