Rashmi B. Prasad

Variation in CDKN2A at 9p21.3 influences childhood acute lymphoblastic leukemia risk

Using data from a genome-wide association study of 907 individuals with childhood acute lymphoblastic leukemia (cases) and 2,398 controls and with validation in samples totaling 2,386 cases and 2,419 controls, we have shown that common variation at 9p21.3 (rs3731217, intron 1 of CDKN2A) influences acute lymphoblastic leukemia risk (odds ratio = 0.71, P = 3.01 × 10−11), irrespective of cell lineage.

Global genomic and transcriptomic analysis of human pancreatic islets reveals novel genes influencing glucose metabolism

Significance We provide a comprehensive catalog of novel genetic variants influencing gene expression and metabolic phenotypes in human pancreatic islets. The data also show that the path from genetic variation (SNP) to gene expression is more complex than hitherto often assumed, and that we need to consider that genetic variation can also influence function of a gene by influencing exon usage or splice isoforms (sQTL), allelic imbalance, RNA editing, and expression of noncoding RNAs, which in turn can influence expression of target genes. Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5′-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.

Genetics of type 2 diabetes-pitfalls and possibilities.

Type 2 diabetes (T2D) is a complex disease that is caused by a complex interplay between genetic, epigenetic and environmental factors. While the major environmental factors, diet and activity level, are well known, identification of the genetic factors has been a challenge. However, recent years have seen an explosion of genetic variants in risk and protection of T2D due to the technical development that has allowed genome-wide association studies and next-generation sequencing. Today, more than 120 variants have been convincingly replicated for association with T2D and many more with diabetes-related traits. Still, these variants only explain a small proportion of the total heritability of T2D. In this review, we address the possibilities to elucidate the genetic landscape of T2D as well as discuss pitfalls with current strategies to identify the elusive unknown heritability including the possibility that our definition of diabetes and its subgroups is imprecise and thereby makes the identification of genetic causes difficult.

Verification of the susceptibility loci on 7p12.2, 10q21.2, and 14q11.2 in precursor B-cell acute lymphoblastic leukemia of childhood

Recent genome-wide association data have implicated genetic variation at 7p12.2 (IKZF1), 10q21.2 (ARIDB5), and 14q11.2 (CEBPE) in the etiology of B-cell childhood acute lymphoblastic leukemia (ALL). To verify and further examine the relationship between these variants and ALL risk, we genotyped 1384 cases of precursor B-cell childhood ALL and 1877 controls from Germany and the United Kingdom. The combined data provided statistically significant support for an association between genotype at each of these loci and ALL risk; odds ratios (OR), 1.69 (P = 7.51 x10(-22)), 1.80 (P = 5.90 x 10(-28)), and 1.27 (P = 4.90 x 10(-6)), respectively. Furthermore, the risk of ALL increases with an increasing numbers of variant alleles for the 3 loci (OR(per-allele) = 1.53, 95% confidence interval, 1.44-1.62; P(trend) = 3.49 x 10(-42)), consistent with a polygenic model of disease susceptibility. These data provide unambiguous evidence for the role of these variants in defining ALL risk underscoring approximately 64% of cases.

Novel subgroups of adult-onset diabetes and their association with outcomes: a data-driven cluster analysis of six variables

BACKGROUND Diabetes is presently classified into two main forms, type 1 and type 2 diabetes, but type 2 diabetes in particular is highly heterogeneous. A refined classification could provide a powerful tool to individualise treatment regimens and identify individuals with increased risk of complications at diagnosis. METHODS We did data-driven cluster analysis (k-means and hierarchical clustering) in patients with newly diagnosed diabetes (n=8980) from the Swedish All New Diabetics in Scania cohort. Clusters were based on six variables (glutamate decarboxylase antibodies, age at diagnosis, BMI, HbA1c, and homoeostatic model assessment 2 estimates of β-cell function and insulin resistance), and were related to prospective data from patient records on development of complications and prescription of medication. Replication was done in three independent cohorts: the Scania Diabetes Registry (n=1466), All New Diabetics in Uppsala (n=844), and Diabetes Registry Vaasa (n=3485). Cox regression and logistic regression were used to compare time to medication, time to reaching the treatment goal, and risk of diabetic complications and genetic associations. FINDINGS We identified five replicable clusters of patients with diabetes, which had significantly different patient characteristics and risk of diabetic complications. In particular, individuals in cluster 3 (most resistant to insulin) had significantly higher risk of diabetic kidney disease than individuals in clusters 4 and 5, but had been prescribed similar diabetes treatment. Cluster 2 (insulin deficient) had the highest risk of retinopathy. In support of the clustering, genetic associations in the clusters differed from those seen in traditional type 2 diabetes. INTERPRETATION We stratified patients into five subgroups with differing disease progression and risk of diabetic complications. This new substratification might eventually help to tailor and target early treatment to patients who would benefit most, thereby representing a first step towards precision medicine in diabetes. FUNDING Swedish Research Council, European Research Council, Vinnova, Academy of Finland, Novo Nordisk Foundation, Scania University Hospital, Sigrid Juselius Foundation, Innovative Medicines Initiative 2 Joint Undertaking, Vasa Hospital district, Jakobstadsnejden Heart Foundation, Folkhälsan Research Foundation, Ollqvist Foundation, and Swedish Foundation for Strategic Research.

MTHFR and MTRR genotype and haplotype analysis and colorectal cancer susceptibility in a case-control study from the Czech Republic

Polymorphic variants in genes involved in one-carbon metabolism, in particular of dietary folate, may modulate the risk for colorectal cancer through aberrant DNA-methylation and altered nucleotide synthesis and repair. In the present study, we have assessed the association of six polymorphisms and relative haplotypes in the MTHFR gene (rs1801133 and rs1801131) and in the MTRR gene (rs1801394, rs1532268, rs162036, and rs10380) with the risk for colorectal cancer in 666 patients and 1377 controls from the Czech Republic. We found that the 677 C>T polymorphism in the MTHFR gene significantly decreased the risk for colorectal cancer in homozygous carriers of the variant allele (OR, 0.58; 95% CI, 0.39-0.87). Also, we noted a significantly different distribution of genotypes between cases and controls for the 66A>G polymorphism in the MTRR gene. In particular, homozygous carriers of the G-containing allele of this polymorphism were at an increased risk for colorectal cancer (OR, 1.39; 95% CI, 1.04-1.85). Haplotype analysis of the two MTHFR polymorphisms showed a moderate difference in the distribution of the TA haplotype between cases and controls. In comparison to the most common haplotype (CA), the TA haplotype was associated with a decreased risk for colorectal cancer (OR, 0.84; 95% CI, 0.71-0.99). No difference in the distribution between cases and controls was observed for the haplotypes based on the four polymorphisms in the MTRR gene. The present study suggests that the 677TT genotype and the TA haplotype in the MTHFR gene may also have a role in colorectal cancer risk in the Czech population, indicating the importance of genes involved in folate metabolism with respect to cancer risk. For MTRR, additional studies on larger populations are needed to clarify the possible role of variation in this gene in colorectal carcinogenesis.

5-Fluorouracil-based chemotherapy for colorectal cancer and MTHFR/MTRR genotypes.

Treatment with 5-fluorouracil (5-FU) is known to improve survival in various cancers. The largest impact of the drug has been reported in colorectal cancer [1]. Active metabolites of 5-FU disrupt both DNA and RNA synthesis through a mechanism involving the folate metabolic pathway [2–4]. The overall response rate to 5-FU in advanced colorectal cancer is limited to 10–15%. Although survival is reported to improve after the addition of irinotecan and oxaliplatin to the 5-FU regimen, toxicity increases [5, 6]. Crucially, response to chemotherapeutics, overall survival (OS) and attendant toxicity is associated with large inter-individual variations [7]. Elevated concentrations of 5,10-methylenetetrahydrofolate (CH2THF), which are critical for the optimal effect of 5-FU, are dependent on methylenetetrahydrofolate reductase (MTHFR), an enzyme that reduces it irreversibly to 5-methyltetrahydrofolate. A link between an improved response to 5-FU treatment and two common polymorphisms in the MTHFR gene, associated with reduced enzymatic activity, has been discerned through in vitro experiments. While an increased sensitivity to the drug was observed in colon and breast cancer cell lines transfected with the variant 677T MTHFR cDNA, 19 different cell lines with the 677 C > T and 1298 A > C variants displayed increased folate concentrations and increased 5-FU efficacy, respectively [8, 9]. The MTHFR activity in tissue is presumed to be a major determinant of clinical response but the pharmacogenetic studies on the effect of the two polymorphisms have not always been consistent [10]. A recent study carried out on 117 colorectal cancer patients showed specific linkage of response to FOLFOX therapy with the two MTHFR polymorphisms [11]. We investigated the role of six genetic variants in the MTHFR and 5-methylenetetrahydrofolate-homocysteine methyltransferase reductase (MTRR) genes on the clinical outcome in 273 colorectal cancer patients who were administered a 5-FU based regimen as first-line post-operative therapy. The patients were treated by an intended curative surgery followed by a 5-FU based adjuvant therapy regimen. The adjuvant therapy consisted of either a Mayo regimen, delivered as a bolus infusion of 5-FU (425 mg m−2) and leucovorin (10 mg m−2) for 5 days every 4 weeks six times or a simplified DeGramond regimen which consisted of a 2 h intravenous (i.v.) infusion of leucovorin (200 mg m−2), then a 5-FU i.v. bolus (400 mg m−2) followed by a 46 h 5-FU continuous i.v. infusion (2400–3000 mg m−2). The patients were diagnosed between September 2004 and August 2009 in the Czech Republic, a country with one of the highest incidence rates, and belonged to a large cohort being investigated for the effect of genetic variability on the disease. The study design was approved by the Ethics Committee of the Institute of Experimental Medicine, Prague, Czech Republic. In this study the outcome variables measured were tumour stage, OS (time from operation till death or censorship) and progression-free survival (PFS, time from operation till progression, death or censorship). The genotyping was carried out using allelic discrimination method and validated by random re-genotyping and direct DNA sequencing. The median survival time of deceased patients was 21 months. Five established prognostic factors (pT, pN and pM, tumour grading and histological type) associated with survival were included as covariates in subsequent Cox regression analyses for assessing the relationship between the polymorphisms and clinical outcome. Patient characteristics like age at diagnosis, duration of therapy, family history and diabetes (self-reported) did not affect OS (probability values higher than 0.13). Out of six polymorphisms investigated, the variant 1298 A > C in MTHFR showed association with PFS. The patients with AC (n = 102) and CC (n = 22) genotypes showed an increased PFS with an adjusted hazard ratio (HR) of 0.52 (95% CI 0.30–0.89) compared with patients with the AA genotype (n = 93). None of the other investigated variants including the 66 A > G, 524 C > T, 1049 A > G and 1793 C > T polymorphisms in the MTRR and the 677 C > T in the MTHFR gene showed any association. Clinical studies on genetic variation in the folate metabolism pathway and colorectal cancer survival have mainly focused on patients undergoing treatment for metastatic colorectal cancer and patients who were administered adjuvant therapy before or after surgery. Critically, most of the reported studies were conducted in a small number of patients (less than 150) and very often the treatment regime consisted of 5-FU and additional anti-neoplastic drugs. In addition to treatment homogeneity, this study, together with two earlier investigations, comprises one of the largest patient groups. While the study by Afzal et al. did not find any modulation in survival due to polymorphisms in patients treated with 5-FU; the study by Boige et al. showed a predictive effect due to the MTHFR 1298 A > C polymorphism in patients receiving oxaliplatin following LV5FU2 [2, 6]. Thus, data from our study coupled with those reported earlier, in particular the one by Etienne-Grimaldi et al. augment an argument for the further investigation of the role of variants in genes involved in folate metabolism, MTHFR in particular, on the effect of 5-FU based chemotherapeutics for treatment of colorectal cancer [11]. 5-FU represents an early success in the relentless endeavours directed towards treatment of cancers and has remained a mainstay treatment. Identification of markers for predicting individual response will be another step towards personalized medicine in the genomic era.

Prevalence and risk factors of gestational diabetes in Punjab, North India: results from a population screening program

OBJECTIVE The World Health Organization (WHO) has in 2013 changed the diagnostic criteria for gestational diabetes mellitus (GDM) to acknowledge the putative effect of mildly elevated fasting plasma glucose (FPG) levels on pregnancy outcomes. We aimed to determine the prevalence and risk factors of GDM comparing the previous WHO 1999 criteria to the WHO 2013 criteria in North India. METHODS In a population-based screening programme, 5100 randomly selected North Indian women were studied using a cross-sectional design with a questionnaire, venous FPG and 2-h capillary plasma glucose (PG) after a 75 g oral glucose tolerance test performed between 24 and 28 weeks of pregnancy. RESULTS The prevalence of GDM was 35% using WHO 2013 criteria vs 9% using WHO 1999 criteria. FPG measurements identified 94% of WHO 2013 GDM cases as opposed to 11% of WHO 1999 GDM cases. In contrast, 2-h PG measurements identified only 13% of WHO 2013 GDM cases compared with 96% of the WHO 1999 GDM cases. Using logistic regression with backward elimination, urban habitat, illiteracy, non-vegetarianism, increased BMI, Hindu religion and low adult height were all independent risk factors of GDM using the 1999 criteria, whereas only urban habitat, low adult height and increased age were independent risk factors of GDM using the 2013 criteria. CONCLUSIONS Intervention studies are needed to justify the WHO 2013 GDM criteria increasing the prevalence four fold to include more than one third of North Indian pregnant women.

Excess maternal transmission of variants in the THADA gene to offspring with type 2 diabetes

Aims/hypothesisGenome-wide association studies (GWAS) have identified more than 65 genetic loci associated with risk of type 2 diabetes. However, the contribution of distorted parental transmission of alleles to risk of type 2 diabetes has been mostly unexplored. Our goal was therefore to search for parent-of-origin effects (POE) among type 2 diabetes loci in families.MethodsFamilies from the Botnia study (n = 4,211, 1,083 families) were genotyped for 72 single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes and assessed for POE on type 2 diabetes. The family-based Hungarian Transdanubian Biobank (HTB) (n = 1,463, >135 families) was used to replicate SNPs showing POE. Association of type 2 diabetes loci within families was also tested.ResultsThree loci showed nominal POE, including the previously reported variants in KCNQ1, for type 2 diabetes in families from Botnia (rs2237895: pPOE = 0.037), which can be considered positive controls. The strongest POE was seen for rs7578597 SNP in the THADA gene, showing excess transmission of the maternal risk allele T to diabetic offspring (Botnia: pPOE = 0.01; HTB pPOE = 0.045). These data are consistent with previous evidence of allelic imbalance for expression in islets, suggesting that the THADA gene can be imprinted in a POE-specific fashion. Five CpG sites, including those flanking rs7578597, showed differential methylation between diabetic and non-diabetic donor islets.Conclusions/interpretationTaken together, the data emphasise the need for genetic studies to consider from which parent an offspring has inherited a susceptibility allele.

MECHANISMS IN ENDOCRINOLOGY: Epigenetic modifications and gestational diabetes: a systematic review of published literature

OBJECTIVE To summarize the current knowledge on epigenetic alterations in mother and offspring subjected to gestational diabetes (GDM) and indicate future topics for research. DESIGN Systematic review. METHODS We performed extensive searches in PubMed, EMBASE and Google scholar, using a combination of the search terms: GDM, gestational diabetes, epigenetic(s), methylation, histone modification, histone methylation, histone acetylation, microRNA and miRNA. Studies that compared women diagnosed with GDM and healthy controls were included. Two authors independently scanned the abstracts, and all included papers were read by at least two authors. The searches were completed on October 31st, 2016. RESULTS We identified 236 articles, of which 43 were considered relevant for this systematic review. Studies published showed that epigenetic alterations could be found in both mothers with GDM and their offspring. However, differences in methodology, diagnostic criteria for GDM and populations studied, together with a limited number of published studies and small sample sizes, preclude clear conclusions about the role of epigenetic modifications in transmitting risk from GDM mothers to their offspring. CONCLUSION The current research literature suggests that GDM may have impact on epigenetic modifications in the mother and offspring. However, larger studies that include multiple cohorts of GDM patients and their offspring are needed.