massively-parallel-splicing-assay-to-examine-splicing-errors-caused

Massively Parallel Splicing Assay to Examine Splicing Errors Caused by Disease-Related Intronic Variants

Splicing errors represent 10-30% of the pathogenic mutations responsible for rare genetic disorders. RNA splicing ensures proper gene expression by ...

Academia Sinica

Cytochrome P450 is an important monooxygenase responsible for the metabolism of a large variety of structurally diverse compounds. The aim of this study was to systematically investigate the DNA sequence variations in 14 cytochrome P450 genes relevant to drug metabolism in the Han Chinese population.

Novel DNA sequence variations of cytochrome P450 genes in the Han Chinese population.

We report the first genome-wide association study of a joint analysis using 795 Han Chinese individuals with treatment-refractory schizophrenia (TRS) and 806 controls. Three loci showed suggestive significant association with TRS were identified. These loci include: rs10218843 (P = 3.04 × 10(-7)) and rs11265461 (P = 1.94 × 10(-7)) are adjacent to signaling lymphocytic activation molecule family member 1 (SLAMF1); rs4699030 (P = 1.94 × 10(-6)) and rs230529 (P = 1.74 × 10(-7)) are located in the gene nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1); and rs13049286 (P = 3.05 × 10(-5)) and rs3827219 (P = 1.66 × 10(-5)) fall in receptor-interacting serine/threonine-protein kinase 4 (RIPK4). One isolated single nucleotide polymorphism (SNP), rs739617 (P = 3.87 × 10(-5)) was also identified to be associated with TRS. The -94delATTG allele (rs28362691) located in the promoter region of NFKB1 was identified by resequencing and was found to associate with TRS (P = 4.85 × 10(-6)). The promoter assay demonstrated that the -94delATTG allele had a significant lower promoter activity than the -94insATTG allele in the SH-SY5Y cells. This study suggests that rs28362691 in NFKB1 might be involved in the development of TRS.

Genome-wide association study of treatment refractory schizophrenia in Han Chinese.

The GLA IVS4+919G>A which is linked to late-onset Fabry disease shows high frequency in Taiwan.

Genetic epidemiological study doesn't support GLA IVS4+919G>A variant is a significant mutation in Fabry disease.

The human genome contains millions of single nucleotide polymorphisms (SNPs); many of these SNPs are intronic and have unknown functional significance. SNPs occurring within intron branchpoint sites, especially at the adenine (A), would presumably affect splicing; however, this has not been systematically studied. We employed a splicing prediction tool to identify human intron branchpoint sites and screened dbSNP for identifying SNPs located in the predicted sites to generate a genome-wide branchpoint site SNP database.

Identification of functional single nucleotide polymorphisms in the branchpoint site.

Full genome analysis of a young girl with deafness, dystonia, central hypomyelination, refractory seizure, and fluctuating liver function impairment revealed a heterozygous, de novo variant in the BCAP31 gene on chromosome Xq28 (NM_001256447.2:c.92G>A), mutations of which caused the X-linked recessive severe neurologic disorder deafness, dystonia, and cerebral hypomyelination. Reverse transcription-polymerase chain reaction of the patient's white blood cells showed the absence of wild-type BCAP31 messenger RNA (mRNA) but the presence of two novel BCAP31 mRNAs. The major alternatively spliced mRNA is due to Exon 2 skipping and the utilization of a new initiation site in Exon 3 that leads to a frameshift and truncated transcript while the minor novel mRNA has a 110 nucleotide insertion to Exon 2. Phasing studies showed that the de novo variant arose in the paternal X chromosome. X chromosome inactivation assay was done and confirmed that the patient's maternal X chromosome was preferentially inactivated, providing evidence that the mutated BCAP31 gene was the one predominantly expressed. According to the American College of Medical Genetics and Genomics guideline, this variant is deemed "pathogenic" (PS2, PS3, PM2, PP3, and PP4) and deleterious. This is the first reported female patient in BCAP31-related syndrome resulted from skewed X-inactivation and a de novo mutation in the active X chromosome.

De novo mutation and skewed X-inactivation in girl with BCAP31-related syndrome.

It is estimated that 10%-30% of disease-associated genetic variants affect splicing. Splicing variants may generate deleteriously altered gene product and are potential therapeutic targets. However, systematic diagnosis or prediction of splicing variants is yet to be established, especially for the near-exon intronic splice region. The major challenge lies in the redundant and ill-defined branch sites and other splicing motifs therein. Here, we carried out unbiased massively parallel splicing assays on 5,307 disease-associated variants that overlapped with branch sites and collected 5,884 variants across the 5' splice region. We found that strong splice sites and exonic features preserve splicing from intronic sequence variation. Whereas the splice-altering mechanism of the 3' intronic variants is complex, that of the 5' is mainly splice-site destruction. Statistical learning combined with these molecular features allows precise prediction of altered splicing from an intronic variant. This statistical model provides the identity and ranking of biological features that determine splicing, which serves as transferable knowledge and out-performs the benchmarking predictive tool. Moreover, we demonstrated that intronic splicing variants may associate with disease risks in the human population. Our study elucidates the mechanism of splicing response of intronic variants, which classify disease-associated splicing variants for the promise of precision medicine.

Mechanism and modeling of human disease-associated near-exon intronic variants that perturb RNA splicing.

Splicing variants are a major class of pathogenic mutations, with their severity equivalent to nonsense mutations. However, redundant and degenerate splicing signals hinder functional assessments of sequence variations within introns, particularly at branch sites. We have established a massively parallel splicing assay to assess the impact on splicing of 11,191 disease-relevant variants. Based on the experimental results, we then applied regression-based methods to identify factors determining splicing decisions and their respective weights.

Article	Year
Novel DNA sequence variations of cytochrome P450 genes in the Han Chinese population. Pharmacogenomics\| PubMed ID: 19290787	2009
Genome-wide association study of treatment refractory schizophrenia in Han Chinese. PloS one\| PubMed ID: 22479419	2012
Genetic epidemiological study doesn't support GLA IVS4+919G>A variant is a significant mutation in Fabry disease. Molecular genetics and metabolism\| PubMed ID: 28377241	2017
Identification of functional single nucleotide polymorphisms in the branchpoint site. Human genomics\| PubMed ID: 29121990	2017
De novo mutation and skewed X-inactivation in girl with BCAP31-related syndrome. Human mutation\| PubMed ID: 32652807	2020
Mechanism and modeling of human disease-associated near-exon intronic variants that perturb RNA splicing. Nature structural & molecular biology\| PubMed ID: 36303034	2022
SpliceAPP: an interactive web server to predict splicing errors arising from human mutations. BMC genomics\| PubMed ID: 38877417	2024

Hung-Lun Chiang

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