Splice Site Single Nucleotide Polymorphism Research Articles

Identification of deleterious single nucleotide polymorphism (SNP)s in the human TBX5 gene & prediction of their structural & functional consequences: An in silico approach

T-box transcription factor 5 gene (TBX5) encodes the transcription factor TBX5, which plays a crucial role in the development of heart and upper limbs. Damaging single nucleotide variants in this gene alter the protein structure, disturb the functions of TBX5, and ultimately cause Holt-Oram Syndrome (HOS). By analyzing the available single nucleotide polymorphism information in the dbSNP database, this study was designed to identify the most deleterious TBX5 SNPs through insilico approaches and predict their structural and functional consequences.Fifty-eight missense substitutions were found damaging by sequence homology-based tools: SIFT and PROVEAN, and structure homology-based tool PolyPhen-2. Various disease association meta-predictors further scrutinized these SNPs. Additionally, conservation profile of the amino acid residues, their surface accessibility, stability, and structural integrity of the native protein upon mutations were assessed. From these analyses, finally 5 SNPs were detected as the most damaging ones: [rs1565941579 (P85S), rs1269970792 (W121R), rs772248871 (V153D), rs769113870 (E208D), and rs1318021626 (I222N)]. Analyses of stop-lost, nonsense, UTR, and splice site SNPs were also conducted.Through integrative bioinformatics analyses, this study has identified the SNPs that are deleterious to the TBX5 protein structure and have the potential to cause HOS. Further wet-lab experiments can validate these findings.

Allelic characterization and protein structure analysis reveals the involvement of splice site mutation for growth habit differences in Lablab purpureus (L.) Sweet

BackgroundInterrelationship between growth habit and flowering played a key role in the domestication history of pulses; however, the actual genes responsible for these traits have not been identified in Indian bean. Determinate growth habit is desirable due to its early flowering, photo-insensitivity, synchronous pod maturity, ease in manual harvesting and short crop duration. The present study aimed to identify, characterize and validate the gene responsible for growth habit by using a candidate gene approach coupled with sequencing, multiple sequence alignment, protein structure prediction and binding pocket analysis. ResultsTerminal flowering locus was amplified from GPKH 120 (indeterminate) and GNIB-21 (determinate) using the primers designed from PvTFL1y locus of common bean. Gene prediction revealed that the length of the third and fourth exons differed between the two alleles. Allelic sequence comparison indicated a transition from guanine to adenine at the end of the third exon in GNIB 21. This splice site single-nucleotide polymorphism (SNP) was validated in germplasm lines by sequencing. Protein structure analysis indicated involvement of two binding pockets for interaction of terminal flowering locus (TFL) protein with other proteins. ConclusionThe splice site SNP present at the end of the third exon of TFL locus is responsible for the transformation of shoot apical meristem into a reproductive fate in the determinate genotype GNIB 21. The splice site SNP leads to absence of 14 amino acids in mutant TFL protein of GNIB 21, rendering the protein non-functional. This deletion disturbed previously reported anion-binding pocket and secondary binding pocket due to displacement of small β-sheet away from an external loop. This finding may enable the modulation of growth habit in Indian bean and other pulse crops through genome editing.

Computational analysis of Cyclin D1 gene SNPs and association with breast cancer.

CCND1 encodes for Cyclin D1 protein and single-nucleotide polymorphisms (SNPs) can modulate its activity. In the present study, the impact of CCND1 SNPs on structure and/or function of Cyclin D1 protein using in silico tools was investigated. Our analysis revealed only one splice site SNP (c.1988+5G<A) can effect CCND1 function. Subsequently, 78 out of 169 missense variants were predicted as pathogenic by Polyphen2, SIFT, PROVEAN, SNPs&GO, and PANTHER, and 4/78 missense SNPs were further evaluated because these four SNPs were found to be reside in highly conserved region of Cyclin D1. However, they did not show any major impact on tertiary structure and domain of Cyclin D1 but overall R15S and A190S has displayed a significant diseased phenotype and an altered molecular mechanism predicted by MutPred, FATHMM, SNPeffect, SNAP2, and PredictSNP. Consistently, A190S, R179L, and R15S may also cause a decrease in stability of Cyclin D1 anticipated by I-Mutant, HOPE and SNP effect. Furthermore, the Kaplan–Meier plotter has explained that high expression of CCND1 is associated with less survival rate of breast cancer patients. Altogether our study suggests that c.1988+5G<A, R15S, R179L, and A190S SNPs could directly or indirectly destabilize Cyclin D1.

Novel genetic susceptibility loci identified by family based whole exome sequencing in Han Chinese schizophrenia patients

Schizophrenia (SCZ) is a highly heritable psychiatric disorder that affects approximately 1% of population around the world. However, early relevant studies did not reach clear conclusions of the genetic mechanisms of SCZ, suggesting that additional susceptibility loci that exert significant influence on SCZ are yet to be revealed. So, in order to identify novel susceptibility genes that account for the genetic risk of SCZ, we performed a systematic family-based study using whole exome sequencing (WES) in 65 Han Chinese families. The analysis of 51 SCZ trios with both unaffected parents identified 22 exonic and 1 splice-site de novo mutations (DNMs) on a total of 23 genes, and showed that 12 genes carried rare protein-altering compound heterozygous mutations in more than one trio. In addition, we identified 26 exonic or splice-site single nucleotide polymorphisms (SNPs) on 18 genes with nominal significance (P < 5 × 10−4) using a transmission disequilibrium test (TDT) in all the families. Moreover, TDT result confirmed a SCZ susceptibility locus on 3p21.1, encompassing the multigenetic region NEK4-ITIH1-ITIH3-ITIH4. Through several different strategies to predict the potential pathogenic genes in silico, we revealed 4 previous discovered susceptibility genes (TSNARE1, PBRM1, STAB1 and OLIG2) and 4 novel susceptibility loci (PSEN1, TLR5, MGAT5B and SSPO) in Han Chinese SCZ patients. In summary, we identified a list of putative candidate genes for SCZ using a family-based WES approach, thus improving our understanding of the pathology of SCZ and providing critical clues to future functional validation.

17-Beta Hydroxysteroid Dehydrogenase 13Is a Hepatic Retinol Dehydrogenase Associated With Histological Features of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. A single-nucleotide polymorphism (SNP), rs6834314, was associated with serum liver enzymes in the general population, presumably reflecting liver fat or injury. We studied rs6834314 and its nearest gene, 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13), to identify associations with histological features of NAFLD and to characterize the functional role of HSD17B13 in NAFLD pathogenesis. The minor allele of rs6834314 was significantly associated with increased steatosis but decreased inflammation, ballooning, Mallory-Denk bodies, and liver enzyme levels in 768 adult Caucasians with biopsy-proven NAFLD and with cirrhosis in the general population. We found two plausible causative variants in the HSD17B13 gene. rs72613567, a splice-site SNP in high linkage with rs6834314 (r2 = 0.94) generates splice variants and shows a similar pattern of association with NAFLD histology. Its minor allele generates simultaneous expression of exon 6-skipping and G-nucleotide insertion variants. Another SNP, rs62305723 (encoding a P260S mutation), is significantly associated with decreased ballooning and inflammation. Hepatic expression of HSD17B13 is 5.9-fold higher (P=0.003) in patients with NAFLD. HSD17B13 is targeted to lipid droplets, requiring the conserved amino acid 22-28 sequence and amino acid 71-106 region. The protein has retinol dehydrogenase (RDH) activity, with enzymatic activity dependent on lipid droplet targeting and cofactor binding site. The exon 6 deletion, G insertion, and naturally occurring P260S mutation all confer loss of enzymatic activity. Conclusion: We demonstrate the association of variants in HSD17B13 with specific features of NAFLD histology and identify the enzyme as a lipid droplet-associated RDH; our data suggest that HSD17B13 plays a role in NAFLD through its enzymatic activity.

Mutation analysis of BRCA1/2 mutations with special reference to polymorphic SNPs in Indian breast cancer patients.

BackgroundGermline mutations BRCA1 and BRCA2 contribute almost equally in the causation of breast cancer (BC). The type of mutations in the Indian population that cause this condition is largely unknown.PurposeIn this cohort, 79 randomized BC patients were screened for various types of BRCA1 and BRCA2 mutations including frameshift, nonsense, missense, in-frame and splice site types.Materials and methodsThe purified extracted DNA of each referral patient was subjected to Sanger gene sequencing using Codon Code Analyzer and Mutation Surveyor and next-generation sequencing (NGS) methods with Ion torrent software, after appropriate care.ResultsThe data revealed that 35 cases were positive for BRCA1 or BRCA2 (35/79: 44.3%). BRCA2 mutations were higher (52.4%) than BRCA1 mutations (47.6%). Five novel mutations detected in this study were p.pro163 frameshift, p.asn997 frameshift, p.ser148 frameshift and two splice site single-nucleotide polymorphisms (SNPs). Additionally, four nonsense and one in-frame deletion were identified, which all seemed to be pathogenic. Polymorphic SNPs contributed the highest percentage of mutations (72/82: 87.8%) and contributed to pathogenic, likely pathogenic, likely benign, benign and variant of unknown significance (VUS). Young age groups (20–60 years) had a high frequency of germline mutations (62/82;75.6%) in the Indian population.ConclusionThis study suggested that polymorphic SNPs contributed a high percentage of mutations along with five novel types. Younger age groups are prone to having BC with a higher mutational rate. Furthermore, the SNPs detected in exons 10, 11 and 16 of BRCA1 and BRCA2 were higher than those in other exons 2, 3 and 9 polymorphic sites in two germline genes. These may be contributory for BC although missense types are known to be susceptible for cancer depending on the type of amino acid replaced in the protein and associated with pathologic events. Accordingly, appropriate counseling and treatment may be suggested.

A Loss-of-Function Variant in a Minor Isoform of ANK3 Protects Against Bipolar Disorder and Schizophrenia

BackgroundAnkyrin-3 (ANK3) was one of the first genes to reach significance in a bipolar disorder genome-wide association study. Many subsequent association studies confirmed this finding and implicated this gene in schizophrenia. However, the exact nature of the role of ANK3 in the pathophysiology remains elusive. In particular, the specific isoforms involved and the nature of the imbalance are unknown. MethodsWe genotyped a Norwegian sample of 402 patients with bipolar disorder, 293 patients with schizophrenia, and 330 healthy control subjects genome-wide with the Illumina Human Exome BeadChip. We performed allelic association tests at the genome-wide and gene levels and found a significantly associated single nucleotide polymorphism in a splice site of ANK3. We replicated this finding in two other samples and studied the functional effect of this single nucleotide polymorphism by performing quantitative polymerase chain reaction on the affected exon junction in complementary DNA from blood total RNA. ResultsThe splice site single nucleotide polymorphism (rs41283526) is located in an alternatively spliced exon of ANK3 and has a strong and significant protective effect against bipolar disorder (odds ratio = .31) and schizophrenia (odds ratio = .21). The minor allele of rs41283526 is a loss-of-function variant that disables the correct splicing of the transcript. Data from the BrainSpan human developmental transcriptome show that the exon bearing this variant is expressed only in a minor isoform of ANK3, the transcription of which is initiated in early adolescence. ConclusionsOur results suggest that an elevated expression of this transcript starting in adolescence may be an important factor in the pathophysiology of schizophrenia and bipolar disorder.

Context-dependent robustness to 5′ splice site polymorphisms in human populations

There has been growing evidence for extensive diversity of alternative splicing in human populations. Genetic variants within the 5' splice site can cause splicing differences among human individuals and constitute an important class of human disease mutations. In this study, we explored whether natural variations of splicing could reveal important signals of 5' splice site recognition. In seven lymphoblastoid cell lines of Asian, European and African ancestry, we identified 1174 single nucleotide polymorphisms (SNPs) within the consensus 5' splice site. We selected 129 SNPs predicted to significantly alter the splice site activity, and quantitatively examined their splicing impact in the seven individuals. Surprisingly, outside of the essential GT dinucleotide position, only ∼14% of the tested SNPs altered splicing. Bioinformatic and minigene analyses identified signals that could modify the impact of 5' splice site polymorphisms, most notably a strong 3' splice site and the presence of intronic motifs downstream of the 5' splice site. Strikingly, we found that the poly-G run, a known intronic splicing enhancer, was the most significantly enriched motif downstream of exons unaffected by 5' splice site SNPs. In TRIM62, the upstream 3' splice site and downstream intronic poly-G runs functioned redundantly to protect an exon from its 5' splice site polymorphism. Collectively, our study reveals widespread context-dependent robustness to 5' splice site polymorphisms in human transcriptomes. Consequently, certain exons are more susceptible to 5' splice site mutations. Additionally, our work demonstrates that genetic diversity of alternative splicing can provide significant insights into the splicing code of mammalian cells.

LACK OF REPLICATION OF ASSOCIATION BETWEEN SCN1A SNP AND FEBRILE SEIZURES

Febrile seizures (FS) affect 3% of children aged 6 months to 6 years and have a strong genetic component with recurrence risk ratios of 3–5 in first-degree relatives.1 In rare families with autosomal dominant FS, mutations in the sodium channel alpha 1 subunit gene, SCN1A , have been identified.2,3 However, FS usually show complex inheritance with a polygenic basis. The search for susceptibility variants has been slow, so the recent report of a common splice site single nucleotide polymorphism (SNP) in SCN1A (IVS5N + 5 G>A, rs3812718) is notable. Two patient groups were studied: the first, adults with focal epilepsy with and without a history of FS (n = 90 and 486 respectively); the second, children with FS (n = 144). Patients homozygous for the A allele were reported to have a genotype relative risk of ∼3.0 for FS.4 The rs3812718 SNP is a plausible candidate for FS as it influences relative expression of neonatal and adult transcripts of SCN1A , which plays a key role in membrane excitability.5 To confirm the association between rs3812718 and FS, independent replication studies are vital.6 We tested the primary hypothesis of the original study, that an AA genotype at this SCN1A polymorphism confers increased risk of FS. For this, we recruited 558 unrelated Australian (predominantly Caucasian) patients from epilepsy clinics at 2 tertiary hospitals in Melbourne. The criteria for a diagnosis of FS were identical to that of the original study. Patients with an afebrile seizure history prior to the onset of FS were excluded. The genotyping methodology has been described previously.7 Approval was obtained from the Human Research Ethics Committees of Austin and Melbourne Health. The cohort of 558 patients comprised 76 (14%) cases with and 482 (86%) without a history of FS. A total of …

A splice site single nucleotide polymorphism of the fatty acid binding protein 4 gene appears to be associated with intramuscular fat deposition in longissimus muscle in Australian cattle

Fatty acid binding protein 4 (FABP4) is a candidate gene affecting fatness traits of mammals. However, its association with fatness traits in cattle and other livestock species is not consistent from one study to another. Here, we sequenced the coding sequence of FABP4 looking for non-synonymous variants. We identified a splice site mutation between the third exon and the third intron of bovine FABP4. We genotyped this SNP, FABP4:g.2502C>G, in 1409 cattle with intramuscular fat measurements from seven breeds. The average allele frequency of the C allele was 0.66 with a range of 0.45 to 0.85. A regression on the number of G alleles shows a statistically significant effect of alpha = 0.11, P = 0.044. This appears to confirm an association between IMF and variation at FABP4, with an effect of 0.3% of the variation in our sample when using this SNP.

No Evidence for Association of OAS1 With Type 1 Diabetes in Unaffected Siblings or Type 1 Diabetic Cases

Type 1 diabetes is a common autoimmune disorder that is strongly clustered in families. As the sharing of alleles of the HLA class II genes cannot explain all of this aggregation, alleles of multiple other loci are involved. Recently, it was reported that an A/G splice-site single nucleotide polymorphism (SNP; rs10774671) in the OAS1 gene, encoding 2'5'-oligoadenylate synthetase, was associated with a protective effect against type 1 diabetes in unaffected siblings, and yet affected siblings showed random transmission. Since this finding is difficult to explain biologically, we genotyped the OAS1 SNP in 1,552 type 1 diabetic families from the U.K., U.S., Romania, and Norway and in 4,287 type 1 diabetic cases and 4,735 control subjects from the U.K. We found no evidence of association in either unaffected (relative risk 1.00; P = 0.94) or affected (1.00; P = 0.96) siblings or in the case-control study (odds ratio 0.99; P = 0.83). These results suggest that additional evidence of association of a low penetrance effect in common disease should be sought when the primary result comes from unaffected siblings in the absence of any effect in cases.

OAS1 Splice Site Polymorphism Controlling Antiviral Enzyme Activity Influences Susceptibility to Type 1 Diabetes

Both genetic and nongenetic factors contribute to the development of type 1 diabetes. Many investigations, including prospective studies of high-risk children, have implicated virus infections as predisposing environmental agents. We previously reported that basal activity of the key antiviral enzyme 2'5'-oligoadenylate synthetase (2'5'AS) was significantly elevated in type 1 diabetic patients compared with healthy control subjects. Recently, we showed that an A/G splice site single nucleotide polymorphism (SNP) in the OAS1 gene encoding 2'5'AS is strongly associated with basal 2'5'AS activity. Basal enzyme activity was highest in individuals with GG genotype and lowest in those with AA genotype. In the present study, we genotyped 835 type 1 diabetic and 401 healthy siblings at the OAS1 splice site polymorphism and (for comparison) at an A/C SNP of the insulin (IDDM2) locus. Results showed that OAS1 GG and GA were significantly increased in diabetic compared with healthy siblings (P = 0.0023). The strength of association was similar to that at IDDM2, where, as expected, the C/C (variable number tandem repeat class I homozygote) genotype was increased in affected compared with healthy siblings (P = 0.0025). The results suggest that host genetic response to virus infection could influence susceptibility to type 1 diabetes.

Variation in GRM3 affects cognition, prefrontal glutamate, and risk for schizophrenia.

GRM3, a metabotropic glutamate receptor-modulating synaptic glutamate, is a promising schizophrenia candidate gene. In a family-based association study, a common GRM3 haplotype was strongly associated with schizophrenia (P = 0.0001). Within this haplotype, the A allele of single-nucleotide polymorphism (SNP) 4 (hCV11245618) in intron 2 was slightly overtransmitted to probands (P = 0.02). We studied the effects of this SNP on neurobiological traits related to risk for schizophrenia and glutamate neurotransmission. The SNP4 A allele was associated with poorer performance on several cognitive tests of prefrontal and hippocampal function. The physiological basis of this effect was assessed with functional MRI, which showed relatively deleterious activation patterns in both cortical regions in control subjects homozygous for the SNP4 A allele. We next looked at SNP4's effects on two indirect measures of prefrontal glutamate neurotransmission. Prefrontal N-acetylaspartate, an in vivo MRI measure related to synaptic activity and closely correlated with tissue glutamate, was lower in SNP4 AA homozygotes. In postmortem human prefrontal cortex, AA homozygotes had lower mRNA levels of the glial glutamate transporter EAAT2, a protein regulated by GRM3 that critically modulates synaptic glutamate. Effects of SNP4 on prefrontal GRM3 mRNA and protein levels were marginal. Resequencing revealed no missense or splice-site SNPs, suggesting that the intronic SNP4 or related haplotypes may exert subtle regulatory effects on GRM3 transcription. These convergent data point to a specific molecular pathway by which GRM3 genotype alters glutamate neurotransmission, prefrontal and hippocampal physiology and cognition, and thereby increased risk for schizophrenia.