Rare oncogenic driver events, particularly affecting the expression or splicing of driver genes, are suspected to substantially contribute to the large heterogeneity of hematologic malignancies. However, their identification remains challenging.
To address this issue, we generated the largest dataset to date of matched whole genome sequencing and total RNA sequencing of hematologic malignancies from 3760 patients spanning 24 disease entities. Taking advantage of our dataset size, we focused on discovering rare regulatory aberrations. Therefore, we called expression and splicing outliers using an extension of the workflow DROP (Detection of RNA Outliers Pipeline) and AbSplice, a variant effect predictor that identifies genetic variants causing aberrant splicing. We next trained a machine learning model integrating these results to prioritize new candidate disease-specific driver genes.
We found a median of seven expression outlier genes, two splicing outlier genes, and two rare splice-affecting variants per sample. Each category showed significant enrichment for already well-characterized driver genes, with odds ratios exceeding three among genes called in more than five samples. On held-out data, our integrative modeling significantly outperformed modeling based solely on genomic data and revealed promising novel candidate driver genes. Remarkably, we found a truncated form of the low density lipoprotein receptor LRP1B transcript to be aberrantly overexpressed in about half of hairy cell leukemia variant (HCL-V) samples and, to a lesser extent, in closely related B-cell neoplasms. This observation, which was confirmed in an independent cohort, suggests LRP1B as a novel marker for a HCL-V subclass and a yet unreported functional role of LRP1B within these rare entities.
Altogether, our census of expression and splicing outliers for 24 hematologic malignancy entities and the companion computational workflow constitute unique resources to deepen our understanding of rare oncogenic events in hematologic cancers.

BACKGROUND: Alport syndrome (AS) is characterised by haematuria, proteinuria, a gradual decline in kidney function, hearing loss, and eye abnormalities. The disease is caused by mutations in COL4An (n = 3, 4, 5) that encodes 3-5 chains of type IV collagen in the glomerular basement membrane. AS has three genetic models: X-linked, autosomal recessive, and autosomal dominant. The most common type of AS is X-linked AS, which is caused by COL4A5.
METHODS: We enrolled children with renal insufficiency and a family history of kidney disorders. The proband was identified using whole-exome sequencing. Sanger sequencing was performed to verify the mutation site. Minigene technology was used to analyse the influence of mutant genes on pre-mRNA shearing, and the Iterative Threading ASSEmbly Refinement (I-TASSER) server was used to analyse the protein structure changes.
RESULTS: The proband, together with her mother and younger brother, displayed microscopic haematuria and proteinuria, Pathological examination revealed mesangial hyperplasia and sclerosis. A novel mutation (NM_000495.5 c.4298-8G > A) in the intron of the COL4A5 gene in the proband was discovered, which was also present in the proband\'s mother, brother, and grandmother. In vitro minigene expression experiments verified that the c.4298-8G > A mutation caused abnormal splicing, leading to the retention of six base pairs at the end of intron 46. The I-TASSER software predicted that the mutation affected the hydrogen-bonding structure of COL4A5 and the electrostatic potential on the surface of the protein molecules.
CONCLUSIONS: Based on the patient\'s clinical history and genetic traits, we conclude that the mutation at the splicing site c.4298-8G > A of the COL4A5 gene is highly probable to be the underlying cause within this particular family. This discovery expands the genetic spectrum and deepens our understanding of the molecular mechanisms underlying AS.

Generalized lipodystrophy is a feature of various hereditary disorders, often leading to a progeroid appearance. In the present study we identified a missense and a frameshift variant in a compound heterozygous state in SUPT7L in a boy with intrauterine growth retardation, generalized lipodystrophy, and additional progeroid features. SUPT7L encodes a component of the transcriptional coactivator complex STAGA. By transcriptome sequencing, we showed the predicted missense variant to cause aberrant splicing, leading to exon truncation and thereby to a complete absence of SUPT7L in dermal fibroblasts. In addition, we found altered expression of genes encoding DNA repair pathway components. This pathway was further investigated and an increased rate of DNA damage was detected in proband-derived fibroblasts and genome-edited HeLa cells. Finally, we performed transient overexpression of wildtype SUPT7L in both cellular systems, which normalizes the number of DNA damage events. Our findings suggest SUPT7L as a novel disease gene and underline the link between genome instability and progeroid phenotypes.

Most pathogenic DMD variants are detectable and interpretable by standard genetic testing for dystrophinopthies. However, approximately 1∼3% of dystrophinopthies patients still do not have a detectable DMD variant after standard genetic testing, most likely due to structural chromosome rearrangements and/or deep intronic pseudoexon-activating variants. Here, we report on a boy with a suspected diagnosis of Becker muscular dystrophy (BMD) who remained without a detectable DMD variant after exonic DNA-based standard genetic testing. Dystrophin mRNA studies and genomic Sanger sequencing were performed in the boy, followed by in silico splicing analyses. We successfully detected a novel deep intronic disease-causing variant in the DMD gene (c.2380 + 3317A > T), which consequently resulting in a new dystrophin pseudoexon activation through the enhancement of a cryptic donor splice site. The patient was therefore genetically diagnosed with BMD. Our case report further emphasizes the significant role of disease-causing splicing variants within deep intronic regions in genetically undiagnosed dystrophinopathies.

BACKGROUND: Pathogenic missense variants in the dystrophin (DMD) gene are rarely reported in dystrophinopathies. Most DMD missense variants are of uncertain significance and their pathogenicity interpretation remains complicated. We aimed to investigate whether DMD missense variants would cause aberrant splicing and re-interpret their pathogenicity based on mRNA and protein studies.
METHODS: Nine unrelated patients who had an elevated serum creatine kinase level with or without muscle weakness were enrolled. They underwent a detailed clinical, imaging, and pathological assessment. Routine genetic testing and muscle-derived mRNA and protein studies of dystrophin and sarcoglycan genes were performed in them.
RESULTS: Three of the 9 patients presented with a Duchenne muscular dystrophy (DMD) phenotype and the remaining 6 patients had a suspected diagnosis of Becker muscular dystrophy (BMD) or sarcoglycanopathy based on their clinical and pathological characteristics. Routine genetic testing detected only 9 predicted DMD missense variants in them, of which 6 were novel and interpreted as uncertain significance. Muscle-derived mRNA studies of sarcoglycan genes didn\'t reveal any aberrant transcripts in them. Dystrophin mRNA studies confirmed that 3 predicted DMD missense variants (c.2380G > C, c.4977C > G, and c.5444A > G) were in fact splicing and frameshift variants due to aberrant splicing. The 9 DMD variants were re-interpreted as pathogenic or likely pathogenic based on mRNA and protein studies. Therefore, 3 patients with DMD splicing variants and 6 patients with confirmed DMD missense variants were diagnosed with DMD and BMD, respectively.
CONCLUSIONS: Our study highlights the importance of muscle biopsy and aberrant splicing for clinical and genetic interpretation of uncertain DMD missense variants.

Detection of aberrantly spliced genes is an important step in RNA-seq-based rare-disease diagnostics. We recently developed FRASER, a denoising autoencoder-based method that outperformed alternative methods of detecting aberrant splicing. However, because FRASER\'s three splice metrics are partially redundant and tend to be sensitive to sequencing depth, we introduce here a more robust intron-excision metric, the intron Jaccard index, that combines the alternative donor, alternative acceptor, and intron-retention signal into a single value. Moreover, we optimized model parameters and filter cutoffs by using candidate rare-splice-disrupting variants as independent evidence. On 16,213 GTEx samples, our improved algorithm, FRASER 2.0, called typically 10 times fewer splicing outliers while increasing the proportion of candidate rare-splice-disrupting variants by 10-fold and substantially decreasing the effect of sequencing depth on the number of reported outliers. To lower the multiple-testing correction burden, we introduce an option to select the genes to be tested for each sample instead of a transcriptome-wide approach. This option can be particularly useful when prior information, such as candidate variants or genes, is available. Application on 303 rare-disease samples confirmed the relative reduction in the number of outlier calls for a slight loss of sensitivity; FRASER 2.0 recovered 22 out of 26 previously identified pathogenic splicing cases with default cutoffs and 24 when multiple-testing correction was limited to OMIM genes containing rare variants. Altogether, these methodological improvements contribute to more effective RNA-seq-based rare diagnostics by drastically reducing the amount of splicing outlier calls per sample at minimal loss of sensitivity.

Introduction: RNA sequence analysis can be effectively used to identify aberrant splicing, and tumor suppressor genes are adequate targets considering their loss-of-function mechanisms. Sanger sequencing is the simplest method for RNA sequence analysis; however, because of its insufficient sensitivity in cases with nonsense-mediated mRNA decay (NMD), the use of cultured specimens with NMD inhibition has been recommended, hindering its wide adoption. Method: The results of Sanger sequencing of peripheral blood RNA without NMD inhibition performed on potential splicing variants of tumor suppressor genes were retrospectively reviewed. For negative cases, in which no change was identified in the transcript, the possibility of false negativity caused by NMD was assessed through a review of the up-to-date literature. Results: Eleven potential splice variants of various tumor suppressor genes were reviewed. Six variants were classified as pathogenic or likely pathogenic based on the nullifying effect identified by Sanger RNA sequencing. Four variants remained as variants of uncertain significance because of identified in-frame changes or normal expression of both alleles. The result of one variant was suspected to be a false negative caused by NMD after reviewing a recent study that reported the same variant as causing a nullifying effect on the affected transcript. Conclusion: Although RNA changes found in the majority of cases were expected to undergo NMD by canonical rules, most cases (10/11) were interpretable by Sanger RNA sequencing without NMD inhibition due to incomplete NMD efficiency or allele-specific expression despite highly efficient NMD.

BACKGROUND: Lipoprotein lipase (LPL) is the rate-limiting enzyme for triglyceride hydrolysis. Homozygous or compound heterozygous LPL variants cause autosomal recessive familial chylomicronemia syndrome (FCS), whereas simple heterozygous LPL variants are associated with hypertriglyceridemia (HTG) and HTG-related disorders. LPL frameshift coding sequence variants usually cause complete functional loss of the affected allele, thereby allowing exploration of the impact of different levels of LPL function in human disease.
METHODS: All exons and flanking intronic regions of LPL were Sanger sequenced in patients with HTG-related acute pancreatitis (HTG-AP) or HTG-AP in pregnancy. Previously reported LPL frameshift coding sequence variants were collated from the Human Gene Mutation Database and through PubMed keyword searching. Original reports were manually evaluated for the following information: zygosity status of the variant, plasma LPL activity of the variant carrier, disease referred for genetic analysis, patient\'s age at genetic analysis, and patient\'s disease history. SpliceAI was employed to predict the potential impact of collated variants on splicing.
RESULTS: Two novel rare variants were identified, and 53 known LPL frameshift coding sequence variants were collated. Of the 51 variants informative for zygosity, 30 were simple heterozygotes, 12 were homozygotes, and 9 were compound heterozygotes. Careful evaluation of the 55 variants with respect to their clinical and genetic data generated several interesting findings. First, we conclude that 6-7% residual LPL function could significantly delay the age of onset of FCS and reduce the prevalence of FCS-associated syndromes. Second, whereas a large majority of LPL frameshift coding sequence variants completely disrupt gene function through their \"frameshift\" nature, a small fraction of these variants may act wholly or partly as \"in-frame\" variants, leading to the generation of protein products with some residual LPL function. Third, we identified two candidate LPL frameshift coding sequence variants that may retain residual function based on genotype-phenotype correlation or SpliceAI-predicted data.
CONCLUSIONS: This study reported two novel LPL variants and yielded new insights into the genotype-phenotype relationship as it pertains to LPL frameshift coding sequence variants.

NUS1 is responsible for encoding of the Nogo-B receptor (NgBR), which is a subunit of cis-prenyltransferase. Over 25 variants in NUS1 have been reported, and these variants have been found to be associated with various phenotypes, such as congenital disorders of glycosylation (CDG) and developmental and epileptic encephalopathy (DEE). We report on the case of a patient who presented with language and motor retardation, epilepsy, and electroencephalogram abnormalities. Upon conducting whole-exome sequencing, we discovered a novel pathogenic variant (chr6:118024873, NM_138459.5: c.791 + 6T>G) in NUS1, which was shown to cause Exon 4 to be skipped, resulting in a loss of 56 amino acids. Our findings strongly suggest that this novel variant of NUS1 is responsible for the development of neurological disorders, including epilepsy. It is believed that the truncation of Nogo-B receptor results in the loss of cis-prenyltransferase activity, which may be the underlying cause of the disease.

Objective: Variants of the polycystic kidney and hepatic disease 1 (PKHD1) gene are associated with autosomal recessive polycystic kidney disease (ARPKD). This study aimed to identify the genetic causes in a Chinese pedigree with ARPKD and design a minigene construct of the PKHD1 gene to investigate the impact of its variants on splicing. Methods: Umbilical cord samples from the proband and peripheral blood samples from his parents were collected, and genomic DNA was extracted for whole-exome sequencing (WES). Bioinformatic analysis was used to identify potential genetic causes, and Sanger sequencing confirmed the existence of variants within the pedigree. A minigene assay was performed to validate the effects of an intronic variant on mRNA splicing. Results: Two variants, c.9455del (p.N3152Tfs*10) and c.2408-13C>G, were identified in the PKHD1 gene (NM_138694.4) by WES; the latter has not been previously reported. In silico analysis predicted that this intronic variant is potentially pathogenic. Bioinformatic splice prediction tools revealed that the variant is likely to strongly impact splice site function. An in vitro minigene assay revealed that c.2408-13C>G can cause aberrant splicing, resulting in the retention of 12 bp of intron 23. Conclusion: A novel pathogenic variant of PKHD1, c.2408-13C>G, was found in a fetus with ARPKD, which enriches the variant spectrum of the PKHD1 gene and provides a basis for genetic counseling and the diagnosis of ARPKD. Minigenes are optimal to determine whether intron variants can cause aberrant splicing.