Large-scale functional screen identifies genetic variants with splicing effects in modern and archaic humans.

Humans coexisted and interbred with other hominins which later became extinct. These archaic hominins are known to us only through fossil records and for two cases, genome sequences. Here, we engineer Neanderthal and Denisovan sequences into thousands of artificial genes to reconstruct the pre-mRNA processing patterns of these extinct populations. Of the 5,169 alleles tested in this massively parallel splicing reporter assay (MaPSy), we report 962 exonic splicing mutations that correspond to differences in exon recognition between extant and extinct hominins. Using MaPSy splicing variants, predicted splicing variants, and splicing quantitative trait loci, we show that splice-disrupting variants experienced greater purifying selection in anatomically modern humans than that in Neanderthals. Adaptively introgressed variants were enriched for moderate-effect splicing variants, consistent with positive selection for alternative spliced alleles following introgression. As particularly compelling examples, we characterized a unique tissue-specific alternative splicing variant at the adaptively introgressed innate immunity gene TLR1, as well as a unique Neanderthal introgressed alternative splicing variant in the gene HSPG2 that encodes perlecan. We further identified potentially pathogenic splicing variants found only in Neanderthals and Denisovans in genes related to sperm maturation and immunity. Finally, we found splicing variants that may contribute to variation among modern humans in total bilirubin, balding, hemoglobin levels, and lung capacity. Our findings provide unique insights into natural selection acting on splicing in human evolution and demonstrate how functional assays can be used to identify candidate causal variants underlying differences in gene regulation and phenotype.

Combining full-length gene assay and SpliceAI to interpret the splicing impact of all possible SPINK1 coding variants.

BACKGROUND: Single-nucleotide variants (SNVs) within gene coding sequences can significantly impact pre-mRNA splicing, bearing profound implications for pathogenic mechanisms and precision medicine. In this study, we aim to harness the well-established full-length gene splicing assay (FLGSA) in conjunction with SpliceAI to prospectively interpret the splicing effects of all potential coding SNVs within the four-exon SPINK1 gene, a gene associated with chronic pancreatitis. RESULTS: Our study began with a retrospective analysis of 27 SPINK1 coding SNVs previously assessed using FLGSA, proceeded with a prospective analysis of 35 new FLGSA-tested SPINK1 coding SNVs, followed by data extrapolation, and ended with further validation. In total, we analyzed 67 SPINK1 coding SNVs, which account for 9.3% of the 720 possible coding SNVs. Among these 67 FLGSA-analyzed SNVs, 12 were found to impact splicing. Through detailed comparison of FLGSA results and SpliceAI predictions, we inferred that the remaining 653 untested coding SNVs in the SPINK1 gene are unlikely to significantly affect splicing. Of the 12 splice-altering events, nine produced both normally spliced and aberrantly spliced transcripts, while the remaining three only generated aberrantly spliced transcripts. These splice-impacting SNVs were found solely in exons 1 and 2, notably at the first and/or last coding nucleotides of these exons. Among the 12 splice-altering events, 11 were missense variants (2.17% of 506 potential missense variants), and one was synonymous (0.61% of 164 potential synonymous variants). Notably, adjusting the SpliceAI cut-off to 0.30 instead of the conventional 0.20 would improve specificity without reducing sensitivity. CONCLUSIONS: By integrating FLGSA with SpliceAI, we have determined that less than 2% (1.67%) of all possible coding SNVs in SPINK1 significantly influence splicing outcomes. Our findings emphasize the critical importance of conducting splicing analysis within the broader genomic sequence context of the study gene and highlight the inherent uncertainties associated with intermediate SpliceAI scores (0.20 to 0.80). This study contributes to the field by being the first to prospectively interpret all potential coding SNVs in a disease-associated gene with a high degree of accuracy, representing a meaningful attempt at shifting from retrospective to prospective variant analysis in the era of exome and genome sequencing.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

A novel homozygous splice-site mutation of JK gene leads to Jk(a-b-) phenotype.

OBJECTIVES: This study aimed to investigate the molecular mechanism of the Jk(a-b-) phenotype in a Chinese transfusion patient. BACKGROUND: Many different mutation types relating to Jk(a-b-) phenotype have been reported. However, the splice-site mutation is relatively rare and the related functional verification is lacking. MATERIALS AND METHODS: In this study, the blood sample was collected from a transfusion patient with the Jk(a-b-) phenotype. Serotyping was performed using routine serological methods. The exons sequences and coding regions of the JK gene were amplified using polymerase chain reaction and directly sequenced. To perform a minigene splicing assay, the intronic mutation sequences were cloned into a pSPL3 splice reporting vector. The splicing reporter minigene assay was performed in HEK 293T cells. RESULTS: The Jk(a-b-) phenotype of the blood sample was identified through serological testing. Sequencing results revealed that the sample had a novel homozygous splice-site mutation JK*02N (NM_015865.7: c.663+3A>C). Further analysis, including cDNA sequencing and minigene splicing assay, confirmed that the novel splice-site mutation resulted in exon skipping. Interestingly, different numbers of exons being skipped were obtained by the two methods. CONCLUSION: This study revealed a novel homozygous splicing-site mutation associated with the Jk(a-b-) phenotype in Chinese population. Our results emphasise the importance of the in vitro functional method minigene splicing assay, while also acknowledging its potential limitations when compared to cDNA sequencing.

Minigene-based splicing analysis and ACMG/AMP-based tentative classification of 56 ATM variants.

The ataxia telangiectasia-mutated (ATM) protein is a major coordinator of the DNA damage response pathway. ATM loss-of-function variants are associated with 2-fold increased breast cancer risk. We aimed at identifying and classifying spliceogenic ATM variants detected in subjects of the large-scale sequencing project BRIDGES. A total of 381 variants at the intron-exon boundaries were identified, 128 of which were predicted to be spliceogenic. After further filtering, we ended up selecting 56 variants for splicing analysis. Four functional minigenes (mgATM) spanning exons 4-9, 11-17, 25-29, and 49-52 were constructed in the splicing plasmid pSAD. Selected variants were genetically engineered into the four constructs and assayed in MCF-7/HeLa cells. Forty-eight variants (85.7%) impaired splicing, 32 of which did not show any trace of the full-length (FL) transcript. A total of 43 transcripts were identified where the most prevalent event was exon/multi-exon skipping. Twenty-seven transcripts were predicted to truncate the ATM protein. A tentative ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme that integrates mgATM data allowed us to classify 29 ATM variants as pathogenic/likely pathogenic and seven variants as likely benign. Interestingly, the likely pathogenic variant c.1898+2T>G generated 13% of the minigene FL-transcript due to the use of a noncanonical GG-5'-splice-site (0.014% of human donor sites). Circumstantial evidence in three ATM variants (leakiness uncovered by our mgATM analysis together with clinical data) provides some support for a dosage-sensitive expression model in which variants producing ≥30% of FL-transcripts would be predicted benign, while variants producing ≤13% of FL-transcripts might be pathogenic. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

De novo variations of ANK1 gene caused hereditary spherocytosis in two Chinese children by affecting pre-mRNA splicing.

BACKGROUND AND AIMS: Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic disorders. Here, two unrelated families with the probands displaying typical manifestations of HS were enrolled. Our study aimed to characterize the effect of two novel variants in HS patients on gene splicing to help minimize the rate of misdiagnosis of HS and enhance clinicians' understanding of the disease. PARTICIPANTS AND METHODS: A retrospective review was conducted. Peripheral blood samples were collected from all the family members, and genomic DNA was extracted for genetic diagnostics. First, high-throughput sequencing technology was used for the preliminary screening of candidate causative variants. Thereafter, the variants were verified via Sanger sequencing. Furthermore, a pathogenicity analysis of the detected variants was performed including in silico prediction and in vitro experiments. We constructed matched wild-type and mutant-type minigene plasmid of ANK1 based on HEK293T cells to address the effects of variants on mRNA splicing. RESULTS: The c.1305 + 2 T > A (family1) and c.1305 + 2del (family2) variants were detected in the ANK1 gene. These two de novo mutations described by us which have not been reported prior to this study. Moreover, the validation results of splicing reporter systems revealed that the intronic mutations resulted in abnormal pre-mRNA splicing. Specifically, the minigene plasmid expressing the c.1305 + 2 T > A variant transcribed the two aberrant transcripts: r.1305_1306ins1305 + 1_1305 + 229 and r.1305_1306ins1305 + 1_1305 + 552. The minigene plasmid expressing c.1305 + 2del transcribed the two aberrant transcripts: r.1305_1306ins1305 + 1_1305 + 228 and r.1305_1306ins1305 + 1_1305 + 551. CONCLUSION: The two de novo variants identified in the ANK1 gene were the genetic etiology of the probands with HS in our study. Our findings further enrich the HS genotype database and provide a basis for genetic counselling and molecular diagnosis.

Identification of a novel TBX5 c.755 + 1 G > A variant and related pathogenesis in a family with Holt-Oram syndrome.

The proband with congenital heart disease and abnormal thumb was clinically diagnosed as Holt-Oram syndrome (HOS). A novel variant, T-box transcription factor 5 (TBX5) c.755 + 1 G > A, was identified in the proband via whole exome sequencing and validated using Sanger sequencing. Pedigree analysis and clinical examinations revealed three/seven individuals over three generations within the family, with features suggestive of HOS. Deep amplicon sequencing confirmed that the allele frequencies of the novel variant in the proband (III-1), her brother (III-2), and her mother (II-2) were 50%, 48.3%, and 38.1%, respectively, indicating that III-1 and III-2 harbored heterozygous variants, while II-2 harbored mosaic heterozygous variants. The minigene splicing assay showed that the novel variant affected the normal splicing of exon 7, resulting in the production of abnormal TBX5 transcripts. Reverse transcription-quantitative polymerase chain reaction and western blot analyses revealed that the novel variant upregulated TBX5 expression at the transcriptional and translational levels. Nuclear localization assay demonstrated impaired nuclear localization of the mutant TBX5. Cell viability assay revealed the inhibition of cell activity by the mutant TBX5. Our findings indicate that the novel variant was potentially induced HOS, probably by causing aberrant splicing, reducing the enrichment of nuclear TBX5 protein, and inhibiting cellular proliferation.

Novel compound heterozygous synonymous and missense variants in the MYO7A gene identified by next-generation sequencing in a Chinese family with nonsyndromic hearing loss.

BACKGROUND: Variants in the MYO7A gene are increasingly identified among patients suffering from Usher syndrome type 1B (USH1B). However, such mutations are less commonly detected among patients suffering from nonsyndromic hearing loss (NSHL), including autosomal recessive deafness (DFNB2) and autosomal dominant deafness (DFNA11). This research attempts to clarify the genetic base of DFNB2 in a Chinese family and determine the pathogenicity of the identified mutations. METHOD: Targeted next-generation sequencing (TGS) of 127 known deafness genes was performed for the 14-year-old proband. Then, Sanger sequencing was performed on the available family members. A minigene splicing assay was performed to verify the impact of the novel MYO7A synonymous variant. After performing targeted next-generation sequencing (TGS) of 127 existing hearing loss-related genes in a 14-year-old proband, Sanger sequencing was carried out on the available family members. Then, to confirm the influence of the novel MYO7A synonymous variants, a minigene splicing assay was performed. RESULTS: Two heteroallelic mutants of MYO7A (NM_000260.3) were identified: a maternally inherited synonymous variant c.2904G > A (p.Glu968=) in exon 23 and a paternally inherited missense variant c.5994G > T (p.Trp1998Cys) in exon 44. The in vitro minigene expression indicated that c.2904G > A may result in skipping of exon 23 resulting in a truncated protein. CONCLUSIONS: We reported a novel missense (c.5994G > T) and identified, for the first time, a novel pathogenic synonymous (c.2904G > A) variant within MYO7A in a patient with DFNB2. These findings enrich our understanding of the MYO7A variant spectrum of DFNB2 and can contribute to accurate genetic counseling and diagnosis of NSHL patients.

5' splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity.

In the human genome, most 5' splice sites (~99%) employ the canonical GT dinucleotide whereas a small minority (~1%) use the noncanonical GC dinucleotide. The functionality and pathogenicity of 5' splice site GT>GC (+2T>C) variants have been extensively studied but we know very little about 5' splice site GC>GT (+2C>T) variants. Herein, we have addressed this deficiency by performing a meta-analysis of reported +2C>T "pathogenic" variants together with a functional analysis of engineered +2C>T substitutions using a cell culture-based full-length gene splicing assay. Our results establish proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and suggest that, in sharp contrast to +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants and understanding the evolutionary switching between GT and GC 5' splice sites in mammalian genomes.

The Experimentally Obtained Functional Impact Assessments of 5' Splice Site GT'GC Variants Differ Markedly from Those Predicted.

INTRODUCTION: 5' splice site GT>GC or +2T>C variants have been frequently reported to cause human genetic disease and are routinely scored as pathogenic splicing mutations. However, we have recently demonstrated that such variants in human disease genes may not invariably be pathogenic. Moreover, we found that no splicing prediction tools appear to be capable of reliably distinguishing those +2T>C variants that generate wild-type transcripts from those that do not. METHODOLOGY: Herein, we evaluated the performance of a novel deep learning-based tool, SpliceAI, in the context of three datasets of +2T>C variants, all of which had been characterized functionally in terms of their impact on pre-mRNA splicing. The first two datasets refer to our recently described "in vivo" dataset of 45 known disease-causing +2T>C variants and the "in vitro" dataset of 103 +2T>C substitutions subjected to full-length gene splicing assay. The third dataset comprised 12 BRCA1 +2T>C variants that were recently analyzed by saturation genome editing. RESULTS: Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of these variants (and smaller datasets of +2T>A and +2T>G variants) revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue was that the impact of those +2T>C (and +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to an almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. CONCLUSION: Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

First estimate of the scale of canonical 5' splice site GT>GC variants capable of generating wild-type transcripts.

It has long been known that canonical 5' splice site (5'SS) GT>GC variants may be compatible with normal splicing. However, to date, the actual scale of canonical 5'SSs capable of generating wild-type transcripts in the case of GT>GC substitutions remains unknown. Herein, combining data derived from a meta-analysis of 45 human disease-causing 5'SS GT>GC variants and a cell culture-based full-length gene splicing assay of 103 5'SS GT>GC substitutions, we estimate that ~15-18% of canonical GT 5'SSs retain their capacity to generate between 1% and 84% normal transcripts when GT is substituted by GC. We further demonstrate that the canonical 5'SSs in which substitution of GT by GC-generated normal transcripts exhibit stronger complementarity to the 5' end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were capable of reliably distinguishing 5'SS GT>GC variants that generated wild-type transcripts from those that did not. Our findings imply that 5'SS GT>GC variants in human disease genes may not invariably be pathogenic.

An in vitro splicing assay reveals the pathogenicity of a novel intronic variant in ATP6V0A4 for autosomal recessive distal renal tubular acidosis.

BACKGROUND: Autosomal recessive distal renal tubular acidosis (dRTA) is a rare hereditary disease caused by pathogenic variants in the ATP6V0A4 gene or ATP6V1B1 gene, and characterized by hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia and nephrocalcinosis. Although several intronic nucleotide variants in these genes have been detected, all of them fell in the apparent splice consensus sequence. In general, transcriptional analysis is necessary to determine the effect on function of the novel intronic variants located out of splicing consensus sequences. In recent years, functional splicing analysis using minigene construction was used to assess the pathogenicity of novel intoronic variant in various field. METHODS: We investigated a sporadic case of dRTA with a compound heterozygous mutation in the ATP6V0A4 gene, revealed by next generation sequencing. One variant was already reported as pathogenic; however, the other was a novel variant in intron 11 (c.1029 + 5G > A) falling outside of the apparent splicing consensus sequence. Expression of ATP6V0A4 was not detected in peripheral leukocytes by RT-PCR analysis. Therefore, an in vitro functional splicing study using minigene construction was conducted to analyze the splicing pattern of the novel variant. RESULTS: A minigene assay revealed that the novel intronic variant leads to a 104 bp insertion immediately following exon 11. In addition, this result was confirmed using RNA extracted from the patient's cultured leukocytes. CONCLUSION: These results proved the pathogenicity of a novel intronic variant in our patient. We concluded that the minigene assay is a useful, non-invasive method for functional splicing analysis of inherited kidney disease, even if standard transcriptional analysis could not detect abnormal mRNA.

Identification of a novel ANK1 gene variant c.1504-9G>A and its mechanism of intron retention in hereditary spherocytosis.

Objective: The objective of this study was to pinpoint pathogenic genes and assess the mutagenic pathogenicity in two pediatric patients with hereditary spherocytosis. Methods: We utilized whole-exome sequencing (WES) for individual analysis (case 1) and family-based trio analysis (case 2). The significance of the intronic mutation was validated through a Minigene splicing assay and supported by subsequent in vitro experiments. Results: Both probands received a diagnosis of hereditary spherocytosis. WES identified a novel ANK1 c.1504-9G>A mutation in both patients, causing the retention of seven nucleotides at the 5' end of intron 13, as substantiated by the Minigene assay. This variant results in a premature stop codon and the production of a truncated protein. In vitro studies indicated a reduced expression of the ANK1 gene. Conclusion: The novel ANK1 c.1504-9G>A variant is established as the causative factor for hereditary spherocytosis, with the c.1504-9G site functioning as a splicing receptor.

Pathogenic relationship between phenotypes of ARPKD and novel compound heterozygous mutations of PKHD1.

Background: To investigate whether the novel mutation of PKHD1 could cause polycystic kidney disease by affecting splicing with a recessive inheritance pattern. Methods: A nonconsanguineous Chinese couple with two recurrent pregnancies showed fetal enlarged echogenic polycystic kidney and oligoamnios were recruited. Pedigree WES, minigene splicing assay experiment and following bioinformatics analysis were performed to verify the effects, and inheritance pattern of diseasing-causing mutations. Results: WES revealed that both fetuses were identified as carrying the same novel mutation c.3592_3628 + 45del, p.? and c.11207 T>C, p.(Ile3736Thr) in the PKHD1 gene (NM_138694.4), which inherited from the father and mother respectively. Both bioinformatic method prediction and minigene splicing assay experience results supported the mutation c.3592_3628 + 45del, p.? affects the splicing of the PKHD1 transcript, resulting in exon 31 skipping. Another missense mutation c.11207 T>C, p.(Ile3736Thr) has a low frequency in populations and is predicted to be deleterious by bioinformatic methods. Conclusion: These findings provide a direct clinical and functional evidence that the truncating mutations of the PKHD1 gene could lead to more severe phenotypes, and cause ARPKD as a homozygous or compound heterozygous pattern. Our study broadens the variant spectrum of the PKHD1 gene and provides a basis for genetic counseling and diagnosis of ARPKD.

Novel genotypes and phenotypes in Snijders Blok-Campeau syndrome caused by CHD3 mutations.

Background: Snijders Blok-Campeau syndrome (SNIBCPS) is a rare genetic disorder characterized by facial abnormalities, hypotonia, macrocephaly, and global developmental delay (GDD) caused by mutations in CHD3 gene. There is limited information on SNIBCPS and few studies on its pathogenic gene CHD3. Methods: We utilized whole-exome sequencing, in vitro minigene splicing assay analysis, and construction of protein models to validate the suspected pathogenic mutation. In addition, the PubMed database was searched using the keywords "Snijders Blok-Campeau syndrome," "CHD3," or "SNIBCPS" to summarize the gene mutations and clinical phenotypic characteristics of children with SNIBCPS. Results: We identified a non-frameshift variant c.3592_c.3606delGCCAAGAGAAAGATG, a splice site variant c.1708-1G>T, and two missense variants, c. 2954G>C (p.Arg985Pro) and c.3371C>T (p.A1124V), in CHD3 variants with SNIBCPS. Importantly, the c.3592_c.3606delGCCAAGAGAAAGATG, c.1708-1G>T, and c.3371C > T (p.A1124V) loci were not reported, and the children in this study also had phenotypic features of unibrow, transverse palmar creases, tracheal bronchus, and hypomelanosis of Ito (HI). The c.1708-1G>T classical splicing mutation leads to abnormal shearing of mRNA, forming a truncated protein that ultimately affects gene function. Conclusion: Our findings have expanded the spectrum of genetic variants and clinical features in children with SNIBCPS. Splicing analysis of CHD3 is an important method to understand the pathogenesis of spliced cells.

A novel variation in DEPDC5 causing familial focal epilepsy with variable foci.

Background: Disheveled, EGL-10, and pleckstrin (DEP) domain-containing protein 5 (DEPDC5) is a component of GTPase-activating protein (GAP) activity toward the RAG complex 1 (GATOR1) protein, which is an inhibitor of the amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. GATOR1 complex variations were reported to correlate with familial focal epilepsy with variable foci (FFEVF). With the wide application of whole exome sequencing (WES), more and more variations in DEPDC5 were uncovered in FFEVF families. Methods: A family with a proband diagnosed with familial focal epilepsy with variable foci (FFEVF) was involved in this study. Whole exome sequencing (WES) was performed in the proband, and Sanger sequencing was used to confirm the variation carrying status of the family members. Mini-gene splicing assay was performed to validate the effect on the alternative splicing of the variation. Results: A novel variant, c.1217 + 2T>A, in DEPDC5 was identified by WES in the proband. This splicing variant that occurred at the 5' end of intron 17 was confirmed by mini-gene splicing assays, which impacted alternative splicing and led to the inclusion of an intron fragment. The analysis of the transcribed mRNA sequence indicates that the translation of the protein is terminated prematurely, which is very likely to result in the loss of function of the protein and lead to the occurrence of FFEVF. Conclusion: The results suggest that c.1217 + 2T>A variations in DEPDC5 might be the genetic etiology for FFEVF in this pedigree. This finding expands the genotype spectrum of FFEVF and provides new etiological information for FFEVF.

Reciprocal abrogation of PKM isoforms: contradictory outcomes and differing impact of splicing signal on CRISPR/Cas9 mediates gene editing in keratinocytes.

The healing of wounded skin is a highly organized process involving a massive cell in- and outflux, proliferation and tissue remodelling. It is well accepted that metabolic constraints such as diabetes mellitus, overweight or anorexia impairs wound healing. Indeed, wound inflammation involves a boost of overall metabolic changes. As wound healing converges inflammatory processes that are also common to transformation, we investigate the functional role of the pro-neoplastic factor pyruvate kinase (PK) M2 and its metabolic active splice variant PKM1 in keratinocytes. Particularly, we challenge the impact of reciprocal ablation of PKM1 or two expression. Here, CRISPR/Cas9 genome editing of the PKM gene in HaCaT reveals an unexpected mutational bias at the 3'SS of exon 9, whereas no preference for any particular kind of mutation at exon 10 3' splice, despite the close vicinity (400 nucleotides apart) and sequence similarity between the two sites. Furthermore, as opposed to transient silencing of PKM2, exclusion splicing of PKM2 via genome editing mutually increases PKM1 mRNA and protein expression and compensates for the absence of PKM2, whereas the reciprocal elimination of PKM1 splicing reduces PKM2 expression and impedes cell proliferation, thus unveiling an essential role for PKM1 in growth and metabolic balance of HaCaT keratinocytes.

High-Throughput Splicing Assays Identify Known and Novel WT1 Exon 9 Variants in Nephrotic Syndrome.

Introduction: Frasier syndrome (FS) is a rare Mendelian form of nephrotic syndrome (NS) caused by variants which disrupt the proper splicing of WT1. This key transcription factor gene is alternatively spliced at exon 9 to produce 2 isoforms ("KTS+" and "KTS-"), which are normally expressed in the kidney at a ∼2:1 (KTS+:KTS-) ratio. FS results from variants that reduce this ratio by disrupting the splice donor of the KTS+ isoform. FS is extremely rare, and it is unclear whether any variants beyond the 8 already known could cause FS. Methods: To prospectively identify other splicing-disruptive variants, we leveraged a massively parallel splicing assay. We tested every possible single nucleotide variant (n = 519) in and around WT1 exon 9 for effects upon exon inclusion and KTS+/- ratio. Results: Splice disruptive variants (SDVs) made up 11% of the tested point variants overall and were tightly concentrated near the canonical acceptor and the KTS+/- alternate donors. Our map successfully identified all 8 known FS or focal segmental glomerulosclerosis (FSGS) variants and 16 additional novel variants which were comparably disruptive to these known pathogenic variants. We also identified 19 variants that, conversely, increased the KTS+/KTS- ratio, of which 2 are observed in unrelated individuals with 46,XX ovotesticular disorder of sex development (46,XX OTDSD). Conclusion: This splicing effect map can serve as functional evidence to guide the clinical interpretation of newly observed variants in and around WT1 exon 9.

Intron retention by a novel intronic mutation in DKC1 gene caused recurrent still birth and early death in a Chinese family.

BACKGROUND: DKC1, the dyskerin encoding gene, functions in telomerase activity and telomere maintenance. DKC1 mutations cause a multisystem disease, dyskeratosis congenita (DC), which is associated with immunodeficiency and bone marrow failure. METHODS: In this research, we reported a novel intronic mutation of DKC1 causing dyskerin functional loss in a Chinese family. Whole exome sequence (WES) of the proband and validation by sanger sequencing help us identify a pathogenic DKC1 mutation. Minigene splicing assays were performed to evaluate functional change of DKC1. RESULTS: A pathogenic DKC1 intronic mutation(c.84 + 7A > G) was identified in the proband, which was inherited from heterozygous mother and not reported before. We detected the novel transcript with a 7 bp intron retention through minigene splicing assay. The newly spliced transcript is so short that would be degraded by nonsense-mediated mRNA decay in vitro and we infer that the novel DKC1 mutation would influences normal physiological function of dyskerin. CONCLUSIONS: Our study identified a novel intronic mutation, which expands the spectrum of pathogenic DKC1 gene mutations and can be used in molecular diagnosis. The mutant allele was transmitted to the next generation with high frequency in the family and causes still birth or early death.

A synonymous mutation in PI4KA impacts the transcription and translation process of gene expression.

Phosphatidylinositol-4-kinase alpha (PI4KIIIα), encoded by the PI4KA gene, can synthesize phosphatidylinositol-4-phosphate (PI-4-P), which serves as a specific membrane marker and is instrumental in signal transduction. PI4KA mutations can cause autosomal recessive diseases involving neurological, intestinal, and immunological conditions (OMIM:619621, 616531, 619708). We detected sepsis, severe diarrhea, and decreased immunoglobulin levels in one neonate. Two novel compound heterozygous mutations, c.5846T>C (p.Leu1949Pro) and c.3453C>T (p.Gly1151=), were identified in the neonate from the father and the mother, respectively. Sanger sequencing and reverse transcription polymerase chain reaction (RT-PCR) for peripheral blood and minigene splicing assays showed a deletion of five bases (GTGAG) with the c.3453C>T variant at the mRNA level, which could result in a truncated protein (p.Gly1151GlyfsTer17). The missense mutation c.5846T>C (p.Leu1949Pro) kinase activity was measured, and little or no catalytic activity was detected. According to the clinical characteristics and gene mutations with functional verification, our pediatricians diagnosed the child with a combined immunodeficiency and intestinal disorder close to gastrointestinal defects and immunodeficiency syndrome 2 (GIDID2; OMIM: 619708). Medicines such as immunomodulators are prescribed to balance immune dysregulation. This study is the first report of a synonymous mutation in the PI4KA gene that influences alternative splicing. Our findings expand the mutation spectrum leading to PI4KIIIa deficiency-related diseases and provide exact information for genetic counseling.