Computational protocol: Phenotype variability in a large Spanish family with Alport syndrome associated with novel mutations in COL4A3 gene

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Protocol publication

[…] This study was conducted according to protocols approved by the Ethical Committee of La Rioja (CEICLAR PI-161), and written informed consent was obtained from each patient for publication. The proband and 18 members of the family (11 males and 8 females), with clinical suspect of AS, were included in this study. Familiar clinical history was performed carefully in each case by reconstruction of the pedigree for at least three generations (Fig. ). Urinalysis and renal function evaluations were carried out in all patients. Audiological and ophthalmic examinations were performed, and, whenever indicated, a renal biopsy was analysed by LM and the ultrastructural study was performed by EM.Fig. 1 Proband (V.I) is a 31-year-old male patient that was referred to the Pediatric Nephrology Service at the age of 8 years for evaluation of persistent hematuria.He fulfilled three of the AS clinical diagnostic criteria: a positive family history of hematuria, proteinuria and hearing loss. Ophthalmic evaluation was normal. The results of audiometric testing revealed a bilateral sensorineural hearing loss. At the age of 13 years, urine analysis revealed proteinuria (1.77 g/24 h), so he underwent a renal biopsy. LM revealed minimal mesangial changes, with no glomeruli or tubulointerstitial lesions, and the immunofluorescence microscopy was negative for IgA, IgG, IgM, C3 and C4. He started treatment with inhibitors of angiotensin converting enzyme (ACE inhibitors). Nevertheless, progressive increase in proteinuria levels (up to 5.2 g/24 h) with clinical and biochemical nephrotic syndrome were found. For this reason, a second renal biopsy was performed. The results were compatible with thin basement membrane disease (TBMD). Antiproteinuric treatment was reinforced with ACE inhibitors and spironolactone. Due to the persistence of nephrotic syndrome, tacrolimus treatment was started, but no response after 10 months of treatment was obtained. At the age of 25 years, he started with progressive renal failure and three years later he received renal transplantation from living related donor. Considering the clinical course of the patient, and keeping in mind the hearing impairment and the family history, molecular genetic testing was carried out. This study was extended to all of family members (10 males and 8 females, age ranges from 25 to 66 years). In order to determine the molecular cause of the disease, blood samples from the proband and his family were collected after genetic counseling and informed consent. Genomic DNA and RNA were isolated from EDTA peripheral blood samples using QIAamp DNA Blood kit and RNeasy Mini kit, respectively, according to the manufacturer’s protocol (Qiagen, http://www.qiagen.com). Mutation analysis of COL4A3 (NM_000091) and COL4A4 (NM_000092) genes was performed by analyzing the entire coding sequences and flanking intronic regions by PCR and subsequent Sanger sequencing in both directions. PCR products were purified with ExoStar™ (GE Healthcare). Sequencing reactions were performed using Big Dye® Terminator v.3.1 Cycle Sequencing kit (Applied Biosystems, Forest City, CA, USA), and purified with Big Dye X-Terminator (Applied Biosystems, Foster City, CA, USA). DNA sequences were analyzed on an ABI PRISM® 3130 automated sequencer (PE Applied Biosystems, Forest City, CA, USA) with Sequencing Analysis Software (Applied Biosystems, Foster City, CA). After that, the obtained sequences were analyzed using SeqScape v2.5 software. The specific primer sequences are available upon request. Pathogenicity of the detected variants was predicted by in silico analysis with bioinformatics tools such as Sorting Intolerant From Tolerant (SIFT), Mutation Taster, Polyphen-2, and Human Splicing Finder (HSF 3.0). ExAC browser of Broad Institute, 1000 Genomes database and dbSNP138. […]

Pipeline specifications

Software tools SeqScape, SIFT, PolyPhen
Application Sanger sequencing