The primers were: COL4A5_SplMutG_Forward primer, 5′-TGGCTATATCCTTTCCCCAGT-3′ and COL4A5_SplMutG_Reverse primer, 5′-GCCACACCTTGTATGCCTTT-3′. Primers flanking the splice region variant were used for PCR amplification and for forward and reverse sequencing. Sanger sequencing was performed on all family members from whom DNA was available. All analyses were based on the human reference sequence UCSC build hg19 (NCBI build 37.2). Analysis and interpretation was performed using DNAstar software. Sequencing was performed on the Illumina MiSeq using a paired-end, 150 base-pair configuration. Target enrichment was performed using oligonucleotide-based targeted capture (Agilent Rapid Capture Enrichment) of whole-genome shotgun sequencing libraries. This assay used next-generation sequencing to analyze all coding exons and at least 10 bp of flanking intronic sequence of a panel of 301 genes to a minimum depth of X15. Larsen, Nephropath/Arkana Laboratories, Little Rock, AR) as a research test for one affected family member. Targeted next-generation sequencing of a customized kidney disease gene panel was performed under IRB approval (Schulman IRB, PI: C. This method could be generally applicable to help diagnose X-linked Alport syndrome.Ĭustom targeted next-generation and direct Sanger sequencing We also tested a novel non-invasive method to characterize the variable deposition of collagen α5(IV) protein in the basement membranes of plucked hair follicles from this family by simple immunofluorescence staining. We examined the consequences of this variant on exon 25 splicing using in silico and in vitro approaches. This report describes a novel splice region variant upstream of COL4A5 exon 25 in a family with both males and females affected. Furthermore, the correlation between genotype and phenotype at the molecular level has not been investigated for the vast majority of variants reported as pathogenic. However, the functional consequences of many splice region variants in AS patients have not been investigated. Hundreds of variants in the AS genes ( COL4A3, COL4A4 and COL4A5) have now been described, and approximately 13 – 16 % are splice site mutations. Early large-scale genetic studies of AS suggested a correlation between mutation type and phenotype. The phenotype can vary in severity and age of presentation, both between and within families. The original descriptions of AS were in males who inherited disease in an X-linked Mendelian pattern, but AS can also be inherited in an autosomal fashion. Alport syndrome (AS) is a hereditary disease with a classical clinical presentation of renal failure, hearing loss, and ocular manifestations.
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