Splice Site Region Research Articles

Mapping the locus for ocular melanosis in Cairn Terriers.

To map the disease locus for familial ocular melanosis (OM) in the Cairn Terrier. Cairn Terriers with OM and normal control dogs. A genome-wide association study (GWAS) was performed using 63 OM-affected and 31 control Cairn Terriers, followed by haplotype analysis. A significantly associated single-nucleotide polymorphism was genotyped in a larger group of OM-affected and control Cairn Terriers. The coding and splice-site regions of genes mapping within the confidence interval were sequenced. A ~9.2 Mb region of chromosome 11 was significantly associated with OM. Haplotype analysis narrowed the region to 1.49 Mb. Genotyping of a SNP within the region showed 86% of OM-affected dogs were homozygous or heterozygous for the risk allele, whereas 78% of unaffected dogs were homozygous for the nonrisk allele. Sequencing of the coding regions and splice sites of four genes (c9orf72, IFNK, the 5' end of MOB3B, and the 3' end of LINGO2) and of a microRNA (MIR876) did not detect any genetic variants unique to OM-affected dogs. OM in Cairn Terriers maps to a 1.49 Mb region of chromosome 11. This accounts for 86% of OM cases in our DNA bank. A second locus may account for the OM phenotype in the remaining 14% of cases. Sequencing of coding regions and splice sites of positional candidate genes and a microRNA did not reveal any genetic variants unique to affected dogs. Further studies are required to elucidate the DNA variant causal for OM in Cairn Terriers and to understand the disease mechanism.

A Splice Site Variant in ADAMTS3 Is the Likely Causal Variant for Pulmonary Hypoplasia with Anasarca in Persian/Persian-Cross Sheep.

Pulmonary hypoplasia with anasarca, or hydrops fetalis, is characterized by stillbirth, diffuse oedema, and generalized lymph node hypoplasia. The enlarged fetus frequently causes dystocia. The disease has been reported in cattle and sheep as an inherited condition with a recessive mode of inheritance. This is the first report of the disease in Persian/Persian-cross sheep in Australia. Affected fetuses were reported from three flocks, and a total of eleven affected, eleven obligate carrier, and 188 related Persian/Persian-cross animals were available for analysis, as well as unrelated control animals. SNP genotyping revealed a region of homozygosity in affected animals on ovine chromosome six, which contained the functional candidate gene ADAMTS3. Whole genome sequencing of two affected fetuses and one obligate carrier ewe revealed a single nucleotide deletion, ENSOARG00000013204:g.87124344delC, located 3 bp downstream from a donor splice site region in the ADAMTS3 gene. Sanger sequencing of cDNA containing this variant further revealed that it is likely to introduce an early splice site in exon 14, resulting in a loss of 6 amino acids at the junction of exon 14 and intron 14/15. A genotyping assay was developed, and the ENSOARG00000013204:g.87124344delC segregated with disease in 209 animals, allowing for effective identification of carrier animals.

A Novel Large Deletion in the EVER1 Gene in a Family With Epidermodysplasia Verruciformis From India.

Epidermodysplasia verruciformis (EV) is a rare autosomal recessive genodermatosis due to mutations in EVER1 and EVER2 genes. The genetic profile of Indian patients with EV has not been previously studied. This report describes the clinical presentation and molecular analysis of a family with EV. Using genomic DNA from two affected probands and healthy controls (two other siblings), conventional polymerase chain reaction (PCR) was conducted with novel primer sets designed to amplify the coding and splice-site regions in the genes EVER1 and EVER 2 . This revealed no amplification with a primer set for exons 16 to 18 in the EVER1 gene of both the probands. Subsequently, long-range PCR spanning the length of exon 15-20 and next-generation sequencing demonstrated a homozygous deletion of 2078 bp in the EVER1 gene ( EVER1 :c.2072_2278del). Screening the family revealed the same homozygous deletion (similar to index cases) in two other affected siblings. The parents and two asymptomatic siblings were heterozygous carriers for the deletion while one healthy sibling was negative. These results were validated with Sanger sequencing. This deletion in exons 17 and 18 of the EVER1 gene results in a frameshift, followed by a premature termination resulting in a severe phenotype. The identification and validation of this large deletion was detected using stepwise amplicon-based target enrichment and long-range PCR, respectively. In this family, this simple strategy greatly enhanced genetic counseling as well as early genetic diagnosis and screening. However, functional assays and larger studies are required to characterize and validate the genetic diversity among Indians with EV.

REEP4 variant analysis in blepharospasm and other neurological disorders.

In preceding work, a deleterious REEP4 variant [GRCh38/hg38, NC_000008.11:g.22140245G>A, NM_025232.4:c.109C>T, p.Arg37Trp] was found to co-segregate with blepharospasm (BSP) in a large African-American pedigree. Other REEP4 variants have been reported in genetic screening studies of dystonia. The REEP4 paralogs, REEP1 and REEP2, are associated with spastic paraplegia. The causal contributions of REEP4 variants to dystonia and other neurological disorders remains indecisive. Sanger sequencing was used to screen subjects (N = 307) with BSP and BSP-plus dystonia affecting additional anatomical segments (BSP+) phenotypes for variants in REEP4. In silico tools were used to examine the deleteriousness of reported (ClinVar) and previously published REEP4 variants. No highly deleterious variant was identified in coding or contiguous splice site regions of REEP4 in our cohort of 307 subjects. In silico analysis identified numerous deleterious REEP4 variants in published screening studies of dystonia and several highly deleterious single nucleotide REEP4 variants in ClinVar. Highly deleterious REEP4 variants are rare in BSP and BSP+ phenotypes.

TOR2A Variants in Blepharospasm.

Genetic factors have been implicated in the pathogenesis of blepharospasm (BSP), a dystonia characterized by excessive blinking and involuntary eyelid closure. Previous research identified a co-segregating deleterious TOR2A variant (GRCh38/hg38, NC_000009.12: g.127733410G>A, NM_001085347.3:c.568C>T, p. Arg190Cys) in three subjects with BSP and three carriers within a multi-generation pedigree. Other TOR2A variants have been reported in patients with dystonia. Sanger sequencing was used to screen a cohort of 307 subjects with isolated BSP or BSP-plus dystonia affecting additional anatomical segments (BSP+). We also utilized computational tools to uniformly assess the deleteriousness and potential pathogenicity of previously reported TOR2A variants. There were no highly deleterious TOR2A variants in the coding or contiguous splice site regions of TOR2A within our cohort of 307 subjects. Highly deleterious variants in TOR2A are rare in patients with BSP/BSP+ phenotypes. Over 300 patients with BSP were screened for variants in TOR2A, a TOR1A (DYT1) homologue. No highly deleterious variants were identified in our cohort. The role of TOR2A in BSP and other forms of dystonia remains indeterminant.

Exon-intron boundary inhibits m6A deposition, enabling m6A distribution hallmark, longer mRNA half-life and flexible protein coding

Regional bias of N6-methyladenosine (m6A) mRNA modification avoiding splice site region, calls for an open hypothesis whether exon-intron boundary could affect m6A deposition. By deep learning modeling, we find that exon-intron boundary represses a proportion (12% to 34%) of m6A deposition at adjacent exons (~100 nt to splice site). Experiments validate that m6A signal increases once the host gene does not undergo pre-mRNA splicing to produce the same mRNA. Inhibited m6A sites have higher m6A enhancers and lower m6A silencers locally and show high heterogeneity at different exons genome-widely, with only a small proportion (12% to 15%) of exons showing strong inhibition, enabling more stable mRNAs and flexible protein coding. m6A is majorly responsible for why mRNAs with more exons be more stable. Exon junction complex (EJC) only partially contributes to this exon-intron boundary m6A inhibition in some short internal exons, highlighting additional factors yet to be identified.

Generation of an induced pluripotent stem cell line from a patient with conduction disease and recurrent ventricular fibrillation with a sodium voltage-gated channel alpha subunit 5 (SCN5A) gene c.392 + 3A > G splice-site variant

Variants in the sodium voltage-gated channel alpha subunit 5 gene (SCN5A) produce variable cardiac phenotypes including Brugada syndrome, conduction disease and cardiomyopathy. These phenotypes can lead to life-threatening arrhythmias, heart failure, and sudden cardiac death. Novel variants in splice-site regions of SCN5A require functional studies to characterise their pathogenicity as they are poorly understood. The generation of an induced pluripotent stem cell line provides a valuable resource to investigate the functional effects of potential splice-disrupting variants in SCN5A.

Somatic Variants in DNA Damage Response Genes in Ovarian Cancer Patients Using Whole-exome Sequencing.

Several clinical trials have investigated homologous recombination deficiency and BRCA1/2 status to select ovarian cancer patients for treatment with poly(ADP-ribose) polymerase-inhibitors (PARPi), but less attention has been given to other DNA-damage response (DDR) pathways. Therefore, we investigated somatic single/multiple nucleotide variants and small insertions/deletions in exonic and splice-site regions of 356 DDR genes to examine whether genes other than BRCA1/2 are altered. Whole-exome sequencing data from eight high-grade serous adenocarcinoma (HGSC) and four clear cell carcinoma (oCCC) patients were analyzed. Forty-two variants (pathogenic, likely pathogenic or variants of uncertain significance) in 28 genes from DDR pathways were identified. Seven out of nine TP53 variants were previously described in The Cancer Genome Atlas Ovarian Cancer; other variants were found in 23 out of 28 unique genes, whereas no variants were reported in FAAP24, GTF2H4, POLE4, RPA3, and XRCC4. As the identified variants were not only limited to well-known TP53, BRCA1/2, and HR-associated genes, our study might contribute to the better understanding of which DDR pathways potentially influence disease progression. Moreover, they may display a potential role as biomarkers to predict platinum-based chemotherapy or PARPi treatment response or disease progression, as differences in disrupted DDR pathways were observed between patients with long and short overall survival in HGSC and oCCC groups.

Identification of a Novel Idiopathic Epilepsy Risk Locus and a Variant in the CCDC85A Gene in the Dutch Partridge Dog

(1) Idiopathic epilepsy (IE) is thought to have a genetic cause in several dog breeds. However, only two causal variants have been identified to date, and few risk loci are known. No genetic studies have been conducted on IE in the Dutch partridge dog (DPD), and little has been reported on the epileptic phenotype in this breed. (2) Owner-filled questionnaires and diagnostic investigations were used to characterize IE in the DPD. A genome-wide association study (GWAS) involving 16 cases and 43 controls was performed, followed by sequencing of the coding sequence and splice site regions of a candidate gene within the associated region. Subsequent whole-exome sequencing (WES) of one family (including one IE-affected dog, both parents, and an IE-free sibling) was performed. (3) IE in the DPD has a broad range in terms of age at onset, frequency, and duration of epileptic seizures. Most dogs showed focal epileptic seizures evolving into generalized seizures. A new risk locus on chromosome 12 (BICF2G630119560; praw = 4.4 × 10-7; padj = 0.043) was identified through GWAS. Sequencing of the GRIK2 candidate gene revealed no variants of interest. No WES variants were located within the associated GWAS region. However, a variant in CCDC85A (chromosome 10; XM_038680630.1: c.689C > T) was discovered, and dogs homozygous for the variant (T/T) had an increased risk of developing IE (OR: 6.0; 95% CI: 1.6-22.6). This variant was identified as likely pathogenic according to ACMG guidelines. (4) Further research is necessary before the risk locus or CCDC85A variant can be used for breeding decisions.

Investigation of the Genetic Etiology in Idiopathic Generalized Epileptic Disorders by Targeted Next-generation Sequencing Technique

Idiopathic generalized epilepsy is the most common group of epilepsy disorders in children and adolescents. Various types of genetic abnormality were identified among the hereditary factors that explain epilepsy. To determine the variations in the etiopathogenesis, treatment protocol planning, and prognosis of idiopathic generalized epilepsy using the next-generation sequencing method. A cross-sectional study. This study included 32 patients with idiopathic generalized epilepsy. Genomic DNA was obtained from peripheral venous blood samples taken from the patients. A total of 18 genes encoding ion channel subunits that are involved in monogenic disorders and are associated with idiopathic generalized epilepsy were included. The targeted custom next-generation sequencing panel was designed to cover all coding exons and all exon/intron splice site regions of 18 genes. We detected 9 (28%) variations, including 1 likely pathogenic (a variant in the SCN1A gene) and 8 of unknown clinical significance (2 in the CLCN2 genes, GABBR2, SCN1B, SLC2A1, SLC4A10 genes, and 2 in the TBC1D24 gene). Study results should be supported by functional advanced studies, with increased existing knowledge in the relevant variations.

Analysis of ProP1 Gene in a Cohort of Tunisian Patients with Congenital Combined Pituitary Hormone Deficiency.

Background: Non-syndromic combined pituitary hormone deficiency (CPHD) occurs due to defects in transcription factors that govern early pituitary development and the specification of hormone-producing cells. The most common mutations are in the Prophet of Pit-1 (ProP1) gene. This work aims to (1) report findings of genetic analyses of Tunisian patients with non-syndromic CPHD and (2) describe their phenotype patterns and their evolution through life. Methods: Fifteen patients from twelve unrelated families with variable clinical phenotypes were included after excluding autoimmune and acquired forms of non-syndromic CPHD. Detailed pedigree charts and auxological, hormonal, radiological, and therapeutic details were recorded. Sanger sequencing was performed, and sequences were analyzed with a specific focus on coding and splice site regions of the ProP1 gene. Retained variants were classified using several in silico pathogenicity prediction tools and the VarSome platform. Results: We identified the common p.Arg73Cys mutation in seven patients from four unrelated pedigrees. We found a novel homozygous mutation (c.340C>T) in one sporadic case. This mutation generates a truncated ProP1 protein, predicted to be non-functional, lacking the last 112 codons (p.(Gln114Ter)). We confirmed by polymerase chain reaction (PCR) the absence of large exon deletions or insertions in the remaining sporadic patients (7/8). Conclusions: We report two mutations {one newly identified [p.(Gln114Ter)] and one previously reported (p.Arg73Cys)} in five unrelated Tunisian families with non-syndromic CPHD. This work is of clinical importance as it reports the high frequency of the p.Arg73Cys mutation in Tunisian CPHD families. Our study also illuminated the involvement of novel gene(s) in the emergence of non-syndromic CPHD.

RF17 | PMON24 The paradox of combined pituitary hormone deficiency and tall stature: a glimpse from exome sequencing

Abstract Introduction An intact GH-IGF-1 axis is known to be essential for proper growth. However, cases in which individuals with growth hormone deficiency (GHD) reach target height without the use of GH suggest that other mechanisms may play a role in the growth process. Out of 209 patients with hypopituitarism followed in the endocrinology clinic at HCFMUSP, 9 presented with normal stature despite GHD and hypogonadotropic hypogonadism. Among them, 1 male patient presented with tall stature. The mechanisms under growth without growth hormone are poorly understood, so we hypothesized that genetic mutations in genes related to tall stature could be a plausible explanation. The aim of the present work was to look for allelic variants in the exome that could be associated with tall stature, combined pituitary hormone deficiency and/or skeletal deformities. The index male patient presented at the age of 28 to a medical assistance due to the lack of pubertal development, leading to the diagnosis of FSH/LH and TSH deficiencies and thus treated accordingly. He was still growing even under testosterone replacement and noticed that his hand and fingers also got bigger in this period. At 32-year-old he presented to our service with a stature of 193.5cm (z score of +2.40) being out of his target height range. An arm span of 195cm was notice together with skeletal deformities such as claw toes, trigger fingers in hands and a barrel chest. At this time ACTH and GH deficiencies were diagnosed and an adult dose of GH was initiated. Over one year treatment he grew 1 cm even presenting complete closed epiphyses in hands but incomplete epiphyseal fusion in the iliac crest (Risser 4). In the skeletal X ray scoliosis, deformities in hands and feet were noticed. Pituitary MRI revealed an ectopic neurohypophysis and a reduced adenohypophysis. Whole exome sequencing was performed and allelic variants in exonic or splice site regions with frequency of less than 1% were prioritized. Out of 26 variants, 13 were classified as VUS or pathogenic in the genes PSG4, ZGPAT, LRP2, LRP5, LRP6, RYR1, REL SPAST, SDHC, EGFR, ARID3B and TGFB2, being the last 5 genes never described in 4 different populational databases (gnomAD, 1000G, ABraOM and SELAdb). Allelic variant in TGFB2 gene has been related to Marfan syndrome. Family members are having these genes in the process of being segregated. Conclusion Mutations in genes related to tall stature may be a mechanism that could explains normal growth among individuals with GHD. Presentation: Sunday, June 12, 2022 12:42 p.m. - 12:47 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Trio-based whole exome sequencing in patients with suspected sporadic inborn errors of immunity: A retrospective cohort study.

<i>De novo</i> variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI). This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI that underwent patient-parent trio-based WES. A (likely) molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic <i>in silico</i> IEI WES gene panel. Systematic evaluation of rare, non-synonymous DNVs in coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were found in IEI genes (<i>NLRP3</i> and <i>RELA</i>) and in potentially novel candidate genes, including <i>PSMB10</i>, <i>DDX1</i>, <i>KMT2C,</i> and <i>FBXW11</i>. The <i>FBXW11</i> canonical splice site DNV was shown to lead to defective RNA splicing, increased NF-κB p65 signalling, and elevated IL-1β production in primary immune cells extracted from the patient with autoinflammatory disease. Our findings in this retrospective cohort study advocate the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we provide functional evidence supporting a causal role for <i>FBXW11</i> loss-of-function mutations in autoinflammatory disease. This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences.

EnsembleSplice: ensemble deep learning model for splice site prediction

BackgroundIdentifying splice site regions is an important step in the genomic DNA sequencing pipelines of biomedical and pharmaceutical research. Within this research purview, efficient and accurate splice site detection is highly desirable, and a variety of computational models have been developed toward this end. Neural network architectures have recently been shown to outperform classical machine learning approaches for the task of splice site prediction. Despite these advances, there is still considerable potential for improvement, especially regarding model prediction accuracy, and error rate.ResultsGiven these deficits, we propose EnsembleSplice, an ensemble learning architecture made up of four (4) distinct convolutional neural networks (CNN) model architecture combination that outperform existing splice site detection methods in the experimental evaluation metrics considered including the accuracies and error rates. We trained and tested a variety of ensembles made up of CNNs and DNNs using the five-fold cross-validation method to identify the model that performed the best across the evaluation and diversity metrics. As a result, we developed our diverse and highly effective splice site (SS) detection model, which we evaluated using two (2) genomic Homo sapiens datasets and the Arabidopsis thaliana dataset. The results showed that for of the Homo sapiens EnsembleSplice achieved accuracies of 94.16% for one of the acceptor splice sites and 95.97% for donor splice sites, with an error rate for the same Homo sapiens dataset, 4.03% for the donor splice sites and 5.84% for the acceptor splice sites datasets.ConclusionsOur five-fold cross validation ensured the prediction accuracy of our models are consistent. For reproducibility, all the datasets used, models generated, and results in our work are publicly available in our GitHub repository here: https://github.com/OluwadareLab/EnsembleSplice

Phenotypic Expression of CFH Rare Variants in Age-Related Macular Degeneration Patients in the Coimbra Eye Study.

PurposeTo determine the association between rare genetic variants in complement factor H (CFH) and phenotypic features in age-related macular degeneration (AMD) patients from the Coimbra Eye Study (CES).MethodsAMD patients from the Incidence CES (NCT02748824) underwent ophthalmologic examination and color fundus photography, spectral-domain optical coherence tomography (SD-OCT), fundus autofluorescence, and near-infrared imaging. Multimodal phenotypic characterization was carried out in a centralized reading center. The coding and splice-site regions of the CFH gene were sequenced through single-molecule molecular inversion probe–based next-generation sequencing in association with the EYE-RISK consortium. Variants with minor allele frequency <0.05 resulting in splice-site or protein change were selected. Differences in phenotypic features between carriers and noncarriers were analyzed using generalized estimated equations logistic regression models, considering intereye correlations.ResultsWe included 39 eyes of 23 patients carrying rare CFH variants and 284 eyes of 188 noncarriers. Carrier status was associated with having higher drusen burden in the macula in the inner Early Treatment Diabetic Retinopathy Study circle (odds ratio [OR], 5.44 [95% confidence interval {CI}, 1.61–18.37]; P = 0.006), outer circle (OR, 4.37 [95% CI, 1.07–17.77]; P = 0.04), and full grid (OR, 4.82 [95% CI, 1.13–20.52]; P = 0.033). In SD-OCT, a lower total macular volume and lower inner retinal layers’ volume (OR, 0.449 [95% CI, 0.226–0.894]; P = 0.023; OR, 0.496 [95% CI, 0.252–0.979]; P = 0.043) and pigment epithelial detachments (PEDs) (OR, 5.24 [95% CI, 1.08–25.44]; P = 0.04) were associated with carrying a rare CFH variant. Carriers with subretinal drusenoid deposits (SDD) had the rare variant P258L in all cases except one.ConclusionsWe identified in our cohort phenotypic differences between carriers and noncarriers of rare variants in the CFH gene. Carriers had more severe disease, namely superior drusen burden, PEDs, and thinner retinas. The rare variant P258L may be associated with SDD. Carriers are probably at increased risk of progression.

Prevalence, parameters, and pathogenic mechanisms for splice-altering acceptor variants that disrupt the AG exclusion zone.

SummaryPredicting the pathogenicity of acceptor splice-site variants outside the essential AG is challenging, due to high sequence diversity of the extended splice-site region. Critical analysis of 24,445 intronic extended acceptor splice-site variants reported in ClinVar and the Leiden Open Variation Database (LOVD) demonstrates 41.9% of pathogenic variants create an AG dinucleotide between the predicted branchpoint and acceptor (AG-creating variants in the AG exclusion zone), 28.4% result in loss of a pyrimidine at the −3 position, and 15.1% result in loss of one or more pyrimidines in the polypyrimidine tract. Pathogenicity of AG-creating variants was highly influenced by their position. We define a high-risk zone for pathogenicity: > 6 nucleotides downstream of the predicted branchpoint and >5 nucleotides upstream from the acceptor, where 93.1% of pathogenic AG-creating variants arise and where naturally occurring AG dinucleotides are concordantly depleted (5.8% of natural AGs). SpliceAI effectively predicts pathogenicity of AG-creating variants, achieving 95% sensitivity and 69% specificity. We highlight clinical examples showing contrasting mechanisms for mis-splicing arising from AG variants: (1) cryptic acceptor created; (2) splicing silencer created: an introduced AG silences the acceptor, resulting in exon skipping, intron retention, and/or use of an alternative existing cryptic acceptor; and (3) splicing silencer disrupted: loss of a deep intronic AG activates inclusion of a pseudo-exon. In conclusion, we establish AG-creating variants as a common class of pathogenic extended acceptor variant and outline factors conferring critical risk for mis-splicing for AG-creating variants in the AG exclusion zone, between the branchpoint and acceptor.

Regulation of alternative splicing of PaFT and PaFDL1, the FT and FD homologs in Platanus acerifolia

Alternative splicing (AS) selects different alternative splice sites and produces a variety of transcripts with different exon/intron combinations, which may result in multiple protein isoforms. The splicing signals include cis-elements and RNA structures; however, the mechanisms of AS regulation in plants have yet to be elucidated. Previous studies have shown that in Platanus acerifolia, the FLOWERING LOCUS T (FT) homolog PaFT has a unique and complex AS pattern, in which most of the splice forms of PaFT involve the first and/or second intron, and the FD homolog PaFDL1 produces two transcripts via AS, whereas the other FT homolog PaFTL is not regulated by AS. In this study, the regulatory mechanism of the AS of PaFT was demonstrated to be conserved in different plant species. To define the distribution of the AS regulatory signals, the intron-swap, site-directed mutagenesis of alternative splice sites, and deletion experiment were performed. For the PaFT gene, all the signals that regulate the AS of the first intron were located within this intron, while the usage of the first alternative splice site in the second intron was determined by the first intron. Meanwhile, the AS of PaFDL1 might be co-regulated by exons and the first intron. Additionally, the first alternative splice site and adjacent region in PaFT intron 1 might contain cis-elements and/or RNA structures that affect the use of the other sites. This study had provided a deeper insight into the distribution of AS signals in plants, namely the AS signals of different splice sites might exist in the intron where the sites were present, and might also be distributed in exons or other introns.

Genetic Risk in Families with Age-Related Macular Degeneration.

PurposeTo determine the contribution of common and rare genetic risk variants in families with age-related macular degeneration (AMD).DesignCase-control study.ParticipantsA family cohort (355 affected and 342 unaffected family members from 144 families with AMD) and an unrelated case-control cohort (1078 patients, 952 controls), recruited from the European Genetic Database.MethodsGenetic data of both cohorts were filtered for carriership of rare genetic variants in the coding and splice-site regions of the complement factor H (CFH) and complement factor I (CFI) genes, and 52 AMD-associated variants were extracted for calculation of genetic risk scores (GRS). To compare GRSs between familial and nonfamilial rare CFH and CFI variant carriers and noncarriers and between AMD disease stages, we performed a 2-way analysis of variance, with Bonferroni correction for multiple testing. Within families with AMD carrying rare CFH and CFI variants, we analyzed segregation patterns by calculating the proportion of affected among carriers.Main Outcome MeasuresGRSs and segregation of rare CFH and CFI variants.ResultsWe observed higher GRSs in familial versus nonfamilial individuals without rare CFH and CFI variants: mean GRS, 1.76 (standard error [SE], 0.08) versus 0.83 (SE, 0.03; P < 0.001). In 51 of 144 families (35.4%), rare CFH and CFI variants were identified. Within the AMD family cohort, carriers of rare CFH and CFI variants showed lower GRSs compared with noncarriers (mean GRS, 1.05 [SE, 0.23] vs. 1.76 [SE, 0.08]; P = 0.02). The proportion of affected family members with a high GRS was 57.3% (176/307). Of the affected family members with a low or intermediate GRS, 40.0% carried rare CFH or CFI variants. Among carriers of 11 rare CFH or CFI variants, the proportion affected by AMD was more than 75%.ConclusionsGenetic risk in families with AMD often is attributed to high GRSs based on common variants. However, in part of the families with a low or intermediate GRS, rare CFH and CFI variants contributed to disease development. We recommend computing GRSs and sequencing the CFH and CFI genes in families with AMD, in particular in the light of ongoing gene-specific clinical trials.

Exploration of Alternative Splicing (AS) Events in MDV-Infected Chicken Spleens.

Marek’s disease (MD) was an immunosuppression disease induced by Marek’s disease virus (MDV). MD caused huge economic loss to the global poultry industry, but it also provided an ideal model for studying diseases induced by the oncogenic virus. Alternative splicing (AS) simultaneously produced different isoform transcripts, which are involved in various diseases and individual development. To investigate AS events in MD, RNA-Seq was performed in tumorous spleens (TS), spleens from the survivors (SS) without any lesion after MDV infection, and non-infected chicken spleens (NS). In this study, 32,703 and 25,217 AS events were identified in TS and SS groups with NS group as the control group, and 1198, 1204, and 348 differently expressed (DE) AS events (p-value < 0.05 and FDR < 0.05) were identified in TS vs. NS, TS vs. SS, SS vs. NS, respectively. Additionally, Function enrichment analysis showed that ubiquitin-mediated proteolysis, p53 signaling pathway, and phosphatidylinositol signaling system were significantly enriched (p-value < 0.05). Small structural variations including SNP and indel were analyzed based on RNA-Seq data, and it showed that the TS group possessed more variants on the splice site region than those in SS and NS groups, which might cause more AS events in the TS group. Combined with previous circRNA data, we found that 287 genes could produce both circular and linear RNAs, which suggested these genes were more active in MD lymphoma transformation. This study has expanded the understanding of the MDV infection process and provided new insights for further analysis of resistance/susceptibility mechanisms.

Nucleotides in both donor and acceptor splice sites are responsible for choice in NAGNAG tandem splice sites.

Among alternative splicing events in the human transcriptome, tandem NAGNAG acceptor splice sites represent an appreciable proportion. Both proximal and distal NAG can be used to produce two splicing isoforms differing by three nucleotides. In some cases, the upstream exon can be alternatively spliced as well, which further increases the number of possible transcripts. In this study, we showed that NAG choice in tandem splice site depends considerably not only on the concerned acceptor, but also on the upstream donor splice site sequence. Using an extensive set of experiments with systematically modified two-exonic minigene systems of AFAP1L2 or CSTD gene, we recognized the third and fifth intronic upstream donor splice site position and the tandem acceptor splice site region spanning from -10 to +2, including NAGNAG itself, as the main drivers. In addition, competition between different branch points and their composition were also shown to play a significant role in NAG choice. All these nucleotide effects appeared almost additive, which explained the high variability in proximal versus distal NAG usage.