Dataset Information

Determination of the pathogenicity of known COL4A5 intronic variants by in vitro splicing assay.

ABSTRACT: X-linked Alport syndrome (XLAS) is a congenital renal disease caused by mutations in COL4A5. In XLAS cases suspected of being caused by aberrant splicing, transcript analysis needs to be conducted to determine splicing patterns and assess the pathogenicity. However, such analysis is not always available. We conducted a functional splicing assay using a hybrid minigene for seven COL4A5 intronic mutations: one was identified by us and six were found in the Human Gene Mutation Database. The minigene assay revealed exon skipping in four variants, exon skipping and a 10-bp insertion in one variant, and no change in one variant, which appeared not to be pathogenic. For one variant, our assay did not work. The results of all three cases for which transcript data were available were consistent with our assay results. Our findings may help to increase the accuracy of genetic test results and clarify the mechanisms causing aberrant splicing.

SUBMITTER: Horinouchi T

PROVIDER: S-EPMC6722096 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Determination of the pathogenicity of known COL4A5 intronic variants by in vitro splicing assay.

Horinouchi Tomoko T Nozu Kandai K Yamamura Tomohiko T Minamikawa Shogo S Nagano China C Sakakibara Nana N Nakanishi Koichi K Shima Yuko Y Morisada Naoya N Ishiko Shinya S Aoto Yuya Y Nagase Hiroaki H Takeda Hiroki H Rossanti Rini R Kaito Hiroshi H Matsuo Masafumi M Iijima Kazumoto K

Scientific reports 20190903 1

X-linked Alport syndrome (XLAS) is a congenital renal disease caused by mutations in COL4A5. In XLAS cases suspected of being caused by aberrant splicing, transcript analysis needs to be conducted to determine splicing patterns and assess the pathogenicity. However, such analysis is not always available. We conducted a functional splicing assay using a hybrid minigene for seven COL4A5 intronic mutations: one was identified by us and six were found in the Human Gene Mutation Database. The minigen ...[more]

PMID: 31481700

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Project description:BackgroundCurrent clinical variant analysis pipelines focus on coding variants and intronic variants within 10-20 bases of an exon-intron boundary that may affect splicing. The impact of newer splicing prediction algorithms combined with in vitro splicing assays on rare variants currently considered Benign/Likely Benign (B/LB) is unknown.MethodsExome sequencing data from 576 pediatric cancer patients enrolled in the Texas KidsCanSeq study were filtered for intronic or synonymous variants absent from population databases, predicted to alter splicing via SpliceAI (>0.20), and scored as potentially deleterious by CADD (>10.0). Total cellular RNA was extracted from monocytes and RT-PCR products analyzed. Subsequently, rare synonymous or intronic B/LB variants in a subset of genes submitted to ClinVar were similarly evaluated. Variants predicted to lead to a frameshifted splicing product were functionally assessed using an in vitro splicing reporter assay in HEK-293T cells.ResultsKidsCanSeq exome data analysis revealed a rare, heterozygous, intronic variant (NM_177438.3(DICER1):c.574-26A>G) predicted by SpliceAI to result in gain of a secondary splice acceptor site. The proband had a personal and family history of pleuropulmonary blastoma consistent with DICER1 syndrome but negative clinical sequencing reports. Proband RNA analysis revealed alternative DICER1 transcripts including the SpliceAI-predicted transcript.Similar bioinformatic analysis of synonymous or intronic B/LB variants (n=31,715) in ClinVar from 61 Mendelian disease genes yielded 18 variants, none of which could be scored by MaxEntScan. Eight of these variants were assessed (DICER1 n=4, CDH1 n=2, PALB2 n=2) using in vitro splice reporter assay and demonstrated abnormal splice products (mean 66%; range 6% to 100%). Available phenotypic information from submitting laboratories demonstrated DICER1 phenotypes in 2 families (1 variant) and breast cancer phenotypes for PALB2 in 3 families (2 variants).ConclusionsOur results demonstrate the power of newer predictive splicing algorithms to highlight rare variants previously considered B/LB in patients with features of hereditary conditions. Incorporation of SpliceAI annotation of existing variant data combined with either direct RNA analysis or in vitro assays has the potential to identify disease-associated variants in patients without a molecular diagnosis.

Dataset Information

Determination of the pathogenicity of known COL4A5 intronic variants by in vitro splicing assay.

Publications

Determination of the pathogenicity of known COL4A5 intronic variants by in vitro splicing assay.

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