Jody Salomon

Barrett associated MHC and FOXF1 variants also increase esophageal carcinoma risk

Barretts esophagus, with gastroesophageal reflux disease and obesity as risk factors, predisposes to esophageal adenocarcinoma (EAC). Recently a British genome wide association study identified two Barretts esophagus susceptibility loci mapping within the major histocompatibility complex (MHC; rs9257809) and closely to the Forkhead‐F1 (FOXF1; rs9936833) coding gene. An interesting issue is whether polymorphisms associated with Barretts esophagus, are also implicated in esophageal carcinoma (EC), and more specifically EAC genesis. Assessing the individual genetic susceptibility can help identify high risk patients more prone to benefit from surveillance programs. Our hypothesis: Barrett associated MHC and FOXF1 variants modify EC risk in Caucasians. In a Dutch case‐control study, 431 patients with EC and 605 healthy controls were included. Polymorphisms at chromosomes 6p21 (MHC) and 16q24 (FOXF1) were determined by means of real‐time polymerase chain reaction (RT‐PCR). Logistic regression analysis was used to calculate odds ratios with 95% confidence intervals. The FOXF1 rs9936833 variant C allele was associated with an increased EAC susceptibility; OR, [95% CI]; 1.21, [0.99–1.47]. A sex‐stratified analysis revealed a similar association in males; 1.24 [1.00–1.55]. The variant MHC rs9257809 G allele as well as the MHC heterozygous AG genotype significantly increased ESCC risk; 1.76 [1.16–2.66] and 1.74 [1.08–2.80], respectively. Sex‐stratification showed that the variant G allele was especially present in female patients; 2.32 [1.04–5.20]. In conclusion, this study provides evidence that MHC rs9257809 and FOXF1 rs9936833 variants, associated with Barretts esophagus, also increase ESCC and EAC susceptibility in Caucasians. FOX proteins are transcription factors involved in organogenesis of the GI tract, while MHC haplotypes are strongly associated with smoking behavior, a crucial risk factor for ESCC. Assessing the individual genetic susceptibility can help identify high risk patients more prone to benefit from (Barrett) surveillance programs.

CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.

TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation.

Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199. V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation.

High enzyme activity UGT1A1 or low activity UGT1A8 and UGT2B4 genotypes increase esophageal cancer risk

Esophageal cancer (EC) has a globally increasing incidence with poor curative treatment options and survival rates. Environmental and dietary factors have crucial roles in esophageal carcinogenesis. Polymorphisms in the UGT genes, a superfamily of enzymes essential for the detoxification of carcinogens, may alter enzyme activity and subsequently may play a role in EC etiology. Rather than solely establishing differences in genotype distribution, we investigated whether functional polymorphisms in UGT genes that can predict enzyme activity in vivo, may influence EC risk. A case-control study including 351 Caucasian EC patients and 592 Caucasian controls was conducted and polymorphisms in seven UGT genes were determined, using the polymerase chain reaction. On the basis of allelic in vitro enzyme activity measurements, genotypes were categorized according to their predicted in vivo enzyme activity into high, medium and low categories. Predicted enzyme activity groups were combined and compared between patients and controls. The UGT1A1 and UGT1A8 predicted high enzyme activity genotypes were significantly more (OR=1.62; 95% CI, 1.02-2.56) and less frequent (OR=0.36; 95% CI, 0.15-0.84) among patients with esophageal squamous cell carcinoma (ESCC), respectively. High (OR=0.42; 95% CI, 0.22-0.84) and medium (OR=0.25; 95% CI, 0.12-0.52) activity UGT2B4 genotypes were significantly less often present in ESCC patients. No association was detected between UGT genotypes and esophageal adenocarcinoma (EAC) risk. Polymorphisms in UGT genes, resulting in altered enzyme activity genotypes, do not seem modifiers of EAC risk. However, the predicted high activity UGT1A1 genotype, associated with low serum levels of the antioxidant bilirubin, was associated with an increased ESCC risk. The UGT1A8 and UGT2B4 genotypes associated with decreased predicted enzyme activities, were significantly associated with an increased risk of ESCC, probably by a decreased detoxification of carcinogens.

Loss of Heterozygosity Is Present in SEC63 Germline Carriers with Polycystic Liver Disease

Polycystic liver disease (PCLD) is an autosomal dominant disorder characterised by multiple fluid filled cysts in the liver. This rare disease is caused by heterozygous germline mutations in PRKCSH and SEC63. We previously found that, in patients with a PRKCSH mutation, over 76% of the cysts acquired a somatic ‘second-hit’ mutation in the wild type PRKCSH allele. We hypothesise that somatic second-hit mutations are a general mechanism of cyst formation in PCLD which also plays a role in PCLD patients carrying a SEC63 germline mutation. We collected cyst epithelial cells from 52 liver cysts from three different SEC63 patients using laser microdissection. DNA samples were sequenced to identify loss of heterozygosity (LOH) mutations and other somatic mutations in cyst epithelial DNA. We discovered somatic SEC63 mutations in patient 3 (1/14 cysts), but not in patient 1 and 2 (38 cysts). Upon review we found that the germline mutation of patient 1 and 2 (SEC63 c.1703_1705delAAG) was present in the same frequency in DNA samples from healthy controls, suggesting that this variant is not causative of PCLD. In conclusion, as somatic second-hit mutations also play a role in cyst formation in patients with a SEC63 germline mutation, this appears to be a general mechanism of cyst formation in PCLD.

No role for glutathione S-transferase genotypes in Caucasian esophageal squamous cell or adenocarcinoma etiology: an European case-control study

BackgroundIdentifying and monitoring high-risk patients can aid the prevention of esophageal cancer (EC). The interaction of environmental risk factor exposure and genetic susceptibility may contribute to the etiology of EC. Biotransformation enzymes such as Glutathione S-Transferases (GSTs ) detoxify mutagenic and genotoxic compounds and therefore control the rate of detoxification of carcinogens. Functional polymorphisms in the genes coding for GSTs alter their enzyme activity in vitro, and were reported to modify EC risk in Asians. We hypothesized that altered enzyme activity GST genotypes influence the susceptibility for esophageal adeno- (EAC) and squamous cell carcinoma (ESCC) in Caucasians.MethodsWe performed a case–control study including 440 Caucasian patients with EC and 592 healthy Caucasian controls matched for age and sex. Functional polymorphisms were selected and genotypes were determined in GST classes Alpha, Mu, Theta and Pi by means of polymerase chain reaction. Genotypes were classified into predicted high, intermediate and low enzyme activity categories based on in vitro activity data. The distribution of the activity genotypes were compared between patients with EAC or ESCC, and controls. Odds ratios (OR) with 95% confidence intervals (CI) were calculated by logistic regression analyses. Gene-gene interactions were tested and for comparison purposes, the predicted low and intermediate activity genotypes were combined. Genotypes with similar risks for EAC or ESCC were combined and analyzed for multiplicative effects.ResultsOur analyses includes 327 patients with EAC and 106 patients with ESCC. Low or intermediate activity enzyme genotypes for GSTM1, GSTA1, GSTP1 I105V and A114V as well as for GSTT1, did not significantly modify the risk for ESCC or EAC in our Dutch population.ConclusionFunctional genotypes in GST genes are not involved in EAC or ESCC susceptibility in Caucasians, in contrast to results on ESCC from Asia or Africa.

Polymorphisms in the insulin-like growth factor axis are associated with gastrointestinal cancer

Introduction Numerous factors influence the development of gastrointestinal (GI) cancer. The insulin-like growth factor (IGF) axis plays a role in embryonic and postnatal growth and tissue repair. Elevated levels of IGFs, low levels of IGF binding proteins (IGFBPs) and over-expression of IGF receptor (IGFR-I) were associated with several stages of cancer. Here, the prevalence of the single nucleotide polymorphisms (SNPs) rs6214 in the IGF type I (IGF-I) gene and rs6898743 in the growth hormone receptor (GHR) gene in patients with GI cancer and controls was studied. Materials & Methods In this Dutch case-control study, DNA isolated from blood of 1,457 GI cancer patients; 438 patients with head and neck cancer (HNC), 475 with esophageal cancer (EC) and 544 with colorectal cancer (CRC) and 1,457 matched controls, was used to determine the rs6214 and rs6898743 genotypes by polymerase chain reaction. The association between these SNPs and GI cancer, HNC, esophageal adenocarcinoma (EAC), esophageal squamous-cell carcinoma (ESCC) and proximal or distal CRC was studied. Odds ratios (ORs) with 95% confidence interval (95% CI) were calculated via unconditional logistic regression. Results Overall for GI cancer, the ORs for SNPs rs6214 and rs6898743 were approximately 1.0 (p-value>0.05), using the most common genotypes GG as reference. An OR of 1.54 (95% CI, 1.05–2.27) was found for EC for genotype AA of rs6214. The ORs for EAC were 1.45 (95% CI, 1.04–2.01) and 1.71 (95% CI, 1.10–2.68), for genotypes GA and AA, respectively. Genotype GC of rs6898743 showed an OR of 0.47 (95% CI, 0.26–0.86) for ESCC. Conclusion The A allele of SNP rs6214 in the IGF-I gene was associated with EAC, and with HNC in women. The GC genotype of rs6898743 in the GHR gene was negatively associated with ESCC.

Genetic polymorphism 609C>T in NAD(P)H:quinone oxidoreductase 1 enhances the risk of proximal colon cancer

Gastrointestinal (GI) cancer is responsible for the majority of deaths among all types of cancer. Lifestyle factors may not only be the main risk factor for GI cancer but reactive oxygen species (ROS) may also be involved. The single-nucleotide polymorphisms (SNPs) 609C>T (rs1800566) and 465C>T (rs1131341) in the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene lead to a decline in NQO1 enzyme activity. NQO1 catalyzes the two-electron reduction of quinones to hydroquinones, thereby preventing the formation of ROS. Such polymorphisms in NQO1 may increase the risk of GI cancer. The aim of this study was to evaluate the influence of the SNPs rs1800566 and rs1131341 in the NQO1 gene on the risk of GI cancer in the Netherlands. Real-time polymerase chain reaction techniques were conducted to determine the NQO1 genotypes of 1457 patients with GI cancer and 1457 age- and gender-matched controls in a case–control study. Binary logistic regression analyses showed no statistically significant difference in genotype distributions between patients and controls: odds ratios (ORs) with 95% confidence interval (CI) for rs1800566 were 1.09 (0.93–1.28) and 1.17 (0.77–1.77) for the CT and TT genotypes, respectively. ORs for rs1131341 CT and TT genotypes were 1.21 (0.90–1.63) and 0.54 (0.05–5.94), respectively. For rs1800566, a significant association between the CT genotype and proximal colon cancer was detected (OR=1.60; 95% CI=1.09–2.35). The NQO1*2 T allele of SNP rs1800566 was found associated with an increased risk for proximal colorectal cancer, whereas SNP rs1131341 was rare in our Dutch population and was not associated with GI cancer.

Polymorphisms in alcohol-metabolizing enzymes and esophageal carcinoma susceptibility : a Dutch Caucasian case-control study

Esophageal cancer (EC), mainly consisting of squamous cell carcinoma (ESCC) in the Eastern world and adenocarcinoma (EAC) in the Western world, is strongly associated with dietary factors such as alcohol use. We aimed to clarify the modifying role in EC etiology in Caucasians of functional genotypes in alcohol-metabolizing enzymes. In all, 351 Caucasian patients with EC and 430 matched controls were included and polymorphisms in CYP2E1, ADH and near ALDH2 genes were determined. In contrast to the results on ESCC in mainly Asian studies, we found that functional genotypes of alcohol-metabolizing enzymes were not significantly associated with EAC or ESCC in an European population.

Urinary Excretion Levels of MMX-Mesalazine as a Tool to Assess Non- Adherence

Objective: 5-Amino salicylicacid (5-ASA) is the cornerstone of ulcerative colitis treatment, with (assessment of) non-adherence as a challenge. Multi-matrix release (MMX)-mesalazine has the advantage of once-daily (OD) dosing. Primarily we assessed urinary (NAc-) 5-ASA excretion, as measured by High-Performance Liquid Chromatography (HPLC), in order to monitor nonadherence, in healthy volunteers taking MMX-mesalazine. Secondly, we established urinary (NAc-)5-ASA cut-off levels for (partial) nonadherence. Method: We studied 25 healthy adult volunteers who used MMX-mesalazine 2400 mg OD (days 1-4), followed by 1200 mg twice daily (BID) (days 8-11), separated by a drug-free interval of 3 days. Daily morning urine spot samples were collected. The cut-off level for adherence was set at the lowest steady state (NAc-)5-ASA urinary concentration level. Results: Stability of urinary 5-ASA and NAc-5-ASA, stored at room temperature during 24 hours was 96.4 ± 8.3% and 96.4 ± 4.1%. Recovery of urinary 5-ASA and NAc-5-ASA was 114.3 ± 10.4% and 107.5 ± 6.4%. The limit of detection and quantification were 1.1 ug/ml and 3.5 ug/ml for NAc-5-ASA and 0.4 ug/ml and 1.3 ug/ml for 5-ASA. The maximal 5-ASA within-run and between-run relative SD were 10.4% and 12.5%. The cut-off level for non-adherence was determined at 9.67 (OD) and at 15.39 (BID) mg/mmol (NAc-) 5-ASA per mmol creatinine. Conclusion: HPLC is a feasible, sensitive and reproducible method to measure urinary (NAc-) 5-ASA excretion in volunteers taking MMX-mesalazine. This study establishes urinary (NAc-) 5-ASA cut-off levels for MMX-mesalazine non-adherence that may be useful in clinical practice and future trials.