Sequence Analysis of the IL28A/IL28B Inverted Gene Duplication That Contains Polymorphisms Associated with Treatment Response in Hepatitis C Patients

Several SNPs located in or around the IL28B gene are associated with response of patients infected with Hepatitis C virus to treatment with pegylated interferon-α +/− ribavirin or with spontaneous clearance of the virus. The results of such studies are so compelling that future treatment approaches are likely to involve clinical decisions being made on the basis of a patient's genotype. Since IL28B is a paralogue of IL28A with greater than 95% sequence identity, it is possible that without genotyping assay specificity, sequences in IL28A may contribute to genotype identification, and potentially confound treatment decisions. This study aimed to 1) examine DNA sequences in IL28B surrounding each of the reported associated SNPs and the corresponding regions in IL28A; and 2) develop a robust assay for rs12979860, the most ‘cosmopolitan’ SNP most strongly associated with treatment response across all global populations studied to date. Bioinformatic analysis of genomic regions surrounding IL28A and IL28B demonstrated that 3 SNPs were unique to IL28B, whereas the remaining 6 SNP regions shared >93% identity between IL28A and IL28B. Using a panel of DNA samples, PCR amplification followed by Sanger sequencing was used to examine IL28B SNPs and the corresponding regions in IL28A. For the overlapping SNPs, all 6 in IL28B were confirmed to be polymorphic whereas the corresponding positions in IL28A were monomorphic. Based upon IL28A and IL28B sequence data, a specific TaqMan® assay was developed for SNP rs12979860 that was 100% concordant to the sequence-derived genotypes. Analysis using a commercial assay identified one discordant result which led to a change in their genotype-calling algorithm. Where future treatment decisions are made upon the results of genotyping assays, it is very important that results are concordant with data from a sequence-based format. This is especially so in situations where designing specific PCR primers is a challenge

Real-world data reveal a diagnostic gap in non-alcoholic fatty liver disease

FP7 Ideas under European Research Council (ERC) award 11537

Body mass index and risk of non-alcoholic fatty liver disease: Two electronic health record prospective studies.

The relationship between rising body mass index (BMI) and prospective risk of non-alcoholic fatty liver disease (NAFLD) / non-alcoholic steatohepatitis (NASH) is virtually absent.Determine the extent of the association between BMI and risk of future NAFLD diagnosis, stratifying by sex and diabetes.Two prospective studies using Humedica and THIN with 1.54 and 4.96 years of follow-up respectively.Electronic health record databases Participants: Patients with had a recorded BMI measurement between 15-60kg/m(2), and smoking status, and one year of active status prior to baseline BMI. Patients with a diagnosis or history of chronic diseases were excluded.None Main Outcome Measure: Recorded diagnosis of NAFLD/NASH during follow-up (Humedica ICD-9 code 571.8, and read codes for NAFLD and NASH in THIN).Hazard ratios (HR) were calculated across BMI categories using BMI of 20-22.5kg/m2 as the reference category, adjusting for age, sex and smoking status. Risk of recorded NAFLD/NASH increased linearly with BMI and was approximately 5-fold higher in Humedica (HR=4.78, 95% CI 4.17-5.47) and 9-fold higher in THIN (HR=8.93, 7.11-11.23) at a BMI of 30-32.5 kg/m(2) rising to around 10-fold higher in Humedica (HR=9.80, 8.49-11.32) and 14-fold higher in THIN (HR=14.32, 11.04-18.57) in the 37.5-40 kg/m(2) BMI category. Risk of NAFLD/NASH was approximately 50% higher in men, and approximately double in those with diabetes.These data quantify the consistent and strong relationships between BMI and prospectively recorded diagnoses of NAFLD/NASH and emphasize the importance of weight reduction strategies for prevention and management of NAFLD

Schematic showing IL28 genes, SNP locations and areas of sequence homology.

<p>Illustration of the relative locations of the <i>IL28A</i> and <i>IL28B</i> genes on the forward and reverse DNA strands on chromosome 19. The shaded boxes indicate regions of the inverted duplication with >95% sequence identity. The direction of the shaded gradient indicates the orientation of the match. SNP identifiers in blue were unique to the <i>IL28B</i> region, whereas SNPs highlighted in other colors were present in the <i>IL28B</i> region as well as a corresponding region in <i>IL28A</i>.</p

Allele frequencies of SNPs unique to <i>IL28B</i> region.

<p>Allele frequencies of SNPs unique to <i>IL28B</i> region.</p

Allelic discrimination plot for the rs12979860 genotyping assay.

<p>Allelic discrimination plot generated by the SDS 2.3 software (Applied Biosystems Inc.) for the rs12979860 genotyping assay using the 48 sample DNA panel. Red and blue circles represent the two homozygous genotypes (T/T and C/C), the green circles represent heterozygous genotype (C/T) and the black squares represent no template control.</p