GC Variants Research Articles

Personalized assessment of the effectiveness of targeted drugs for the treatment of cystic fibrosis in a patient with c.264_268delATATT and c.3139+1G>C rare variants of the <i>CFTR</i> gene

BACKGROUND: When new, most often rare CFTR gene variants, are detected in patients with cystic fibrosis, intestinal organoids are used, which allows for the assessment of the residual functional activity of the CFTR channel and the effect of targeted drugs to determine the possibility of further pathogenetic therapy. Clinical trials of the effectiveness of the targeted drugs in patients with rare CFTR variants are expensive, and patient groups are extremely small. A forskolin-induced swelling assay on a patient’s intestinal organoids allows for a personalized approach when studying rare or even single CFTR variants. AIM: To examine the effect of the CFTR potentiator ivacaftor and the combination of ivacaftor with the CFTR correctors lumacaftor, tezacaftor, and elexacaftor on the restoration of CFTR channel functions on a culture of intestinal organoids obtained from a patient with two rare CFTR variants c.264_268delATATT and c.3139+1GC. MATERIALS AND METHODS: To assess the activity of the CFTR channel, the forskolin-induced swelling assay on organoids and intestinal current measurements on rectal biopsy samples method were used. The clinical picture of a patient with the c.264_268delATATT/c.3139+1GC genotype was described. RESULTS: In vitro, the genetic variants c.264_268delATATT and c.3139+1GC lead to a complete loss of the functional CFTR protein, whereas CFTR modulators do not positively affect and do not lead to the restoration of CFTR function, in contrast from F508del/F508del control. The disease severity in a child is consistent with the results of functional tests. CONCLUSION: Both CFTR variants are classified as “severe” and cause a complete loss of chloride channel activity. No effective CFTR modulators were found for the c.264_268delATATT and c.3139+1GC variants; thus, targeted therapy cannot be recommended to the patient.

Association between Interleukin-6 Gene Polymorphism (rs1800795 and rs1800796) and Type 2 Diabetes Mellitus in a Ghanaian Population: A Case-Control Study in the Ho Municipality.

There is no conclusive evidence on the association between interleukin- (IL-) 6 gene polymorphism and type 2 diabetes mellitus (type 2 DM). Thus, this study is aimed at evaluating the role of rs1800795 and rs1800796 polymorphisms in the pathogenesis of type 2 DM among Ghanaians in the Ho Municipality. We recruited into this hospital-based case-control study 174 patients with type 2 DM (75 DM alone and 99 with DM+HTN) and 149 healthy individuals between 2018 and 2020. Demographic, lifestyle, clinical, anthropometric, and haemodynamic variables were obtained. Fasting blood samples were collected for haematological, biochemical, and molecular analyses. Genomic DNA was extracted, amplified using Tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) technique, and genotyped for IL-6 gene polymorphism. Logistic regression analyses were performed to assess the association between IL-6 gene polymorphism and type 2 DM. The minor allele frequency (MAF) of the rs1800795 and rs1800796 polymorphisms was higher in DM alone (57.5%, 62.0%) and DM with HTN groups (58.3%, 65.3%) than controls (33.1%, 20.0%). Carriers of the rs1800795GC genotype (aOR = 2.35, 95% CI: 1.13-4.90, p = 0.022) and mutant C allele (aOR = 2.41, 95% CI: 1.16-5.00, p = 0.019) as well as those who carried the rs1800796GC (aOR = 8.67, 95% CI: 4.00-18.90, p < 0.001) and mutant C allele (aOR = 8.84, 95% CI: 4.06-19.26, p = 0.001) had increased odds of type 2 DM. For both polymorphisms, carriers of the GC genotype had comparable levels of insulin, HOMA-IR, and fasting blood glucose (FBG) with those who carried the GG genotype. IL-6 levels were higher among carriers of the rs1800796GC variant compared to carriers of the rs1800796GG variant (p = 0.023). The rs1800796 polymorphism, dietary sugar intake, and exercise status, respectively, explained approximately 3% (p = 0.046), 3.2% (p = 0.038, coefficient = 1.456), and 6.2% (p = 0.004, coefficient = -2.754) of the variability in IL-6 levels, suggesting weak effect sizes. The GC genotype and mutant C allele are risk genetic variants associated with type 2 DM in the Ghanaian population. The rs1800796 GC variant, dietary sugar intake, and exercise status appear to contribute significantly to the variations in circulating IL-6 levels but with weak effect sizes.

Screening of Type 2 Diabetes Patients of Khyber Pakhtunkhwa for CDKAL1 Variant (rs10946398)

Diabetes Mellitus Type 2 (T2DM) is a metabolic disorder influenced by environmental and genetic factors, with varied genetic predispositions across populations. Research on T2DM's genetic risk factors in the Pakistani community is limited. Objective: To explore the association between the CDKAL1 gene variant (rs10946398) and T2DM in the Khyber Pakhtunkhwa population in Pakistan. Methods: The study involved 100 T2DM patients and 100 controls, matched by age and gender, following specific inclusion and exclusion criteria. Sociodemographic data were collected alongside 3ml of whole blood for DNA extraction. The CDKAL1 gene was analyzed using PCR-based Sequence Restriction Fragment Length Polymorphism (RFLP), and the data were processed with SPSS version 26.0s. Results: Findings showed that 47% of cases were aged 39 to 59, with 94% having a family history of T2DM and 85% leading a sedentary lifestyle. A significant association was observed between the CDKAL1 rs10946398 variant and T2DM. The GC variant was linked to a fourfold increase in risk (OR: 4.833, CI: 1.197-19.509, P=0.027), and the CC variant had a twofold association (OR: 2.788, CI: 1.545-5.033, P=0.001). These associations persisted after adjusting for family history, age, socioeconomic factors, exercise, and diet. Conclusions: The study identified a significant correlation between the CDKAL1 rs10946398 variants and T2DM susceptibility in the Khyber Pakhtunkhwa population, emphasizing the role of genetic factors in the disease's prevalence within this community.

Genomic profiling of six human somatic histone H1 variants denotes that H1X accumulates at recently incorporated transposable elements.

Histone H1, a vital component in chromatin structure, binds to linker DNA and regulates nuclear processes. We have investigated the distribution of histone H1 variants in a breast cancer cell line using ChIP-Seq. Two major groups of variants are identified: H1.2, H1.3, H1.5and H1.0 are abundant in low GC regions (B compartment), while H1.4 and H1X preferentially localize in high GC regions (A compartment). Examining their abundance within transposable elements (TEs) reveals that H1X and H1.4 are enriched in recently-incorporated TEs (SVA and SINE-Alu), while H1.0/H1.2/H1.3/H1.5 are more abundant in older elements. Notably, H1X is particularly enriched in SVA families, while H1.4 shows the highest abundance in young AluY elements. Although low GC variants are generally enriched in LINE, LTR and DNA repeats, H1X and H1.4 are also abundant in a subset of recent LINE-L1 and LTR repeats. H1X enrichment at SVA and Alu is consistent across multiple cell lines. Further, H1X depletion leads to TE derepression, suggesting its role in maintaining TE repression. Overall, this study provides novel insights into the differential distribution of histone H1 variants among repetitive elements, highlighting the potential involvement of H1X in repressing TEs recently incorporated within the human genome.

Long-term dynamic shifts in genomic base content and evolutionary trajectories of SARS-CoV-2 variants.

The rapid spread and remarkable mutations of SARS-CoV-2 variants, particularly Omicron, necessitate an understanding of their evolutionary characteristics. In this study, we analyzed representative high-quality whole-genome sequences of 2008 SARS-CoV-2 variants to explore long-term dynamic changes in genomic base (especially GC) content and variations during viral evolution. Our results demonstrated a highly negative correlation between GC content and variant emergence time (r = -0.765, p < 2.22e-16). Major gene partitions (S, N, ORF1ab) displayed similar trends. Omicron exhibited a significantly lower GC content than non-Omicron variants (p < 2.22e-16). Notably, we observed a robust negative correlation between C and T content (r = -0.778, p < 2.22e-16) and between G and A content (r = -0.773, p < 2.22e-16). Among all strains, Omicron showed the greatest base variation, with C->T mutations being the most frequent (median [interquartile range [IQR]]: 29 (27, 31), 37.67%), succeeded by G->A mutations (11 (9, 13), 14.63%). Over a 3-year span, an annual decline rate of 0.12% in SARS-CoV-2 GC content was observed and could become more pronounced in future emerging variants. These findings provided insights into the evolutionary trajectory of SARS-CoV-2, underscoring the significance of continuous genomic surveillance for effective prediction of and response to future variants.

The Role of Polymorphism in the Endothelial Homeostasis and Vitamin D Metabolism Genes in the Severity of Coronary Artery Disease.

Coronary artery disease (CAD) remains one of the leading causes of cardiovascular morbidity and mortality worldwide. The maintenance of endothelial homeostasis and vitamin D metabolism play an important role in CAD pathogenesis. This study aimed to determine the association of endothelial homeostasis and vitamin D metabolism gene polymorphism with CAD severity. A total of 224 low-risk patients (SYNTAX score ≤ 31) and 36 high-risk patients (SYNTAX score > 31) were recruited for this study. The serum level of E-, L- and P-selectins; endothelin; eNOS; 25OH; and 1.25-dihydroxy vitamin D was measured using an enzyme-linked immunosorbent assay (ELISA). Polymorphic variants in SELE, SELP, SELPLG, END1, NOS3, VDR and GC were analyzed using a polymerase chain reaction (PCR). We found no differences in the serum levels of the studied markers between high- and low-risk patients. Three polymorphic variants associated with CAD severity were discovered: END1 rs3087459, END1 rs5370 and GC rs2298849 in the log-additive model. Moreover, we discovered a significantly decreased serum level of 1.25-dihydroxy vitamin D in high-risk CAD patients with the A/A-A/G genotypes of the rs2228570 polymorphism of the VDR gene, the A/A genotype of the rs7041 polymorphism of the GC gene and the A/A genotype of the rs2298849 polymorphism of the GC gene.

Use of whole exome sequencing for identification of genetic variants related to Growth Hormone Deficiency and Short Stature: A Family-Based Study.

Genetic polymorphisms in genes involved in growth process and Vitamin-D metabolism form a significant etiology behind growth hormone deficiency and short stature. The aim of this study was to explore for known and unknown genes and variants related to growth hormone and short stature in a family based study using whole exome sequencing (WES). This family-based study included a family with members diagnosed with growth hormone deficiency, short stature and Vitamin-D deficiency (four boys affected and four boys non-affected). The participants were recruited from King Abdulaziz University Hospital (Jeddah, Saudi Arabia) and referred to King Fahad Centre for Medical Research (Jeddah, Saudi Arabia from April 2022 to June 2022. The consanguineous parents and one of the affected boys (aged 16 years old) underwent WES. Several variants in RNPC3, ACAN, GC, VDR and LRP2 were identified in index cases but not in controls. Novel frameshift and splice region variants in RNPC3 (c.358dupA, p.Arg120fs) were detected. Other missense variants were also observed including variants in ACAN (c.2591C>T, c.2789G>T, c.2815T>A, c.4207A>G, c.4523A>C and c.7119C>G), GC (rs4588 and rs7041) and LRP2 (rs2075252 and rs1991517). A start loss variant in VDR (rs2228570) with high impact was also observed. Our findings suggest a potential association of these variants with growth hormone deficiency and short stature. In this study, novel pathogenic variants in RNPC3 were revealed as well as other variants in ACAN and in genes related to Vitamin-D metabolism (GC, VDR and LRP2) that some or all might be associated with growth hormone deficiency. Further large-scale studies are required to address the association of these variants with growth hormone deficiency and its subsequent short stature.

Association of ACE ID, MTHFR C677T, and MIF-173GC variants with the clinical course of COVID-19 patients

The course of coronavirus disease-2019 (COVID-19) differs from person to person. The relationship between the genetic variations of the host and the course of COVID-19 has been a matter of interest. In this study, we investigated whether Angiotensin-Converting Enzyme (ACE) ID, Methylenetetrahydrofolate Reductase (MTHFR) C677T, and Macrophage Migration Inhibitory Factor (MIF)-173GC variants are risk factors for the clinical course of COVID-19 disease in Turkish patients. One hundred COVID-19 patients were included in the study. The diagnosis of COVID-19 was made using Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Chest Computed Tomography (CT). The patients were evaluated in 3 groups: intensive care, service, and outpatient treatment. ACE ID, MTHFR C677T, and MIF-173GC variants were genotyped by PCR-Restriction Fragment Length Polymorphism (RFLP) methods. When the genotype distribution between the groups was examined, it was found that the frequency of the ACE DD genotype and the D allele was higher in the intensive care group compared to the hospitalized and outpatient groups. MTHFR C677T CT genotype T allele and MIF-173GC, CC genotype C allele were more prevalent in the intensive care group compared to other groups. Patients with PCR-positive results had a higher MTHFR C677T C/C genotype and C allele. In CT-positive patients, the MTHFR C677T CT genotype and the MIF-173GC, G allele were more common. It is predicted that genetic predisposition may contribute to COVID-19 morbidity and mortality. Our results show that ACE ID, MTHFR C677T, and MIF-173GC variants affect the course of COVID-19 disease in the Turkish population.

Is acute low-dose gamma irradiation an effective elicitor for secondary metabolism in Leontopodium alpinum (Cass.) callus culture?

Leontopodium alpinum Cass. (Edelweiss) is a protected species in some European countries and has become of growing interest for exploitation by the food supplements and cosmetics industry, due to its valuable composition in bioactive and biocompatible compounds. Small scale field cultivation and in vitro biotechnological approaches are economically viable alternatives to the production via chemical synthesis. In this study, we aimed to compare two performant L. alpinum callus culture lines and to investigate the effect of acute low dose gamma irradiation on biomass accumulation (Gi), total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (DPPH) and secondary metabolites’ profile. A purple, pigmented line (PC) was obtained under lightning conditions and in the absence of light, an unpigmented greenish line (GC) was established. Both lines displayed impressive proliferative capacity after 14 days of culture, with an 18.31 ( ± 1.83) fold mass increase for PC and a 13.27 ( ± 2.42) for GC. In respect to Gi, TPC, TFC and DPPH, the purple callus line significantly outperformed the unpigmented line. According to the UPLC-HRMS analysis of the methanolic extracts, both callus lines displayed similar metabolic profiles, having similar quantities of 3,5-dicaffeoylquinic acid, leontopodic acids A and B, but 60% higher concentration of chlorogenic acid in the GC line. In the methanolic callus extracts, 9 economically important phenylpropanoid derivatives were identified. The two callus lines responded differently to low dose gamma irradiation. Although the control values measured for Gi, TPC, TFC and DPPH were significantly higher in the PC line, this line was generally negatively impacted by the gamma treatments. On the contrary, higher, but not significant, values were registered in some GC variants post irradiation with 25, 30 and 40 Gy. UPLC-HRMS investigations emphasized the overproduction of 7 compounds, post irradiation with all doses, for the PC line, while for the unpigmented line only one compound was stimulated by all irradiation treatments. The highest stimulative effect was obtained for the 35 and 40 Gy experimental variants, in the GC line, where dihydroxybenzoic acid glucoside accumulation was increased by 160%. The results of this study showed, for the first time, the different responses of two distinct L. alpinum callus lines using gamma irradiation elicitation method, which can be successfully applied to stimulate the production of certain industrially important compounds as chlorogenic acid, dicaffeoylquinic acid isomers or characteristic glucaric acids.

Building a quantitative and predictive model of 5'SS selection by human U1 snRNP using RNA-map.

Alternative splicing of pre-mRNA greatly expands the protein coding potential of our genome and accounts for extensive cell and tissue-specific variation in gene expression. Spliceosome assembly depends on the recognition of the 5′ splice site (5′SS) at the exon-intron boundary by the U1 small nuclear ribonucleoprotein (U1 snRNP). Critically, mutations in either U1 or the 5′SS can lead to human disease, including cancer. Despite decades of investigation, accurately predicting 5′SS selection and alternative splicing outcomes is difficult, suggesting new approaches are needed. Here, we present our progress in developing a quantitative and predictive model of 5′SS selection by human U1 snRNP grounded in biochemical understanding. We used the RNA on a massively parallel array (RNA-MaP) platform to measure equilibrium binding constants of reconstituted human U1 snRNP to >60,000 unique RNAs. Our designed RNA library features all possible GU and GC variants in addition to thousands of identified mutations, allowing us to build a comprehensive and predictive model of 5′SS/U1 interactions at the transcriptome scale. Overall, our results will provide a first principles understanding of 5′SS selection and will serve as a foundation for future work to systematically determine the effects of other splicing factors and splice site usage in vivo.

GC1f Vitamin D Binding Protein Isoform as a Marker of Severity in Autism Spectrum Disorders.

Autism spectrum disorders (ASD) are characterized by a wide spectrum of clinical, behavioral, and cognitive manifestations. It is, therefore, crucial to investigate possible biomarkers associated with specific ASD phenotypes. Ample literature suggests a possible role for vitamin D (VD) in influencing ASD clinical phenotypes. We analyzed three vitamin D binding protein gene (DBP) functional polymorphisms (rs2282679, rs7041, and rs4588), which are involved in the modulation of vitamin D serum concentration in 309 ASD children and 831 healthy controls. Frequency comparisons of single nucleotide polymorphisms (SNPs) alleles, genotypes, and GC isoforms (GC1f, G1s, and GC2)—generated by the combination of rs7041 and rs4588 alleles—were correlated with ASD diagnostic, behavioral, and functioning scales. The GC1f isoform was significantly more frequent in ASD compared with controls (18.6% vs. 14.5% pc = 0.02). Significantly higher scores for item 15 of the Childhood Autism Rating Scale (CARS) and lower ones for the Children’s Global Assessment Scale (CGAS) functioning scales were seen in ASD carrying the GC1f isoform. In GC phenotype analysis, a gradient of severity for overall CARS scores and CARS item 15 was observed, with scores decreasing according to the presence of GC1f-GC1f > GC1f-GC1s > GC1s-GC1s > GC1f-GC2 > GC2-GC2 isoforms. Similarly, lower CGAS scores were seen in carriers of the GC1f-GC1f isoform, whereas higher scores were present in those carrying GC2-GC2 (p = 0.028). This is the first study to evaluate possible relationships between GC variants and the different aspects of ASD in Italian ASD children. Results, although needing to be validated in ampler cohorts, suggest that the GC1f isoform could be a marker of severity in ASD that may be useful in establishing the intensity of therapeutic and rehabilitative protocols.

Haplotypes in the GC, CYP2R1 and CYP24A1 Genes and Biomarkers of Bone Mineral Metabolism in Older Adults.

Candidate gene studies have analyzed the effect of specific vitamin D pathway genes on vitamin D availability; however, it is not clear whether genetic variants also affect overall bone metabolism. This study evaluated the association between genetic polymorphisms in GC, CYP2R1 and CYP24A1 and serum levels of total 25(OH)D, iPTH and other mineral metabolism biomarkers (albumin, total calcium and phosphorus) in a sample of 273 older Spanish adults. We observed a significant difference between CYP2R1 rs10741657 codominant model and total 25(OH)D levels after adjusting them by gender (p = 0.024). In addition, the two SNPs in the GC gene (rs4588 and rs2282679) were identified significantly associated with iPTH and creatinine serum levels. In the case of phosphorus, we observed an association with GC SNPs in dominant model. We found a relationship between haplotype 2 and 25(OH)D levels, haplotype 4 and iPTH serum levels and haplotype 7 and phosphorus levels. In conclusion, genetic variants in CYP2R1 and GC could be predictive of 25(OH)D and iPTH serum levels, respectively, in older Caucasian adults. The current study confirmed the role of iPTH as one of the most sensitive biomarkers of vitamin D activity in vivo.

Splicing Outcomes of 5' Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays.

Combining data derived from a meta-analysis of human disease-associated 5′ splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15–18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.

Association of GC Variants with Bone Mineral Density and Serum VDBP Concentrations in Mexican Population.

Vitamin D-binding protein (VDBP) is encoded by the GC gene and is an active participant in the control of bone metabolism. However, the effect of its major variants on VDBP concentration and bone mineral density (BMD) remains unclear. Our aim was to analyze the effect of major GC variants on serum VDBP concentration and BMD. We recruited individuals from the Health Workers Cohort Study, which includes employees of the Mexican Institute of Social Security (IMSS). A total of 1853 adults were included. The single nucleotide polymorphisms (SNPs) rs7041 and rs4588 were genotyped to identify the three best characterized haplotypes of GC. Serum VBDP, 25(OH)D and BMD were also measured. Among women, the G allele of rs7041 was associated with higher VDBP and BMD compared to homozygous TT. The A allele of rs4588 was associated with lower VDBP and BMD compared to CC homozygous. In men, GC variants were only associated with VDBP levels. We did not observe an association between free/bioavailable 25(OH)D and BMD in men and women. Our results support an association of VDBP in bone health. The G and C alleles, from rs7041 and rs4588, respectively, are associated with high concentrations of VDBP and BMD in this sample of Mexican postmenopausal women.

Prevalence and predictors of vitamin D deficiency in young African children

BackgroundChildren living in sub-Saharan Africa have a high burden of rickets and infectious diseases, conditions that are linked to vitamin D deficiency. However, data on the vitamin D status of young African children and its environmental and genetic predictors are limited. We aimed to examine the prevalence and predictors of vitamin D deficiency in young African children.MethodsWe measured 25-hydroxyvitamin D (25(OH)D) and typed the single nucleotide polymorphisms, rs4588 and rs7041, in the GC gene encoding the vitamin D binding protein (DBP) in 4509 children aged 0–8 years living in Kenya, Uganda, Burkina Faso, The Gambia and South Africa. We evaluated associations between vitamin D status and country, age, sex, season, anthropometric indices, inflammation, malaria and DBP haplotypes in regression analyses.ResultsMedian age was 23.9 months (interquartile range [IQR] 12.3, 35.9). Prevalence of vitamin D deficiency using 25(OH)D cut-offs of < 30 nmol/L and < 50 nmol/L was 0.6% (95% CI 0.4, 0.9) and 7.8% (95% CI 7.0, 8.5), respectively. Overall median 25(OH)D level was 77.6 nmol/L (IQR 63.6, 94.2). 25(OH)D levels were lower in South Africa, in older children, during winter or the long rains, and in those with afebrile malaria, and higher in children with inflammation. 25(OH)D levels did not vary by stunting, wasting or underweight in adjusted regression models. The distribution of Gc variants was Gc1f 83.3%, Gc1s 8.5% and Gc2 8.2% overall and varied by country. Individuals carrying the Gc2 variant had lower median 25(OH)D levels (72.4 nmol/L (IQR 59.4, 86.5) than those carrying the Gc1f (77.3 nmol/L (IQR 63.5, 92.8)) or Gc1s (78.9 nmol/L (IQR 63.8, 95.5)) variants.ConclusionsApproximately 0.6% and 7.8% of young African children were vitamin D deficient as defined by 25(OH)D levels < 30 nmol/L and < 50 nmol/L, respectively. Latitude, age, season, and prevalence of inflammation and malaria should be considered in strategies to assess and manage vitamin D deficiency in young children living in Africa.

A new mechanism for a familiar mutation \u2013 bovine DGAT1 K232A modulates gene expression through multi-junction exon splice enhancement

BackgroundThe DGAT1 gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes. Since first described over 15 years ago, a protein-coding variant K232A has been assumed as the causative variant underlying these effects, following in-vitro studies that demonstrated differing levels of triglyceride synthesis between the two protein isoforms.ResultsWe used a large RNAseq dataset to re-examine the underlying mechanisms of this large milk production QTL, and hereby report novel expression-based functions of the chr14 g.1802265AA > GC variant that encodes the DGAT1 K232A substitution. Using expression QTL (eQTL) mapping, we demonstrate a highly-significant mammary eQTL for DGAT1, where the K232A mutation appears as one of the top associated variants for this effect. By conducting in vitro expression and splicing experiments in bovine mammary cell culture, we further show modulation of splicing efficiency by this mutation, likely through disruption of an exon splice enhancer as a consequence of the allele encoding the 232A variant.ConclusionsThe relative contributions of the enzymatic and transcription-based mechanisms now attributed to K232A remain unclear; however, these results suggest that transcriptional impacts contribute to the diversity of lactation effects observed at the DGAT1 locus.

Assessing vitamin D related genetic variants, status, and influence factors in pregnant women in Eastern and Central China.

Vitamin D deficiency has recently become a global public health problem. However, it is still unclear if gene polymorphisms in the vitamin D pathway influence vitamin D levels among pregnant women in Eastern and Central China. The objective of this study was to assess factors influencing vitamin D levels in pregnant women. A total of 326 participants in Shandong and Henan provinces in China were enrolled from August 2017 to April 2019. Serum 25(OH)D levels and single nucleotide polymorphisms (SNPs) in the vitamin D pathway were measured using the blood samples collected in the first trimester, second trimester, and third trimester. Data on demographics, lifestyle, and health behavior were collected using a questionnaire. Statistical analyses were performed using the R software. The prevalence of 25(OH)D deficiency was significantly more severe in pregnant women. The average 25(OH)D value of all enrolled pregnant women was 14.57 ± 7.21 ng/ml (deficiency). Only 15 (4.60%) participants had a 25(OH)D concentration ≥30 ng/ml (sufficient). The prevalence of four ranks of vitamin D levels from severe 25(OH)D deficiency to 25(OH)D sufficiency (<10, 10–20, 20–30, and ≥30 ng/ml) was 29.14%, 52.45%, 13.80%, and 4.60%, respectively. Variants of GC (rs1155563) and CYP24A1 (rs6013897) were significantly associated with both 25(OH)D concentrations and vitamin D deficiency among pregnant women, respectively. Our findings suggest that pregnant women in Eastern and Central China are at high risk of vitamin D deficiency. Genetic mutants in the vitamin D pathway (GC and CYP24A1) were significantly associated with 25(OH)D levels in pregnant women in Eastern and Central China.

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

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

Effect of genetic factors on the response to vitamin D3 supplementation in the VIDARIS randomized controlled trial.

Supplementation provides the best means of improving vitamin D status; however, individual responses vary partly owing to genetics. The aim of this study was to determine whether 28 single nucleotide polymorphisms (SNPs) in six key vitamin D pathway genes (GC, DHCR7, CYP2 R1, CYP24 A1, CYP27 B1, VDR) were associated with differences in response to supplementation. Participants (N=313; n=160 vitamin D, n=153 placebo) were part of VIDARIS (Vitamin D and Acute Respiratory Infections Study), a double-blind, randomized controlled trial involving oral monthly supplementation of either vitamin D3 (200 000 IU each for the first 2 mo, thereafter 100 000 IU monthly) or placebo for 18 mo. Circulating 25-hydroxyvitamin D (25[OH]D) concentrations at baseline and 2, 6, 12, and 18 mo, and vitamin D binding protein (Gc-globulin) and calculated free 25(OH)D concentrations at baseline and 2 mo were obtained. Multiple regression was used to model associations between genetic variants and 25(OH)D, Gc-globulin, and free 25(OH)D concentrations. SNPs within GC, CYP2 R1, and CYP27 B1 were associated with 25(OH)D concentrations following supplementation. However, only two GC gene SNPs (rs2282679, rs1155563) were significant after adjustment for multiple testing. This effect disappeared after more than 2 mo of supplementation. None of the SNPs were significantly associated with Gc-globulin concentrations; however, there was a significant interaction with one SNP in DHCR7 (rs12785878), which was associated with reduced free 25(OH)D concentrations in the supplemented arm. Only variants of GC were associated with 25(OH)D concentrations after supplementation. This effect was modest and disappeared after >2 mo of supplementation, suggesting it may be time/dose-dependent and saturable.

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

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