PMC5453569_01
Identification of candidate COL4A5 and COL4A3 variants segregating with Alport syndrome in three Italian families.. 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. M, mutant; W, wild type; R, A or G; S, G or C; Y, C or T; CKD, chronic kidney disease.
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The variation was found in the heterozygous state in the mother (II5), who suffers from micro-hematuria (Fig 1C). Since the three female carriers of the branch-point NM_000495.4:c.2245-40A>G variant showed different severity of phenotypic manifestations (Fig 1A), we investigated the possibility that these differences might be due to skewed X-inactivation.
PMC5453569_02
Identification of candidate COL4A5 and COL4A3 variants segregating with Alport syndrome in three Italian families.. 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. M, mutant; W, wild type; R, A or G; S, G or C; Y, C or T; CKD, chronic kidney disease.
pone.0178630.g001.jpg
PMC5453569_03_pone.0178630.g001.jpg
pone.0178630.g001
The variation was found in the heterozygous state in the mother (II5), who suffers from micro-hematuria (Fig 1C). Since the three female carriers of the branch-point NM_000495.4:c.2245-40A>G variant showed different severity of phenotypic manifestations (Fig 1A), we investigated the possibility that these differences might be due to skewed X-inactivation.
PMC5453569_03
Identification of candidate COL4A5 and COL4A3 variants segregating with Alport syndrome in three Italian families.. 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. M, mutant; W, wild type; R, A or G; S, G or C; Y, C or T; CKD, chronic kidney disease.
pone.0178630.g001.jpg
PMC5453569_03_pone.0178630.g001.jpg
pone.0178630.g001
The variation was found in the heterozygous state in the mother (II5), who suffers from micro-hematuria (Fig 1C). Since the three female carriers of the branch-point NM_000495.4:c.2245-40A>G variant showed different severity of phenotypic manifestations (Fig 1A), we investigated the possibility that these differences might be due to skewed X-inactivation.
In-vitro analysis of the impact of c.2245-40A>G variant on COL4A5 pre-mRNA splicing.. (A) Schematic representation of the hybrid pBS-KS-COL4A5_ex29 minigene where alpha-globin exons are represented by light grey boxes, fibronectin (FN1) exons by white boxes, whereas introns are shown as black lines (not to scale). Exon 29 of COL4A5 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-HaeIII). 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 x 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.
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Given the unavailability of a suitable biological specimen from the patient to extract RNA, COL4A5 exon 29, with the surrounding intronic sequences, was cloned, either in the wild-type or in the mutant version, into a pBS-KS_modified hybrid minigene vector (Fig 2A). Transfection with the mutant vector originated two aberrant products: the most abundant (93.9%) resulting from the skipping of exon 29, and the other one (6.1%) resulting from the inclusion of a shorter exon 29, caused by the activation of an exonic cryptic 3' acceptor site (Fig 2B).
Functional characterization of the signal peptide deletion in COL4A3.. 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 mum.
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Our results show a clear difference in hybEGFP subcellular localization in the presence of the deleted COL4A3 signal peptide compared to the wild type (Fig 3). In particular, hybEGFP fused with the mutant peptide lacking 8 amino acids displays a diffuse and uniform localization in the cytoplasm and in the nucleus, similarly to the soluble EGFP (Fig 3).