Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis

<div><p>Alport syndrome (AS) is an inherited progressive renal disease caused by mutations in <i>COL4A3</i>, <i>COL4A4</i>, and <i>COL4A5</i> genes. Despite simultaneous screening of these genes being widely available, mutation detection still remains incomplete in a non-marginal portion of patients. Here, we applied whole-exome sequencing (WES) in 3 Italian families negative after candidate-gene analyses. In Family 1, we identified a novel heterozygous intronic variant (c.2245-40A>G) -outside the conventionally screened candidate region for diagnosis- potentially disrupting <i>COL4A5</i> exon29 splicing. Using a minigene-based approach in HEK293 cells we demonstrated that this variant abolishes exon29 branch site, causing exon skipping. Moreover, skewed X-inactivation of the c.2245-40A>G allele correlated with disease severity in heterozygous females. In Family 2, WES highlighted a novel <i>COL4A5</i> hemizygous missense mutation (p.Gly491Asp), which segregates with the phenotype and impacts on a highly-conserved residue. Finally, in Family 3, we detected a homozygous 24-bp in-frame deletion in <i>COL4A3</i> exon1 (NM_000091.4:c.30_53del:p.Val11_Leu18del or c.40_63del24:p.Leu14_Leu21del), which is ambiguously annotated in databases, although it corresponds to a recurrent AS mutation. Functional analyses showed that this deletion disrupts COL4A3 signal peptide, possibly altering protein secretion. In conclusion, WES -together with functional studies- was fundamental for molecular diagnosis in 3 AS families, highlighting pathogenic variants that escaped previous screenings.</p></div

<i>In-vitro</i> analysis of the impact of c.2245-40A>G variant on <i>COL4A5</i> pre-mRNA splicing.

<p>(A) Schematic representation of the hybrid pBS-KS-COL4A5_ex29 minigene where α-globin exons are represented by light grey boxes, fibronectin (<i>FN1</i>) exons by white boxes, whereas introns are shown as black lines (not to scale). Exon 29 of <i>COL4A5</i> is represented by a dark grey box. The c.2245-40A>G mutation in intron 28 is indicated by a star. Primers used in RT-PCR assays are also indicated. (B) On the left, agarose gel (2%) electrophoresis of RT-PCR products obtained from RNA of HEK293 cells transfected with the wild-type (wt) or mutant (mut) minigene vector. M: molecular weight marker (pUC9-<i>Hae</i>III). In the middle, GeneMapper windows show fluorescence peaks corresponding to the molecular species amplified by RT-PCR. Grey shaded peaks correspond to the RT-PCR-labeled products, whose relative quantitation is reported on the right of the panel (%). Unshaded peaks represent the size standard (ROX-500 HD). The <i>x</i> axis indicates fluorescence units. On the right, schematic representation of the splicing products, as verified by Sanger sequencing. The length of each fragment is shown.</p

<i>In-vitro</i> analysis of the impact of c.2245-40A>G variant on <i>COL4A5</i> pre-mRNA splicing.

<p>(A) Schematic representation of the hybrid pBS-KS-COL4A5_ex29 minigene where α-globin exons are represented by light grey boxes, fibronectin (<i>FN1</i>) exons by white boxes, whereas introns are shown as black lines (not to scale). Exon 29 of <i>COL4A5</i> is represented by a dark grey box. The c.2245-40A>G mutation in intron 28 is indicated by a star. Primers used in RT-PCR assays are also indicated. (B) On the left, agarose gel (2%) electrophoresis of RT-PCR products obtained from RNA of HEK293 cells transfected with the wild-type (wt) or mutant (mut) minigene vector. M: molecular weight marker (pUC9-<i>Hae</i>III). In the middle, GeneMapper windows show fluorescence peaks corresponding to the molecular species amplified by RT-PCR. Grey shaded peaks correspond to the RT-PCR-labeled products, whose relative quantitation is reported on the right of the panel (%). Unshaded peaks represent the size standard (ROX-500 HD). The <i>x</i> axis indicates fluorescence units. On the right, schematic representation of the splicing products, as verified by Sanger sequencing. The length of each fragment is shown.</p

Functional characterization of the signal peptide deletion in COL4A3.

<p>Single confocal sections of HEK293 cells expressing EGFP N-terminus fused either with the entire COL4A3 signal peptide (SP-wt-hybEGFP, top panels) or the 8-amino-acid deleted signal peptide (SP-del-hybEGFP, middle panels). Positive control cells, expressing a soluble EGFP (pEGFP-N1) are also shown (bottom panels). DAPI, 4',6-diamidino-2-phenylindole; EGFP, Enhanced green fluorescent protein. Scale bar: 10 μm.</p

Identification of candidate <i>COL4A5</i> and <i>COL4A3</i> variants segregating with Alport syndrome in three Italian families.

<p>Pedigrees of Family 1 (A, X-linked), 2 (B, X-linked), and 3 (C, autosomal recessive) showing the segregation of the identified variants with AS. Individuals analyzed by WES are pointed by an arrow. The genotype of available individuals from each family is indicated below the corresponding symbols and illustrative electropherograms are shown on the right. <u>M</u>, mutant; W, wild type; R, A or G; S, G or C; Y, C or T; CKD, chronic kidney disease.</p

Patient characteristics.

<p>Patient characteristics.</p

Living conditions scoring system.

<p>Living conditions scoring system.</p

Socioeconomic factors as related to the cumulative outcome of mortality and lost to follow-up.

<p>Socioeconomic factors as related to the cumulative outcome of mortality and lost to follow-up.</p

The social profile of the cohort of 257 CKD paediatric patients.

<p>The social profile of the cohort of 257 CKD paediatric patients.</p

Survival curves.

<p>Owing to the great variability of the characteristics of the cohort (from age, to duration of disease, to disease stage at entry, to the different robustness of the socioeconomic markers), only a fully adjusted multivariable analysis of the whole cohort could provide a synthetic view of the determinants of the combined outcome end-point of the cohort as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153963#pone.0153963.t005" target="_blank">Table 5</a>.</p