X-Linked Alport Syndrome in Women: Genotype and Clinical Course in 24 Cases

Objectives: X-linked Alport syndrome (XLAS) females are at risk of developing proteinuria and chronic kidney damage (CKD). The aim of this study is to evaluate the genotype-phenotype correlation in this rare population.Materials and Methods: This is a prospective, observational study of XLAS females, confirmed by a pathogenic mutation in COL4A5 and renal ultrastructural evaluation. Proteinuria, renal function and extrarenal involvement were monitored during follow-up. Patients were divided in 2 groups, according to mutations in COL4A5: missense (Group 1) and non-missense variants (Group 2).Results: Twenty-four XLAS females, aged 10.6 +/- 10.4 years at clinical onset (mean follow-up: 13.1 +/- 12.6 years) were recruited between 2000 and 2017 at a single center. In group 1 there were 10 patients and in group 2, 14 (mean age at the end of follow-up: 24.9 +/- 13.6 and 23.2 +/- 13.8 years, respectively). One patient in Group 1 and 9 in Group 2 (p = 0.013) developed proteinuria during follow-up. Mean eGFR at last follow-up was lower in Group 2 (p = 0.027), where two patients developed CKD. No differences in hearing loss were documented among the two groups. Two patients in Group 2 carried one mutation in both COL4A5 and COL4A3 (digenic inheritance) and were proteinuric. In one family, the mother presented only hematuria while the daughter was proteinuric and presented a greater inactivation of the X chromosome carrying the wild-type allele.Conclusions: The appearance of proteinuria and CKD is more frequent in patients with severe variants. Carrying digenic inheritance and skewed XCI seem to be additional risk factors for proteinuria in XLAS females

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

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

<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

Patient characteristics.

<p>Patient characteristics.</p