A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.

An infant case of pseudohypoaldosteronism type1A caused by a novel NR3C2 variant

Pseudohypoaldosteronism type1A (PHA1A) is the renal form of pseudohypoaldosteronism with autosomal dominant inheritance. PHA1A is caused by haploinsufficiency of the mineralocorticoid receptor, which is encoded by NR3C2. We encountered an infant who was diagnosed with PHA1A due to hyponatremia, hyperkalemia, and poor weight gain in the neonatal period. She carried a novel heterozygous mutation (NM_000901.5: c.1757 + 1 G > C) in the splice donor site of IVS-2 in NR3C2.

Turnover of sex defining mutation provides an insight into evolution of sex chromosomes in the golden pompano (Trachinotus ovatus)

The golden pompano (Trachinotus ovatus) is a marine fish species in the family Carangidae. We constructed a chromosome-level genome assembly of a male golden pompano. QTL-mapping and GWAS analysis showed that this species has a ZZ/ZW sex determination system and a sex defining SNP (Chr16:18219150:G/A), located on the splice donor site (GT-AG) of the first intron of Hsd17b1, was exclusively associated with the phenotypic sex. The W-linked coding sequences of Hsd17b1 were conserved across vertebrates, while Z-linked coding sequences introduced extra 64 bases and were malfunctional. The golden pompano and the greater amberjack (Seriola dumerili), divergent in 57 million years ago in the same family, share the same features of sex determination, including the same sex determining gene, malfunctional Z-linked haplotypes, undifferentiated sex chromosomes except that the sex defining SNPs are different. In simulation analysis, turnover of sex determining mutation, single mutation dominating sex determination and undifferentiated sex chromosomes were also observed. We proposed a hypothesis that W-linked haplotypes of the sex determining gene of Hsd17b1 were under purifying selection, Z-linked haplotypes may evolve near neutrally, recurrent and directional transformations from W-linked haplotypes to Z-linked haplotypes caused by inactivating mutations, relatively strong forces of drift and recombination comprehensively contributed to turnover of sex defining mutation and undifferentiation of sex chromosome. We also established zebrafish mutants and homozygous mutants were “all male”, which indirectly supported this hypothesis.

Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

A novel synonymous ABCA3 variant identified in a Chinese family with lethal neonatal respiratory failure

Background Lethal respiratory failure is primarily caused by a deficiency of pulmonary surfactant, and is the main cause of neonatal death among preterm infants. Pulmonary surfactant metabolism dysfunction caused by variants in the ABCA3 gene is a rare disease with very poor prognosis. Currently, the mechanisms associated with some ABCA3 variants have been determined, including protein mistrafficking and impaired phospholipid transport. However, some novel variants and their underlying pathogenesis has not been fully elucidated yet. In this study we aimed to identify the genetic features in a family with lethal respiratory failure. Methods We studied members of two generations of a Chinese family, including a female proband, her parents, her monozygotic twin sister, and her older sister. Trio whole exome sequencing (WES) were used on the proband and her parents to identify the ABCA3 variants. Sanger sequencing and real-time quantitative polymerase chain reaction (PCR) were used on the monozygotic twin sister of proband to validate the ABCA3 synonymous variant and exon deletion, respectively. The potential pathogenicity of the identified synonymous variant was predicted using the splice site algorithms dbscSNV11_AdaBoost, dbscSNV11_RandomForest, and Human Splicing Finder (HSF). Results All patients showed severe respiratory distress, which could not be relieved by mechanical ventilation, supplementation of surfactant, or steroid therapy, and died at an early age. WES analysis revealed that the proband had compound heterozygous ABCA3 variants, including a novel synonymous variant c.G873A (p.Lys291Lys) in exon 8 inherited from the mother, and a heterozygous deletion of exons 4–7 inherited from the father. The synonymous variant was consistently predicted to be a cryptic splice donor site that may lead to aberrant splicing of the pre-mRNA by three different splice site algorithms. The deletion of exons 4–7 of the ABCA3 gene was determined to be a likely pathogenic variant. The variants were confirmed in the monozygotic twin sister of proband by Sanger sequencing and qPCR respectively. The older sister of proband was not available to determine if she also carried both ABCA3 variants, but it is highly likely based on her clinical course. Conclusions We identified a novel synonymous variant and a deletion in the ABCA3 gene that may be responsible for the pathogenesis in patients in this family. These results add to the known mutational spectrum of the ABCA3 gene. The study of ABCA3 variants may be helpful for the implementation of patient-specific therapies.

Mutation analysis of the SLC26A4 gene in patients in Yakutia with inner ear abnormalities: IP-I, IP-II (Mondini) and/or EVA

Мутации гена SLC26A4 могут приводить как к формированию аутосомно-рецессивной тугоухости 4 типа (DFNB4, OMIM #600791), так и к синдрому Пендреда (PDS, OMIM #274600), при котором нейросенсорная потеря слуха сочетается с дисфункцией щитовидной железы, клинически проявляющейся во второй декаде жизни. Обе формы могут сопровождаться специфическими аномалиями внутреннего уха: IP-I, IP-II (Mondini) и/или EVA. В Якутии аудиологическими, рентгенологическими и молекулярно-генетическими методами обследовано 165 пациентов с врожденным нарушением слуха. При компьютерной томографии пирамиды височных костей у 9 из 165 (5,5%) пациентов были обнаружены аномалии IP-I, IP-II (Mondini) и/или EVA. Методом прямого секвенирования по Сэнгеру у этих 9 пациентов было проведено определение нуклеотидной последовательности гена SLC26A4 (21 экзон). В гене SLC26A4 обнаружено 5 ранее известных вариантов, среди которых 4 варианта относились к миссенс-заменам: c.85G>C p.(Glu29Gln), c.441G>A p.(Met147Ile), c.757A>G p.(Ile253Val), c.2027T>A p.(Leu676Gln) и один вариант затрагивал донорный сайт сплайсинга - c.2089+1G>A (IVS18+1G>A). У 4-х из 9 пациентов патогенные варианты гена SLC26A4 обнаружены в гомозиготном или компаунд-гетерозиготном состоянии. Доля биаллельных мутаций гена SLС26A4 у пациентов с IP-I, IP-II (Mondini) и/или EVA составила 44,4%. Пациенты с биаллельными мутациями гена SLC26A4 имели тяжелые врожденные нарушения слуха (двусторонняя нейросенсорная тугоухость от III степени до глухоты), при этом доминирующим типом аномалий были IP-II (Mondini)+EVA (62,5%), аномалии IP-I не были выявлены ни у одного пациента. По совокупности полученных клинических и молекулярно-генетических данных у трех пациентов форма заболевания классифицирована как аутосомно-рецессивная тугоухость 4 типа (DFNB4), а у одной пациентки с двусторонней аномалией EVA, нейросенсорной тугоухостью III степени и узловым зобом (оперирован) подтвержден синдром Пендреда. Mutations in the SLC26A4 gene can lead to both the formation of autosomal recessive deafness type 4 (DFNB4, OMIM#600791), and to Pendred’s syndrome (PDS, OMIM#274600), in which sensorineural hearing loss is combined with thyroid dysfunction, with both forms can be accompanied by specific anomalies of the inner ear: IP-I, IP-II (Mondini) and/or EVA. Using audiological, radiological and molecular genetics methods, 165 patients with congenital hearing impairment in Yakutia were examined. Computed tomography revealed IP-I, IP-II (Mondini) and/or EVA abnormalities in 9 of 165 (5,5%) patients. Then, using direct Sanger sequencing in these 9 patients, the nucleotide sequence of the coding regions of the SLC26A4 gene (21 exons) was determined. In total, 5 previously known variants were found in the SLC26A4 gene, among which 4 variants were missense substitutions: c.85G>C p.(Glu29Gln), c.441G>A p.(Met147Ile), c.757A>G p.(Ile253Val), c.2027T>A p.(Leu676Gln) and one variant affected the splice donor site - c.2089+1G>A (IVS18+1G>A). In 4 out of 9 patients, pathogenic variants of the SLC26A4 gene were found in a homozygous or compound heterozygous state. The total contribution of biallelic mutations in the SLC26A4 gene among patients with inner ear anomalies was 44,4%. Patients with biallelic SLC26A4-mutations had several to profound bilateral sensorineural hearing loss. In patients with biallelic SLC26A4-mutations, the dominant type of anomaly was IP-II (Mondini)+EVA (62,5%), IP-I anomalies were not detected in any patient. In three patients we were able to confirm the diagnosis of DFNB4, and in one patient, due to the sum of phenotypic features (operated on for nodular goiter, autosomal recessive deafness with EVA), Pendred’s syndrome was diagnosed.

PRKG2 Splice Site Variant in Dogo Argentino Dogs with Disproportionate Dwarfism

Dwarfism phenotypes occur in many species and may be caused by genetic or environmental factors. In this study, we investigated a family of nine Dogo Argentino dogs, in which two dogs were affected by disproportionate dwarfism. Radiographs of an affected dog revealed a decreased level of endochondral ossification in its growth plates, and a premature closure of the distal ulnar physes. The pedigree of the dogs presented evidence of monogenic autosomal recessive inheritance; combined linkage and homozygosity mapping assigned the most likely position of a potential genetic defect to 34 genome segments, totaling 125 Mb. The genome of an affected dog was sequenced and compared to 795 control genomes. The prioritization of private variants revealed a clear top candidate variant for the observed dwarfism. This variant, PRKG2:XM_022413533.1:c.1634 + 1G>T, affects the splice donor site and is therefore predicted to disrupt the function of the PKRG2 gene encoding protein, kinase cGMP-dependent type 2, a known regulator of chondrocyte differentiation. The genotypes of the PRKG2 variant were perfectly associated with the phenotype in the studied family of dogs. PRKG2 loss-of-function variants were previously reported to cause disproportionate dwarfism in humans, cattle, mice, and rats. Together with the comparative data from other species, our data strongly suggest PRKG2:c.1634+1G>T to be a candidate causative variant for the observed dwarfism phenotype in Dogo Argentino dogs.

Contribution of Noncanonical Splice Variants to TTN Truncating Variants Cardiomyopathy

Background: Heterozygous TTN truncating variants cause 10% to 20% of idiopathic dilated cardiomyopathy (DCM). Although variants which disrupt canonical splice signals (ie, invariant dinucleotide of splice donor site, invariant dinucleotide of the splice acceptor site) at exon-intron junctions are readily recognized as TTN truncating variants, the effects of other nearby sequence variations on splicing and their contribution to disease is uncertain. Methods: Rare variants of unknown significance located in the splice regions of highly expressed TTN exons from 203 DCM cases, 3329 normal subjects, and clinical variant databases were identified. The effects of these variants on splicing were assessed using an in vitro splice assay. Results: Splice-altering variants of unknown significance were enriched in DCM cases over controls and present in 2% of DCM patients ( P =0.002). Application of this method to clinical variant databases demonstrated 20% of similar variants of unknown significance in TTN splice regions affect splicing. Noncanonical splice-altering variants were most frequently located at position +5 of the donor site ( P =4.4×10 7 ) and position -3 of the acceptor site ( P =0.002). SpliceAI, an emerging in silico prediction tool, had a high positive predictive value (86%–95%) but poor sensitivity (15%–50%) for the detection of splice-altering variants. Alternate exons spliced out of most TTN transcripts frequently lacked the consensus base at +5 donor and −3 acceptor positions. Conclusions: Noncanonical splice-altering variants in TTN explain 1-2% of DCM and offer a 10-20% increase in the diagnostic power of TTN sequencing in this disease. These data suggest rules that may improve efforts to detect splice-altering variants in other genes and may explain the low percent splicing observed for many alternate TTN exons.

Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Case Report: A Novel Mutation in NFKB1 Associated With Pyoderma Gangrenosum

Pyoderma gangrenosum (PG) is a rare, destructive inflammatory skin disease of which a painful nodule or pustule breaks down to form a progressively enlarging ulcer. Ulcerations associated with PG may occur after trauma or injury to the skin. The etiology has not been clearly elucidated. Our report described a PG patient with a heterozygous splice-donor-site mutation in NFKB1 (c.730+5G>A) causing the absence of exon 8 and the formation of truncated p105 (p.Asp191_Lys244delinsGlu; p105delEx8), which led to distinct symptoms of high fever and excessive inflammation in wound area after routine surgical procedures. The functional analysis showed that the variant caused reduced phosphorylation of p105 and resulted in the decreased processing of p105 to p50. We conclude that the patient's symptoms were caused by dysregulation of the NF-κB signaling pathway.