Improving mutation screening in familial hematuric nephropathies through next generation sequencing

Abstract

Alport syndrome is an inherited nephropathy associated with mutations in genes encoding type IV collagen chains present in the glomerular basement membrane. COL4A5 mutations are associated with the major X-linked form of the disease, and COL4A3 and COL4A4 mutations are associated with autosomal recessive and dominant forms (thought to be involved in 15% and 1%-5% of the families, respectively) and benign familial hematuria. Mutation screening of these three large genes is time-consuming and expensive. Here, we carried out a combination of multiplex PCR, amplicon quantification, and next generation sequencing (NGS) analysis of three genes in 101 unrelated patients. We identified 88 mutations and 6 variations of unknown significance on 116 alleles in 83 patients. Two additional indel mutations were found only by secondary Sanger sequencing, but they were easily identified retrospectively with the web-based sequence visualization tool Integrative Genomics Viewer. Altogether, 75 mutations were novel. Sequencing the three genes simultaneously was particularly advantageous as the mode of inheritance could not be determined with certainty in many instances. The proportion of mutations in COL4A3 and COL4A4 was notably high, and the autosomal dominant forms of Alport syndrome appear more frequently than reported previously. Finally, this approach allowed the identification of large COL4A3 and COL4A4 rearrangements not described previously. We conclude that NGS is efficient, reduces screening time and cost, and facilitates the provision of appropriate genetic counseling in Alport syndrome.

Keywords: Alport syndrome; genetic renal disease; molecular genetics.

Figure 1.

Mutation screening strategy. Schematic visualization of the workflow used for variation screening. CGH, comparative genomic hybridization; CNV, copy number variation; NGS, next generation sequencing; PGM, personal genome machine.

Figure 2.

Large COL4A3-COL4A4 rearrangements. Characterization of rearrangements detected by NGS. (A) GeneScan analysis of part of one multiplex PCR amplification product in patient 41 (red) and control DNA (blue) showing a heterozygous deletion of COL4A3 exon 22, whereas the signal for COL4A3 exon 25 appears normal. (B) Haplotype analysis of single nucleotide variants (SNVs) localized in COL4A3 intron 15 and exons 21–23, 25, and 46 in patient 41 and her parents showing no maternal contribution for SNVs in exons 21–23 but a biparental contribution to the SNVs in intron 15, exon 25 (there is no SNV in COL4A3 exon 24), and exon 46. (C) Array CGH profile of chromosome 2 showing a duplication of most of the COL4A3 gene in case 35. Results are shown as log2 intensity ratios of patient 35 to reference. Size of the duplication, which includes 11 probes, is between 99 and 113 kb. (D) MLPA analysis of DNA of patient 42 (left panel), his father (center panel), and his mother (right panel). Probes for COL4A3 and COL4A4 exons are indicated above their respective peaks. Three control probes (not labeled) are used for normalization. MLPA shows a duplication of COL4A3 exons 44–47 in the homozygous state in the patient and the heterozygous state in his parents. The same customized MLPA approach confirmed the rearrangements in cases 24, 42, 54, and 68.

Figure 3.

Autosomal dominant forms of AS appear more frequently than reported previously. Pedigrees of four families with autosomal dominant Alport syndrome. The mode of inheritance was certain in family 24 because of father to son transmission of the disease, but it may have been either autosomal dominant or X-linked in the three other families. In all cases, probands (shown by arrows) present without renal failure, but their parents and/or grandparents have developed renal failure with or without deafness.