Toby Johnson

Effect of five genetic variants associated with lung function on the risk of chronic obstructive lung disease, and their joint effects on lung function

RATIONALE Genomic loci are associated with FEV1 or the ratio of FEV1 to FVC in population samples, but their association with chronic obstructive pulmonary disease (COPD) has not yet been proven, nor have their combined effects on lung function and COPD been studied. OBJECTIVES To test association with COPD of variants at five loci (TNS1, GSTCD, HTR4, AGER, and THSD4) and to evaluate joint effects on lung function and COPD of these single-nucleotide polymorphisms (SNPs), and variants at the previously reported locus near HHIP. METHODS By sampling from 12 population-based studies (n = 31,422), we obtained genotype data on 3,284 COPD case subjects and 17,538 control subjects for sentinel SNPs in TNS1, GSTCD, HTR4, AGER, and THSD4. In 24,648 individuals (including 2,890 COPD case subjects and 13,862 control subjects), we additionally obtained genotypes for rs12504628 near HHIP. Each allele associated with lung function decline at these six SNPs contributed to a risk score. We studied the association of the risk score to lung function and COPD. MEASUREMENTS AND MAIN RESULTS Association with COPD was significant for three loci (TNS1, GSTCD, and HTR4) and the previously reported HHIP locus, and suggestive and directionally consistent for AGER and TSHD4. Compared with the baseline group (7 risk alleles), carrying 10-12 risk alleles was associated with a reduction in FEV1 (β = -72.21 ml, P = 3.90 × 10(-4)) and FEV1/FVC (β = -1.53%, P = 6.35 × 10(-6)), and with COPD (odds ratio = 1.63, P = 1.46 × 10(-5)). CONCLUSIONS Variants in TNS1, GSTCD, and HTR4 are associated with COPD. Our highest risk score category was associated with a 1.6-fold higher COPD risk than the population average score.

IL8 polymorphisms and overall survival in pazopanib- or sunitinib-treated patients with renal cell carcinoma

Background:We evaluated germline single nucleotide polymorphisms (SNPs) for association with overall survival (OS) in pazopanib- or sunitinib-treated patients with advanced renal cell carcinoma (aRCC).Methods:The discovery analysis tested 27 SNPs within 13 genes from a phase III pazopanib trial (N=241, study 1). Suggestive associations were then pursued in two independent datasets: a phase III trial (COMPARZ) comparing pazopanib vs sunitinib (N=729, study 2) and an observational study of sunitinib-treated patients (N=89, study 3).Results:In study 1, four SNPs showed nominally significant association (P≤0.05) with OS; two of these SNPs (rs1126647, rs4073) in IL8 were associated (P≤0.05) with OS in study 2. Because rs1126647 and rs4073 were highly correlated, only rs1126647 was evaluated in study 3, which also showed association (P≤0.05). In the combined data, rs1126647 was associated with OS after conservative multiple-test adjustment (P=8.8 × 10−5; variant vs reference allele hazard ratio 1.32, 95% confidence interval: 1.15–1.52), without evidence for heterogeneity of effects between studies or between pazopanib- and sunitinib-treated patients.Conclusions:Variant alleles of IL8 polymorphisms are associated with poorer survival outcomes in pazopanib- or sunitinib-treated patients with aRCC. These findings provide insight in aRCC prognosis and may advance our thinking in development of new therapies.

Hyperbilirubinemia in pazopanib- or sunitinib-treated patients in COMPARZ is associated with UGT1A1 polymorphisms

Pazopanib and sunitinib are angiogenesis inhibitors approved for treatment of advanced renal cell carcinoma. Treatmentassociated elevations in alanine aminotransferase (ALT) and bilirubin have been reported for both therapies [1]. UGT1A1 polymorphisms are known to cause reduced expression/ activity of UGT1A1 and predispose individuals to Gilbert’s syndrome, a benign form of episodic jaundice [2, 3]. Our analyses of previous pazopanib clinical trials found that UGT1A1 *28 was associated with on-treatment hyperbilirubinemia [4]. This study extended our previous analysis of UGT1A1*28 to additional alleles (*36, *37, and *6) and investigated their association with on-therapy serum total bilirubin in COMPARZ (NCT00720941 and NCT01147822), a phase III, randomized, clinical trial comparing pazopanib versus sunitinib for the treatment of advanced renal cell carcinoma [1]. Of 1110 randomized participants, on-therapy hyperbilirubinemia [total bilirubin ≥1.5 × upper limit of the normal range (ULN)] was observed in 16% (89/557) of pazopanib-arm patients and 9% (49/553) of sunitinib-arm patients. In the pharmacogenetic population (patients who received at least one dose of study treatment, consented, and were successfully genotyped, n = 719), the incidence of hyperbilirubinemia was 17% (62 of 369) for pazopanib and 10% (34 of 350) for sunitinib. The incidence of hyperbilirubinemia varies by UGT1A1 genotypes for both therapies (Figure 1). Logistic regression adjusted for ancestry principal components was used to compare hyperbilirubinemia cases (pazopanib n = 62; sunitinib n = 34) against controls (pazopanib n = 211; sunitinib n = 212). Controls were defined as patients with all on-therapy bilirubin ≤1 × ULN and treatment exposure equal to or greater than median exposure until bilirubin elevation in cases (defined in the supplementary materials, available at Annals of Oncology online). Patients with predicted reduced UGT1A1 function (homozygous or inferred compound heterozygous for *28, *37, and *6) had higher baseline bilirubin and were more likely to experience hyperbilirubinemia when receiving either pazopanib (P = 7.7 × 10) or sunitinib (P = 1.7 × 10), with odds ratios (95% confidence interval) 9.97 (4.13–24.03) and 5.83 (2.04–16.68), respectively. After adjusting for baseline bilirubin, patients with predicted, reduced UGT1A1 function remained more likely to experience hyperbilirubinemia when receiving pazopanib (P = 0.012) or sunitinib (P = 0.024), with odds ratios (95% confidence interval) 3.65 (1.31–10.16) and 4.51 (1.26–16.11), respectively. UGT1A1 genotypes were not significantly associated with ALT ≥3 × ULN in pazopanib-treated patients. A borderline significant association (P = 0.030) was seen between UGT1A1 genotypes with reduced function (predicted) and decreased incidence of ALT ≥3 × ULN in sunitinib-treated patients; additional studies are required to confirm this observation. Pazopanib is a potent inhibitor of UGT1A1 in vitro [4]. Our data suggest that hyperbilirubinemia in pazopanibtreated patients may be the result of inhibition of UGT1A1 combined with effects of genetic variants in the UGT1A1 gene. We expect this would result in higher levels of unconjugated hyperbilirubinemia, usually associated with a benign clinical course. Sunitinib, however, does not inhibit UGT1A1 [5]. To our knowledge, this is the first study reporting the association between UGT1A1 genotype and hyperbilirubinemia in patients receiving sunitinib. Our data suggest that some instances of hyperbilirubinemia in patients treated with pazopanib or sunitinib may be benign manifestations of Gilbert’s syndrome. Bilirubin fractionation or, if not available, UGT1A1 genotyping, would enable further characterization of liver safety risk and help in making treatment decisions.

Gene-centric meta-analyses for central adiposity traits in up to 57,412 individuals of European descent confirm known loci and reveal several novel associations

Waist circumference (WC) and waist-to-hip ratio (WHR) are surrogate measures of central adiposity that are associated with adverse cardiovascular events, type 2 diabetes and cancer independent of body mass index (BMI). WC and WHR are highly heritable with multiple susceptibility loci identified to date. We assessed the association between SNPs and BMI-adjusted WC and WHR and unadjusted WC in up to 57 412 individuals of European descent from 22 cohorts collaborating with the NHLBIs Candidate Gene Association Resource (CARe) project. The study population consisted of women and men aged 20-80 years. Study participants were genotyped using the ITMAT/Broad/CARE array, which includes ∼50 000 cosmopolitan tagged SNPs across ∼2100 cardiovascular-related genes. Each trait was modeled as a function of age, study site and principal components to control for population stratification, and we conducted a fixed-effects meta-analysis. No new loci for WC were observed. For WHR analyses, three novel loci were significantly associated (P < 2.4 × 10(-6)). Previously unreported rs2811337-G near TMCC1 was associated with increased WHR (β ± SE, 0.048 ± 0.008, P = 7.7 × 10(-9)) as was rs7302703-G in HOXC10 (β = 0.044 ± 0.008, P = 2.9 × 10(-7)) and rs936108-C in PEMT (β = 0.035 ± 0.007, P = 1.9 × 10(-6)). Sex-stratified analyses revealed two additional novel signals among females only, rs12076073-A in SHC1 (β = 0.10 ± 0.02, P = 1.9 × 10(-6)) and rs1037575-A in ATBDB4 (β = 0.046 ± 0.01, P = 2.2 × 10(-6)), supporting an already established sexual dimorphism of central adiposity-related genetic variants. Functional analysis using ENCODE and eQTL databases revealed that several of these loci are in regulatory regions or regions with differential expression in adipose tissue.

The epigenetic signature of systemic insulin resistance in obese women

Aims/hypothesisInsulin resistance (IR) links obesity to type 2 diabetes. The aim of this study was to explore whether white adipose tissue (WAT) epigenetic dysregulation is associated with systemic IR by genome-wide CG dinucleotide (CpG) methylation and gene expression profiling in WAT from insulin-resistant and insulin-sensitive women. A secondary aim was to determine whether the DNA methylation signature in peripheral blood mononuclear cells (PBMCs) reflects WAT methylation and, if so, can be used as a marker for systemic IR.MethodsFrom 220 obese women, we selected a total of 80 individuals from either of the extreme ends of the distribution curve of HOMA-IR, an indirect measure of systemic insulin sensitivity. Genome-wide transcriptome and DNA CpG methylation profiling by array was performed on subcutaneous (SAT) and visceral (omental) adipose tissue (VAT). CpG methylation in PBMCs was assayed in the same cohort.ResultsThere were 647 differentially expressed genes (false discovery rate [FDR] 10%) in SAT, all of which displayed directionally consistent associations in VAT. This suggests that IR is associated with dysregulated expression of a common set of genes in SAT and VAT. The average degree of DNA methylation did not differ between the insulin-resistant and insulin-sensitive group in any of the analysed tissues/cells. There were 223 IR-associated genes in SAT containing a total of 336 nominally significant differentially methylated sites (DMS). The 223 IR-associated genes were over-represented in pathways related to integrin cell surface interactions and insulin signalling and included COL5A1, GAB1, IRS2, PFKFB3 and PTPRJ. In VAT there were a total of 51 differentially expressed genes (FDR 10%); 18 IR-associated genes contained a total of 29 DMS.Conclusions/interpretationIn individuals discordant for insulin sensitivity, the average DNA CpG methylation in SAT and VAT is similar, although specific genes, particularly in SAT, display significantly altered expression and DMS in IR, possibly indicating that epigenetic regulation of these genes influences metabolism.

BRCA1/2 mutations associated with progression-free survival in ovarian cancer patients in the AGO-OVAR 16 study

OBJECTIVE AGO-OVAR 16 demonstrated that pazopanib maintenance therapy significantly increased progression-free survival (PFS) in patients with ovarian cancer whose disease had not progressed after first-line therapy. In a sub-study, we evaluated the effect of clinically important germline BRCA1 and BRCA2 mutations on PFS. METHODS Of 940 AGO-OVAR 16 participants, 664 had BRCA1/2 exon sequencing data (pazopanib, n=335; placebo, n=329). A Cox model was used to test the association between genetic variants and PFS. RESULTS Ninety-seven of 664 patients (15%) carried clinically important BRCA1/2 mutations (BRCA1/2 carriers: pazopanib 14%, placebo 16%). Median PFS was longer in BRCA1/2 mutation carriers than in BRCA1/2 non-carriers in the placebo arm (30.3 vs 14.1 months, hazard ratio, 0.48; 95% confidence interval [CI]: 0.29-0.78; P=0.0031); a similar non-significant trend was noted with pazopanib (30.2 vs 17.7 months, hazard ratio, 0.64; 95% CI: 0.40-1.03; P=0.069). Among BRCA1/2 non-carriers, PFS was longer for pazopanib-treated patients than placebo-treated patients (17.7 vs 14.1 months, hazard ratio, 0.77; 95% CI: 0.62-0.97; P=0.024). Among BRCA1/2 carriers, there was no significant PFS difference between treatments, although numbers were small (pazopanib, 46; placebo, 51), resulting in a wide CI (hazard ratio, 1.36; 95% CI: 0.66-2.82). CONCLUSIONS Patients with clinically important BRCA1/2 mutations had better prognosis. BRCA1/2 mutation status might be added as strata in future trials in primary ovarian cancer.

Different effects of the BIM deletion polymorphism on treatment of solid tumors by the tyrosine kinase inhibitors (TKI) pazopanib, sunitinib, and lapatinib

FISH ‘borderline’ tumors (i.e. 15%–20% of rearranged nuclei) emphasizing potential issues in ALK FISH analysis [2]. They especially reported the particular cases of two FISH− IHC+ tumors with ALK amplification (i.e. high copy number corresponding to more than six copies per nucleus). Within 26 ALK FISH+ tumors (i.e. with more than 15% of rearranged FISH nuclei using the FDA-approved ALK break-apart FISH probe), no tumor presented ALK amplification. ALK copy number gains have been described in NSCLC. Although ALK amplification in ALK-rearranged tumors seems to be rare, few studies have been conducted in these tumors [3– 5]. We report here a case of ALK-rearranged and amplified tumor that was ALK IHC negative. We identified a single case of both ALK-rearranged and amplified tumor among ∼70 cases of ALK FISH positive NSCLC (∼7% of the NSCLC samples analyzed in our institutional cancer molecular genetics platform). This represents <2% of ALK-rearranged NSCLC and 0.1% of unselected NSCLC in our institution. The sample was a metastasis from the abdominal wall in a patient with a voluminous lung tumor. The ALK rearrangement consisted in both FISH split and single 30 signals in 30% of tumor cells (mean 2.8 split and 1.5 single 30 signals per nucleus). There was a mean number of 4.5 fused signals per nucleus. Adding spit and single 30 signals resulted in a mean number of 8.8 ALK copies per nucleus whereas the mean number of chromosome 2 per nucleus was 3.5 with an ALK/chromosome 2 ratio of 2.5. This was consistent with an ALK amplification. The ALK IHC analysis (clone 5A4, 1:25, Clinisciences) was negative (score 0) (Figure 1). To date, this 46-year-old smoker woman, with KRAS, EGFR and BRAF nonmutated status, has not been treated by crizotinib. This particular case illustrates some challenges in ALK rearrangement screening. First, ALK amplification, with FISH clusters in some cells as encountered in our case can impair the detection of positive signals within tumor cells and in this manner requires careful examination of these cases. Second, if ALK copy number gains are frequent in ALK nonrearranged tumors, ALK-amplified tumors are rare in ALK-rearranged tumors, as previously described [1, 3–5]. Finally, as previously mentioned in some studies, we agree with Ilie et al. and think that both IHC and FISH analyses should be carried out to avoid ALK status misdiagnosis [1, 2]. We should be aware of these particular cases because this rare ALK double alteration would have been missed by an IHC-based prescreening, as proposed in some studies.

Phenome-wide association study using research participants’ self-reported data provides insight into the Th17 and IL-17 pathway

A phenome-wide association study of variants in genes in the Th17 and IL-17 pathway was performed using self-reported phenotypes and genetic data from 521,000 research participants of 23andMe. Results replicated known associations with similar effect sizes for autoimmune traits illustrating self-reported traits can be a surrogate for clinically assessed conditions. Novel associations controlling for a false discovery rate of 5% included the association of the variant encoding p.Ile684Ser in TYK2 with increased risk of tonsillectomy, strep throat occurrences and teen acne, the variant encoding p.Arg381Gln in IL23R with a decrease in dandruff frequency, the variant encoding p.Asp10Asn in TRAF3IP2 with risk of male-pattern balding, and the RORC regulatory variant (rs4845604) with protection from allergies. This approach enabled rapid assessment of association with a wide variety of traits and investigation of traits with limited reported associations to overlay meaningful phenotypic context on the range of conditions being considered for drugs targeting this pathway.

Erratum to: The epigenetic signature of systemic insulin resistance in obese women

The number of differentially methylated sites (DMS) found to display directionally consistent differences in methylation in the cohorts studied by both Nilsson et al [10] and Arner et al was incorrectly reported to be 592, not 591. The sentence concerned should read: ‘Nilsson et al reported, in a cohort of 56 individuals, 15,627 DMS (q<0.15) in WAT associated with type 2 diabetes [10]; 671 of the DMS overlapped with those in the present study, of which 591 displayed directionally consistent differences in methylation in both cohorts (p<2.7×10) (ESM Table 4) [10].’ The second footnote to ESM Table 4 incorrectly referred to transcriptome profiles not methylome profiles. It should have read: ‘b. comparison with published methylome profiles on SAT; see references in main manuscript for details.’

Chapter 44 – Hypertension

Hypertension is heritable and is a major global health problem, but identifying genetic variants contributing to the disorder has been challenging. In the last few years, there have been major advances in our understanding of the genetic basis of hypertension and continuous blood pressure (BP) variation, yet total heritability estimates imply that much remains to be discovered. Genome-wide association studies (GWAS), mostly with effectively very large sample sizes achieved by meta-analyzing smaller GWAS, have led to the discovery of 40 distinct BP loci. In this chapter we describe the association studies and their contribution to our understanding of disease pathology, and we review the potential of these discoveries for translation into new and more effective therapeutic agents. Finally, we assess the potential utility of BP genetic variants for predicting risk of hypertension and cardiovascular disease.