End Of Intron Research Articles

Rare occurrence of cryptic 5' splice sites by downstream 3' splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana.

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta.

A novel COL4A5 splicing mutation causes alport syndrome in a Chinese family

BackgroundAlport 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.MethodsWe 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.ResultsThe 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.ConclusionsBased 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.

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.

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

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. 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. 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. 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.

Distinct Pattern of ABL1 Genomic Breakpoints in Chronic Myeloid Leukemia and BCR::ABL1-Positive Acute Lymphoblastic Leukemia: Analysis of 884 Patients with Minor and Major BCR::ABL1 Fusion

The BCR::ABL1 fusion gene is a hallmark of chronic myeloid leukemia (CML) but is also found in patients with acute lymphoblastic leukemia (ALL). There are two main breakpoint clusters in BCR - “minor” (between exons 1-2, resulting in p190 fusion protein, prevalent in ALL and scarce in CML) and “Major” (between exons 13-15, resulting in p210 protein). On the ABL1 side, the breakpoints are mostly localized between exons 1-3. Due to large intronic areas where the breakpoints occur, only a few papers have been published focusing on the structure of BCR::ABL1 fusions. Moreover, with a single exception, these studies included only the Major- BCR::ABL1 patients. Here, we focused on the analysis of BCR::ABL1 breakpoints involving CML and ALL, children and adults, and Major and minor forms of fusion. To our knowledge, we present data on the largest cohort of patients with BCR::ABL1 fusion identified at the DNA level described to date. Genomic breakpoints were found in 884 patients with BCR::ABL1+ ALL (n=463) and CML (n=421) by multiplex long distance PCR/Sanger sequencing or by NGS Custom Target Enrichment. The RSS database, MEME software and RepeatMasker were used to search RSS, specific motifs known for mediation of DNA breaks (59 motifs) and interspersed and other types of repeats within particular DNA areas. The uniformity test performed on breakpoint distribution within the minor (n=356) and Major (n=528) BCR areas revealed a non-random pattern (p=2.34e-21 and p=2.56e-10, respectively) with breakpoints accumulated in the second halves of the intron 1 (minor BCR) and intron 13 (Major BCR). Within the ABL1 area, breakpoint sites were distributed more randomly (p=9.28e-03); however, the breakpoint distribution within the ABL1 differed significantly between patients with CML and ALL (p=7.43e-05; see Figure) with higher accumulation of breakpoints near the 5‘ end of the ABL1 intron 1 in CML and near the 3‘ end of the intron 1 in ALL. The difference was not driven by the type of fusion (minor vs. Major BCR) as it was still visible even if only Major BCR::ABL1+ patients were analysed (414 CML and 108 ALL; p=2.28e-03). Comparison between various groups of patients (females vs. males; adults vs. children; various age groups, minor vs. Major BCR fusion [in ALL patients]), did not reveal a significantly distinct breakpoint distribution in any BCR or ABL1 area. Analysis of genomic breakpoints showed that fusions are mostly formed in loci with short homologies (49%; 1-71bp, median=2bp), by blunt end junctions (36%) or by a junction with insertion of random nucleotides (13%; 1-42bp, median=3bp) strongly suggesting NHEJ mechanism of double-strand break repair in vast majority of cases. In 313 patients we compared BCR::ABL1 and the reciprocal ABL1::BCR fusion. Vast majority of the BCR::ABL1 and ABL1::BCR breakpoints (81% and 79% on BCR and ABL1 area, respectively) were located within ±100bp window, showing almost precise reciprocal translocation, with negligible losses/gains of DNA resulting from the fusion. Analysis of the association of breakpoints with DNA motifs and chromatin structure did not reveal any significant association with the localization of breakpoints. In conclusion, we show different breakpoint distribution in ABL1 between CML and BCR::ABL1+ ALL. The double-strand breaks are repaired by NHEJ; however, we have not found any DNA or epigenetic motif, which might be responsible for the breakpoints. Our data suggest that the differences in breakpoint distribution are due to different chromatin accessibility and/or different relative positions of the BCR and ABL1 genes throughout the cell development, reflecting the origin of CML in earlier stages of hematopoiesis compared to ALL. Supported by AZV (NU21-03-00128) and NICR No. LX22NPO5102.

Barley SS2a single base mutation at the splicing site led to obvious change in starch

Starch biosynthesis is a complex process that relies on the coordinated action of multiple enzymes. Resistant starch is not digested in the small intestine, thus preventing the rapid rise of the glycemic index. Starch synthase 2a (SS2a), a key enzyme in amylopectin biosynthesis, has significant effects on starch structure and properties. In this study, we identified an ss2a null mutant (M3-1413) with a single base mutation from an ethyl methane sulfonate (EMS)-mutagenized population of barley. The mutation was located at the 3´ end of the first intron of the RNA splicing receptor (AG) site, resulting in abnormal RNA splicing and two abnormal transcripts of ss2a, which caused the inactivation of the SS2a gene. The starch structure and properties were significantly altered in the mutant, with M3-1413 containing decreased total starch and increased amylose and resistant starch levels. This study sheds light the effect of barley ss2a null mutations on starch properties and helps to guide new applications of barley starch to develop nutritious food products.

Purifying selection against spurious splicing signals contributes to the base composition evolution of the polypyrimidine tract.

Among eukaryotes, the major spliceosomal pathway is highly conserved. While long introns may contain additional regulatory sequences, the ones in short introns seem to be nearly exclusively related to splicing. Although these regulatory sequences involved in splicing are well-characterized, little is known about their evolution. At the 3' end of introns, the splice signal nearly universally contains the dimer AG, which consists of purines, and the polypyrimidine tract upstream of this 3' splice signal is characterized by over-representation of pyrimidines. If the over-representation of pyrimidines in the polypyrimidine tract is also due to avoidance of a premature splicing signal, we hypothesize that AG should be the most under-represented dimer. Through the use of DNA-strand asymmetry patterns, we confirm this prediction in fruit flies of the genus Drosophila and by comparing the asymmetry patterns to a presumably neutrally evolving region, we quantify the selection strength acting on each motif. Moreover, our inference and simulation method revealed that the best explanation for the base composition evolution of the polypyrimidine tract is the joint action of purifying selection against a spurious 3' splice signal and the selection for pyrimidines. Patterns of asymmetry in other eukaryotes indicate that avoidance of premature splicing similarly affects the nucleotide composition in their polypyrimidine tracts.

Sequence variations affect the 5' splice site selection of plant introns.

Introns are noncoding sequences spliced out of pre-mRNAs by the spliceosome to produce mature mRNAs. The 5' ends of introns mostly begin with GU and have a conserved sequence motif of AG/GUAAGU that could base-pair with the core sequence of U1 snRNA of the spliceosome. Intriguingly, ∼ 1% of introns in various eukaryotic species begin with GC. This occurrence could cause misannotation of genes; however, the underlying splicing mechanism is unclear. We analyzed the sequences around the intron 5' splice site (ss) in Arabidopsis (Arabidopsis thaliana) and found sequences at the GC intron ss are much more stringent than those of GT introns. Mutational analysis at various positions of the intron 5' ss revealed that although mutations impair base pairing, different mutations at the same site can have different effects, suggesting that steric hindrance also affects splicing. Moreover, mutations of 5' ss often activate a hidden ss nearby. Our data suggest that the 5' ss is selected via a competition between the major ss and the nearby minor ss. This work not only provides insights into the splicing mechanism of intron 5' ss but also improves the accuracy of gene annotation and the study of the evolution of intron 5' ss.

A novel nonsense variant in RHAG underlies a Nordic Rhnull phenotype.

The extremely rare Rhnull phenotype is characterized by the absence of all Rh antigens on erythrocytes. It is divided into the regulator and amorph types based on the underlying genetic background. The more common regulator type depends on critical variants silencing RHAG, which encodes RhAG glycoprotein, necessary for RhD/RhCE expression. Rhnull cells have altered expression of glycophorin B and LW glycoprotein. Four unrelated Rhnull individuals were investigated. Serological testing was performed according to standard blood bank practice. RHD/RHCE and S/s allele-specific Polymerase chain reaction (PCR) genotyping was done on genomic DNA using in-house PCR assays. RHAG, and in some cases also RHD/RHCE, were sequenced. Initial s phenotyping results triggered additional serological investigation. Anti-Rh29 was identified in all four individuals. Extended typing with anti-S and anti-s showed that the three samples predicted to type as s+ failed to react with 2 of 5 anti-s. Sequence analysis of all 10 RHAG exons and the immediate intron/exon boundaries revealed a single nucleotide variant in the 3'-end of intron 6, c.946 -2a>g in all samples. RHD/RHCE showed no alterations. A novel Nordic Rhnull allele was identified. In addition, it was shown that s+ Rhnull red blood cells are not only U- but also have qualitative changes in their s antigen expression.

Maize β-amylase7 encodes two proteins using alternative transcriptional start sites: nuclear BAM7 and plastidic BAM2.

An unusual β-amylase7 (BAM7) gene in some angiosperms, including grasses such as maize (Zea mays), appears to encode two functionally distinct proteins; a nuclear-localized transcription factor (BAM7) and a plastid-localized starch hydrolase (BAM2). In Arabidopsis (Arabidopsis thaliana), these two proteins are encoded by separate genes on different chromosomes but their physiological functions are not well established. Using the maize BAM7 gene as a model, we detected two populations of transcripts by 5'-RACE that encode the predicted proteins. The two transcripts are apparently synthesized independently using separate core promoters about 1 kb apart, the second of which is located in the first intron of the full-length gene. The N-terminus of the shorter protein, ZmBAM7-S, begins near the 3' end of the first intron of ZmBAM7-L and starts with a predicted chloroplast transit peptide. We previously showed that ZmBAM7-S is catalytically active with properties like those of AtBAM2. Here, we report that ZmBAM7-S targets green fluorescent protein to plastids. The transcript encoding the longer protein, ZmBAM7-L, encodes an additional DNA-binding domain containing a functional nuclear localization signal. This putative dual-function gene originated at least 400 Mya, prior to the emergence of ferns, and has persisted in some angiosperms that lack a separate BAM2 gene. It appears to have been duplicated and subfunctionalized in at least four lineages of land plants, resulting in two genes resembling Arabidopsis BAM2 and BAM7. Targeting of two products from a single gene to different subcellular locations is not uncommon in plants, but it is unusual when they are predicted to serve completely different functions in the two locations.

A splice site mutation in the FvePHP gene is associated with leaf development and flowering time in woodland strawberry.

Leaves and flowers are crucial for the growth and development of higher plants. In this study we identified a mutant with narrow leaflets and early flowering (nlef) in an ethyl methanesulfonate-mutagenized population of woodland strawberry (Fragaria vesca) and aimed to identify the candidate gene. Genetic analysis revealed that a single recessive gene, nlef, controlled the mutant phenotype. We found that FvH4_1g25470, which encodes a putative DNA polymerase α with a polymerase and histidinol phosphatase domain (PHP), might be the candidate gene, using bulked segregant analysis with whole-genome sequencing, molecular markers, and cloning analyses. A splice donor site mutation (C to T) at the 5' end of the second intron led to an erroneous splice event that reduced the expression level of the full-length transcript of FvePHP in mutant plants. FvePHP was localized in the nucleus and was highly expressed in leaves. Silencing of FvePHP using the virus-induced gene silencing method resulted in partial developmental defects in strawberry leaves. Overexpression of the FvePHP gene can largely restore the mutant phenotype. The expression levels of FveSEP1, FveSEP3, FveAP1, FveFUL, and FveFT were higher in the mutants than those in 'Yellow Wonder' plants, probably contributing to the early flowering phenotype in mutant plants. Our results indicate that mutation in FvePHP is associated with multiple developmental pathways. These results aid in understanding the role of DNA polymerase in strawberry development.

A seven-transmembrane protein-TM7SF3, resides in nuclear speckles and regulates alternative splicing

SummaryThe seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates cellular stress and the unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This unexpected location for a trans-membranal protein enables formation of stable complexes between TM7SF3 and pre-mRNA splicing factors including DHX15, LARP7, HNRNPU, RBM14, and HNRNPK. Indeed, TM7SF3 regulates alternative splicing of >330 genes, mainly at the 3′end of introns by directly modulating the activity of splicing factors such as HNRNPK. These effects are observed both in cell lines and primary human pancreatic islets. Accordingly, silencing of TM7SF3 results in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident protein of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing.

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

SUMMARYN6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

S233: THERAPEUTIC GENE EDITING OF T CELLS CORRECTS CTLA4 INSUFFICIENCY.

Background: Heterozygous mutations in CTLA4 result in an inborn error of immunity (IEI) (also known as primary immunodeficiency) with a severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic stem cell transplantation. The mutational landscape and requirement for tight regulation of CTLA4 make viral gene addition approaches unappealing. Gene editing strategies permit alteration of CTLA4 while retaining the endogenous gene control machinery. Aims: We set out to devise a CRISPR/Cas9/AAV6 gene editing strategy to correct CTLA4 insufficiency in T cells. Methods: We designed several homology directed repair (HDR) editing strategies that would correct the genetic defect. We first assessed correction of an individual point mutation. We then evaluated several universal strategies that enable correction of most disease-causing mutations with a single edit; the first that inserts the CTLA4 cDNA in exon 1, and a second that inserts the CTLA4 cDNA at the 3’end of the first intron of CTLA4. All AAV6 HDR donor templates included a GFP reporter gene to enable easy identification of the edited cells. Results: Superior editing efficiencies were obtained with the intronic approach compared to the other editing strategies and this strategy was then further evaluated. CTLA4 function and expression kinetics were assessed following editing using flow cytometry-based assays. Functional studies using CTLA4 transendocytosis (TE) assays, demonstrated restoration of CD80 and CD86 internalization in the edited CD4+ T cells. Following gene editing, transgene expression kinetics were comparable to healthy control CD4+ T cells. Gene editing of T cells isolated from patients with CTLA4 insufficiency restored CTLA4 expression and rescued transendocytosis of CD80 and CD86 in vitro. Using a similar approach, gene corrected T cells from CTLA4-/- mice engrafted in immunodeficient mice at clinically relevant frequencies and tail vein bleeds were performed 1, 3 and 4 weeks post adoptive transfer. In the mice which received the GFP+ edited cells, a stable population of GFP+ cells was detectable at all timepoints demonstrating in vivo persistence as well as genetic stability. All mice were sacrificed 4 weeks after cell transfer. To assess lymphoproliferation, the cellularity of peripheral lymph nodes and spleen weight were analyzed. Spleen and lymph node size, lymph node cell counts, and spleen weight were all significantly lower in mice which received the edited cells (n=5) compared to non-edited controls (n=5) and there was no significant difference in these parameters between mice who had received edited CTLA4-/- T cells and those who received WT T cells (n=4). Analysis of lymph node and spleen cells confirmed persistence of CTLA4 expression in the edited (GFP+) cells and revealed that a higher proportion of Treg than Tconv had been successfully edited. Summary/Conclusion: Together these data demonstrated that CTLA4 edited T cells survived in vivo, expressed CTLA4 and were able to control the clinical phenotype of CTLA4 insufficiency, providing a powerful proof-of-principle of our T cell GT approach. Our data provide proof-of-concept that gene editing can restore CTLA4 function in T cells demonstrating the potential of this approach to treat CTLA4 insufficiency. A similar approach could be used in other IEIs that are caused by multiple heterozygous mutations.

GFI1-Dependent Repression of SGPP1 Increases Multiple Myeloma Cell Survival.

Simple SummaryNew therapies have greatly improved the progression-free and overall survival for patients with “standard risk” multiple myeloma (MM). However, patients with “high risk” MM, in particular patients whose MM cells harbor non-functional p53, have very short survival times because of the early relapse and rapid development of highly therapy-resistant MM. In this report, we identify a novel mechanism responsible for Growth Factor Independence-1 (GFI1) regulation of the growth and survival of MM cells through its modulation of sphingolipid metabolism, regardless of their p53 status. We identify the Sphingosine-1-Phosphate Phosphatase (SGPP1) gene as a novel direct target of GFI1 transcriptional repression in MM cells, thus increasing intracellular sphingosine-1-phosphate levels, which stabilizes c-Myc. Our results support GFI1 as an attractive therapeutic target for all types of MM, including the “high risk” patient population with non-functional p53, as well as a possible therapeutic approach for other types of cancers expressing high levels of c-Myc.Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5’ end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.

An intron mutation of HNF1A causes abnormal splicing and impairs its activity as a transcription factor

Mutations in HNF1A are associated with Maturity Onset Diabetes of the Young type 3 (MODY3) and most of them are in the coding region. Herein, we identified an intron mutation at the 6th nucleotide upstream of the end of intron 7 of HNF1A, named IVS7-6G > A, in a patient with early-onset diabetes. The “minigene” assay showed that IVS7-6G > A produced two aberrant mRNA variants translating into two truncated proteins: L502S fs* and G437A fs*, both affecting HNF1A transactivation domain (TAD). To determine functional consequences of IVS7-6G > A mutation, we made plasmids encoding truncated HNF1A containing different portions of HNF1A TAD and found that the TAD of HNF1A is important not only for its regulatory activities, but also for its nuclearization, and the residues 282–501 was more essential than 502–631. Our data suggested IVS7-6G > A impaired HNF1A splicing and may contribute to the pathogenesis of MODY3.

Haplotype structures and polymorphisms of dog leukocyte antigen (DLA) class I loci shaped by intralocus and interlocus recombination events.

The dog leukocyte antigen (DLA) class I genomic region is located on chromosome 12, and the class I genomic region is composed of at least two distinct haplotypic gene structures, DLA-88-DLA-12 and DLA-88-DLA-88L. However, detailed information of the genomic differences among DLA-88, DLA-12, and DLA-88L are still lacking at the full-length gene level, and therefore, DLA allelic sequences classified for each of these loci are limited in number so far. In this study, we determined the DNA sequence of a 95-kb DLA class I genomic region including DLA-88, DLA-12/88L, and DLA-64 with three DLA homozygous dogs and of 37 full-length allelic gene sequences for DLA-88 and DLA-12/88L loci in 26 DLA class I homozygous dogs. Nucleotide diversity profiles of the 95-kb regions and sequence identity scores of the allelic sequences suggested that DLA-88L is a hybrid gene generated by interlocus and/or intralocus gene conversion between DLA-88 and DLA-12. The putative minimum conversion tract was estimated to be at least an 850-bp segment in length located from the 5´flanking untranslated region to the end of intron 2. In addition, at least one DLA-12 allele (DLA-12*004:01) was newly generated by interlocus gene conversion. In conclusion, the analysis for the occurrence of gene conversion within the dog DLA class I region revealed intralocus gene conversion tracts in 17 of 27 DLA-88 alleles and two of 10 DLA-12 alleles, suggesting that intralocus gene conversion has played an important role in expanding DLA allelic variations.

Germ-line mutations in WDR77 predispose to familial papillary thyroid cancer

The inheritance of predisposition to nonsyndromic familial nonmedullary thyroid cancer (FNMTC) remains unclear. Here, we report six individuals with papillary thyroid cancer (PTC) in two unrelated nonsyndromic FNMTC families. Whole-exome sequencing revealed two germ-line loss-of-function variants occurring within a 28-bp fragment of WDR77, which encodes a core member of a transmethylase complex formed with the protein arginine methyltransferase PRMT5 that is responsible for histone H4 arginine 3 dimethylation (H4R3me2) in frogs and mammals. To date, the association of WDR77 with susceptibility to cancer in humans is unknown. A very rare heterozygous missense mutation (R198H) in WDR77 exon 6 was identified in one family of three affected siblings. A heterozygous splice-site mutation (c.619+1G > C) at the 5' end of intron 6 is present in three affected members from another family. The R198H variant impairs the interaction of WDR77 with PRMT5, and the splice-site mutation causes exon 6 skipping and results in a marked decrease in mutant messenger RNA, accompanied by obviously reduced H4R3me2 levels in mutation carriers. Knockdown of WDR77 results in increased growth of thyroid cancer cells. Whole-transcriptome analysis of WDR77 mutant patient-derived thyroid tissue showed changes in pathways enriched in the processes of cell cycle promotion and apoptosis inhibition. In summary, we report WDR77 mutations predisposing patients to nonsyndromic familial PTC and link germ-line WDR77 variants to human malignant disease.

Interpretable prioritization of splice variants in diagnostic next-generation sequencing

A critical challenge in genetic diagnostics is the computational assessment of candidate splice variants, specifically the interpretation of nucleotide changes located outside of the highly conserved dinucleotide sequences at the 5' and 3' ends of introns. To address this gap, we developed the Super Quick Information-content Random-forest Learning of Splice variants (SQUIRLS) algorithm. SQUIRLS generates a small set of interpretable features for machine learning by calculating the information-content of wild-type and variant sequences of canonical and cryptic splice sites, assessing changes in candidate splicing regulatory sequences, and incorporating characteristics of the sequence such as exon length, disruptions of the AG exclusion zone, and conservation. We curated a comprehensive collection of disease-associated splice-altering variants at positions outside of the highly conserved AG/GT dinucleotides at the termini of introns. SQUIRLS trains two random-forest classifiers for the donor and for the acceptor and combines their outputs by logistic regression to yield a final score. We show that SQUIRLS transcends previous state-of-the-art accuracy in classifying splice variants as assessed by rank analysis in simulated exomes, and is significantly faster than competing methods. SQUIRLS provides tabular output files for incorporation into diagnostic pipelines for exome and genome analysis, as well as visualizations that contextualize predicted effects of variants on splicing to make it easier to interpret splice variants in diagnostic settings.

High-Resolution Architecture and Partner Genes of MYC Rearrangements in Lymphoma with DLBCL Morphology

High-Resolution Architecture and Partner Genes of MYC Rearrangements in Lymphoma with DLBCL Morphology