Association of IRS1 genetic variants with glucose control and insulin resistance in type 2 diabetic patients from Bosnia and Herzegovina

Background Previous studies reported conflicting results regarding association of insulin receptor substrate 1 (IRS1) gene variation with type 2 diabetes (T2D) and insulin resistance (IR) in different ethnic groups. We examined the association of rs7578326, rs2943641, and rs4675095 in the IRS1 gene with T2D and related traits in a population from Bosnia and Herzegovina, which is one of the European countries with the highest T2D prevalence of 12.5%. Methods Our study included 390 T2D patients and 252 control subjects. Biochemical parameters, including fasting glucose (FG), fasting insulin (FI), homeostasis model assessment insulin resistance index (HOMA-IR), and HbA1c were measured in all participants. Genotyping analysis was performed by Mass Array Sequenom iPlex platform. Results Our results demonstrated that rs7578326 and rs4675095 variants were associated with increased FG levels. The rs7578326 was also associated with higher FI, HOMA-IR (B = 0.08, 95% CI [0.01, 0.15], padd = 0.025; B = 0.079, 95% CI [0.006, 0.150], padd = 0.033, respectively) in T2D, and with HbA1c (B = 0.034, 95% CI [0.003, 0.065], pdom = 0.035) in non-drug-treated T2D. In contrast, rs2943641 C allele was associated with lower FG levels in control subjects (B = −0.17, 95% CI [−0.03, −0.002], padd = 0.030) and HbA1c (B = 0.03, 95% CI [0.002, 0.06], pdom = 0.040) in non-drug-treated T2D. Conclusions We report the association between common variants in IRS1 gene with insulin resistance, glucose, and HbA1c levels in Bosnia and Herzegovina’s population.

Absence of kdr resistance alleles in the Union of the Comoros, East Africa

Knockdown resistance (kdr) and CYP9K1 genotypes were detected by a MOLDI-TOF based SNP genotyping assay (Sequenom iPLEX) in samples of Anopheles gambiae collected at 13 sites throughout the Union of the Comoros and Dar es Salaam, Tanzania during February and March 2011. All A. gambiae specimens collected in the Comoros were homozygous for the susceptible kdr alleles (+/+) while 96% of A. gambiae from Dar es Salaam were homozygous for the East African kdr resistant genotype (E/E). In contrast, all specimens from Dar es Salaam and the Comoros were homozygous for the cyp3 allele (c3/c3) at the CYP9K1 locus; the locus has been implicated in metabolic resistance against pyrethroid insecticides in West Africa. All specimens had typical A. gambiae genotypes for SNPs within the divergence Islands on all three chromosomes. Although further spatial and temporal studies are needed, the distribution of kdr genotypes between the Comoros and Tanzania further supports isolation of the Comoros populations from A. gambiae populations on mainland Africa.

Genetic variation in immunomodulatory genes as markers of melanoma recurrence-free and overall survival.

9021 Background: Small reported studies have provided some evidence implicating immune related genes in melanoma susceptibility and prognosis; however candidate selection of these prior efforts has been limited. In this study, we performed an analysis of germline variants in immuno-modulatory genes for their association with melanoma survival in a well characterized cohort of prospectively accrued melanoma patients. Methods: Germline DNA isolated from blood samples of 817 melanoma patients was genotyped for 94 SNPs tagging 55 immuno-modulatory genes using Sequenom iPLEX. Cox models were used to test associations between each SNP and recurrence-free and overall survival (RFS and OS), with adjustments for age, gender, subtype, thickness, ulceration, and anatomic site. ROC curves were constructed from different SNP/clinical covariate combinations and the area under the curve (AUC) was used to assess their utility in the classification of 3-year recurrence. Results: The SNP rs2796817 in TGFB2 had strong associations with both RFS (HR=3.8, CI 95%: 1.3-11, p=0.02) and OS (HR=5.5, CI 95%: 1.6-19, p=0.029). Other interesting associations with OS came from IRF8 (rs4843861, HR=0.62, CI 95%: 0.39-0.99, p=0.017), CCL5 (rs4796120, HR=7.6, CI 95%: 2.3-25, p=0.035), and CD8A (rs3810831, HR=2.4, CI 95%: 0.91-6.2, p=0.048). A multivariate model including stage, subtype, and one of the SNPs (rs3810831 from CD8A), was shown to improve the AUC when compared to a model including only stage and subtype (0.77 vs. 0.79). Conclusions: We identified several immune-related loci associated with melanoma RFS and OS. The strongest association, rs2796817, maps in TGFB2, which among other functions suppresses IL-2 dependent T-cell growth. In addition to other associations found in the study these findings provide evidence for the involvement of immuno-modulatory genes in melanoma prognosis and suggest further investigations of immune related genes in disease progression. This is currently underway in the second stage validation analysis, which includes an expanded set of immune target genes as well as an additional independent cohort of melanoma patients.

High-throughput single-nucleotide polymorphism-based typing of shared Pseudomonas aeruginosa strains in cystic fibrosis patients using the Sequenom iPLEX platform

Shared strains of Pseudomonas aeruginosa are now well recognized in people with cystic fibrosis (CF), and suitable P. aeruginosa laboratory typing tools are pivotal to understanding their clinical significance and guiding infection control policies in CF clinics. We therefore compared a single-nucleotide polymorphism (SNP)-based typing method using Sequenom iPLEX matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) with typing methods used routinely by our laboratory. We analysed 617 P. aeruginosa isolates that included 561 isolates from CF patients collected between 2001 and 2009 in two Brisbane CF clinics and typed previously by enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as 56 isolates from non-CF patients analysed previously by multilocus sequence typing (MLST). The isolates were tested using a P. aeruginosa Sequenom iPLEX MALDI-TOF (PA iPLEX) method comprising two multiplex reactions, a 13-plex and an 8-plex, to characterize 20 SNPs from the P. aeruginosa housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. These 20 SNPs were employed previously in a real-time format involving 20 separate assays in our laboratory. The SNP analysis revealed 121 different SNP profiles for the 561 CF isolates. Overall, there was at least 96 % agreement between the ERIC-PCR and SNP analyses for all predominant shared strains among patients attending our CF clinics: AUST-01, AUST-02 and AUST-06. For the less frequently encountered shared strain AUST-07, 6/25 (24 %) ERIC-PCR profiles were misidentified initially as AUST-02 or as unique, illustrating the difficulty of gel-based analyses. SNP results for the 56 non-CF isolates were consistent with previous MLST data. Thus, the PA iPLEX format provides an attractive high-throughput alternative to ERIC-PCR for large-scale investigations of shared P. aeruginosa strains.

The role of functionally defective rare germline variants of sialic acid acetylesterase in autoimmune Addison's disease

BackgroundAutoimmune Addison's disease (AAD) is a rare condition with a complex genetic basis. A panel of rare and functionally defective genetic variants in the sialic acid acetylesterase (SIAE) gene has recently been implicated in several common autoimmune conditions. We performed a case–control study to determine whether these rare variants are associated with a rarer condition, AAD.MethodWe analysed nine SIAE gene variants (W48X, M89V, C196F, C226G, R230W, T312M, Y349C, F404S and R479C) in a United Kingdom cohort of 378 AAD subjects and 387 healthy controls. All samples were genotyped using Sequenom iPlex chemistry to characterise primer extension products.ResultsA heterozygous rare allele at codon 312 (312*M) was found in one AAD patient (0.13%) but was not detected in the healthy controls. The commoner, functionally recessive variant at codon 89 (89*V) was found to be homozygous in two AAD patients but was only found in the heterozygous state in controls. Taking into account all nine alleles examined, 4/378 (1.06%) AAD patients and 1/387 (0.25%) healthy controls carried the defective SIAE alleles, with a calculated odds ratio of 4.13 (95% CI 0.44–97.45, two-tailed P value 0.212, NS).ConclusionWe demonstrated the presence of 89*V homozygotes and the 312*M rare allele in the AAD cohort, but overall, our analysis does not support a role for rare variants in SIAE in the pathogenesis of AAD. However, the relatively small collection of AAD patients limits the power to exclude a small effect.

Highly Multiplexed Genotyping of Thiopurine S-Methyltransferase Variants Using MALDI-TOF Mass Spectrometry: Reliable Genotyping in Different Ethnic Groups

Background: To avoid severe hematotoxicity in patients, determination of the TPMT (thiopurine S-methyltransferase) genotype before commencing thiopurine therapy has become accepted. Methods: We used MALDI-TOF mass spectrometry (MS) based on Sequenom iPLEX® technology to develop novel multiplex assays for comprehensive testing of TPMT. Two assays, a 15-plex and a 7-plex assay, consisting of multiplex PCR, shrimp alkaline phosphatase treatment, primer extension, and MALDI-TOF MS analysis, allow detection of all currently known functionally relevant 24 TPMT alleles (TPMT*2 to *18, *20 to *23). Previously identified variant DNA samples and newly constructed synthetic templates were used as quality controls. Results: Assay evaluation performed on a panel of 586 genomic DNA samples previously genotyped by other methods (denaturing HPLC, sequencing) resulted in 100% agreement. Analyses of the distribution of TPMT alleles in 116 samples from a Ghanaian population revealed a TPMT*8 allele frequency of 3.4%. In a Korean population of 118 unrelated individuals, we found a TPMT*6 allele frequency of 1.3%. Conclusions: The newly developed multiplex MALDI-TOF MS assay allows efficient genotyping for all currently known functional TPMT variants. To achieve the most accurate prediction of TPMT phenotype, molecular diagnosis of TPMT should include all these variants.