Genetic and epigenetic risks of intracytoplasmic sperm injection method

Pregnancies achieved by assisted reproduction technologies, particularly by intracytoplasmic sperm injection （ICSI） procedures, are susceptible to genetic risks inherent to the male population treated with ICSI and additional risks inherent to this innovative procedure. The documented, as well as the theoretical, risks are discussed in the present review study. These risks mainly represent thatconsequences of the genetic abnormalities underlying male subfertility （or infertility） and might become stimulators for the development of novel approaches and applications in the treatment of infertility. In addition, risks with a polygenic background appearing at birth as congenital anomalies and other theoretical or stochastic risks are discussed. Recent data suggest that assisted reproductive technology might also affect epigenetic characteristics of the male gamete, the female gamete, or might have an impact on early embryogenesis. It might be also associated with an increased risk for genomic imprinting abnormalities.

CYP4F2 polymorphism as a genetic risk factor for major hemorrhagic complications in Chinese patients on warfarin therapy

Warfarin is a commonly used anticoagulant with a narrow therapeutic range and risk of hemorrhagic complications. After CYP2C9 and VKORC1, CYP4F2 was confirmed as the third principle genetic determinant of warfarin dose variability.

Single-nucleotide polymorphisms based genetic risk score in the prediction of pancreatic cancer risk

BACKGROUND Disease-related single nucleotide polymorphisms(SNPs)based genetic risk score(GRS)has been proven to provide independent inherited risk other than family history in multiple cancer types.AIM To evaluate the potential of GRS in the prediction of pancreatic cancer risk.METHODS In this case-control study(254 cases and 1200 controls),we aimed to evaluate the association between GRS and pancreatic ductal adenocarcinoma(PDAC)risk in the Chinese population.The GRS was calculated based on the genotype information of 18 PDAC-related SNPs for each study subject(personal genotyping information of the SNPs)and was weighted by external odd ratios(ORs).RESULTS GRS was significantly different in cases and controls(1.96±3.84 in PDACs vs 1.09±0.94 in controls,P<0.0001).Logistic regression revealed GRS to be associated with PDAC risk[OR=1.23,95%confidence interval(CI):1.13-1.34,P<0.0001].GRS remained significantly associated with PDAC(OR=1.36,95%CI:1.06-1.74,P=0.015)after adjusting for age and sex.Further analysis revealed an association of increased risk for PDAC with higher GRS.Compared with low GRS(<1.0),subjects with high GRS(2.0)were 99%more likely to have PDAC(OR:1.99,95%CI:1.30-3.04,P=0.002).Participants with intermediate GRS(1.0-1.9)were 39%more likely to have PDAC(OR:1.39,95%CI:1.03-1.84,P=0.031).A positive trend was observed(P trend=0.0006).CONCLUSION GRS based on PDAC-associated SNPs could provide independent information on PDAC risk and may be used to predict a high risk PDAC population.

Association of sleep quality with myopia based on different genetic risk levels

AIM: To analyse the association of sleep quality with myopia under different genetic risk(GR) levels.METHODS: A cross-sectional survey of students aged 9-14 y in Wenzhou, China, was conducted. Refraction without cycloplegia and ocular parameters were measured. Sleep quality was assessed with the Pittsburgh Sleep Quality Index(PSQI). Seventeen single nucleotide polymorphisms(SNPs) were replicated by association analysis and used to compute the GR score(GRS). Possible confounders were assessed by a questionnaire that collected information about the children and their parents. Generalized linear models were used to analyse the sleep quality, the GR, and their interaction effects on the risk of myopia.RESULTS: Out of 1354 children included in this study, 353(26.07%) had sleep disturbances. The GRS ranged from 4.49 to 12.89 with a mean of 7.74±1.23, and the participants were divided into a low GR group, a moderate GR group and a high GR group according to the GRS quartile. In the generalized linear model, the children with sleep disturbances and high GR had a higher risk of myopia than those without sleep disturbances and with low GR(OR=1.59, 95%CI: 1.12-2.25;OR=1.88, 95%CI: 1.23-2.88, respectively). Compared to those with low GR and SDs, children with high GR with or without SDs had a higher risk of myopia(OR=4.88, 95%CI: 2.03-11.71;OR=1.70, 95%CI: 1.06-2.72, respectively).CONCLUSION: The prevalence of sleep disturbances in elementary school students in Wenzhou was 26.07%. There is a significant interaction between sleep disturbances and a high GR of myopia, suggesting that a high GR of myopia may increase children’s sensitivity to sleep disturbances. This study indicates that children with a high GR of myopia need to achieve adequate sleep duration and excellent sleep quality.

Multi-locus genetic risk score predicts risk for Crohn's disease in Slovenian population

AIM: To develop a risk model for Crohn&#x02019;s disease (CD) based on homogeneous population.METHODS: In our study were included 160 CD patients and 209 healthy individuals from Slovenia. The association study was performed for 112 single nucleotide polymorphisms (SNPs). We generated genetic risk scores (GRS) based on the number of risk alleles using weighted additive model. Discriminatory accuracy was measured by area under ROC curve (AUC). For risk evaluation, we divided individuals according to positive and negative likelihood ratios (LR) of a test, with LR &#x0003e; 5 for high risk group and LR &#x0003c; 0.20 for low risk group.RESULTS: The highest accuracy, AUC of 0.78 was achieved with GRS combining 33 SNPs with optimal sensitivity and specificity of 75.0% and 72.7%, respectively. Individuals with the highest risk (GRS &#x0003e; 5.54) showed significantly increased odds of developing CD (OR = 26.65, 95%CI: 11.25-63.15) compared to the individuals with the lowest risk (GRS &#x0003c; 4.57) which is a considerably greater risk captured than in one SNP with the highest effect size (OR = 3.24). When more than 33 SNPs were included in GRS, discriminatory ability was not improved significantly; AUC of all 74 SNPs was 0.76.CONCLUSION: The authors proved the possibility of building accurate genetic risk score based on 33 risk variants on Slovenian CD patients which may serve as a screening tool in the targeted population.

Stair climbing,genetic predisposition,and the risk of incident type 2 diabetes:A large population-based prospective cohort study

Background:Cross-sectional evidence and small-scale trials suggest positive effects of stair climbing on cardiometabolic disease and glucose regulation.However,few studies have examined the long-term association between stair climbing and the incidence of type 2 diabetes(T2D).We aimed to prospectively evaluate the association of stair climbing with T2D and assess modifications by genetic predisposition to T2D.Methods:We included 451,699 adults(mean age=56.3±8.1 years,mean±SD;55.2%females)without T2D at baseline in the UK Biobank and followed up to March 31,2021.Stair climbing information was collected through the touchscreen questionnaire.Genetic risk score for T2D consisted of 424 single nucleotide polymorphisms.Results:During a median follow up of 12.1 years,14,896 T2D cases were documented.Compared with participants who reported no stair climbing,those who climbed stairs regularly had a lower risk of incident T2D(10-50 steps/day:hazard ratio(HR)=0.95,95%confidence interval(95%CI):0.89-1.00;60-100 steps/day:HR=0.92,95%CI:0.87-0.98;110-150 steps/day:HR=0.86,95%CI:0.80-0.91;>150 steps/day:HR=0.93,95%CI:0.87-0.99,p for trend=0.0007).We observed a significant interaction between stair climbing and genetic risk score on the subsequent T2D risk(p for interaction=0.0004),where the risk of T2D showed a downward trend in subjects with low genetic risk and those who reported stair climbing activity of 110-150 steps/day appeared to have the lowest overall T2D risk among those with intermediate to high genetic risk.Conclusion:A higher number of stairs climbed at home was associated with lower T2D incidence risk,especially among individuals with a low genetic predisposition to T2D.These findings highlight that stair climbing,as incidental physical activity,offers a simple and low-cost complement to public health interventions for T2D prevention.

Genetic variants involved in gallstone formation and capsaicin metabolism,and the risk of gallbladder cancer in Chilean women

AIM:To determine the effects of genetic variants associated with gallstone formation and capsaicin (a pungent component of chili pepper) metabolism on the risk of gallbladder cancer (GBC).METHODS: A total of 57 patients with GBC, 119 patients with gallstones, and 70 controls were enrolled in this study. DNA was extracted from their blood or paraffi n block sample using standard commercial kits. The statuses of the genetic variants were assayed using Taqman SNP Genotyping Assays or Custom Taqman SNP Genotyping Assays.RESULTS:The non-ancestral T/T genotype of apolipoprotein B rs693 polymorphism was associated with a decreased risk of GBC (OR:0.14,95% CI:0.03-0.63). The T/T genotype of cholesteryl ester transfer protein (CETP) rs708272 polymorphism was associated with an increased risk of GBC (OR:5.04,95% CI:1.43-17.8).CONCLUSION: Genetic variants involved in gallstone formation such as the apolipoprotein B rs693 and CETP rs 708272 polymorphisms may be related to the risk of developing GBC in Chilean women.

Population-standardized genetic risk score： the SNP-based method of choice for inherited risk assessment of prostate cancer

Several different approaches are available to clinicians for determining prostate cancer （PCa） risk. The clinical validity of various PCa risk assessment methods utilizing single nucleotide polymorphisms （SNPs） has been established; however, these SNP-based methods have not been compared. The objective of this study was to compare the three most commonly used SNP-based methods for PCa risk assessment. Participants were men （n = 1654） enrolled in a prospective study of PCa development. Genotypes of 59 PCa risk-associated SNPs were available in this cohort. Three methods of calculating SNP-based genetic risk scores （GRSs） were used for the evaluation of individual disease risk such as risk allele count （GRS-RAC）, weighted risk allele count （GRS-wRAC）, and population-standardized genetic risk score （GRS-PS）. Mean GRSs were calculated, and performances were compared using area under the receiver operating characteristic curve （AUC） and positive predictive value （PPV）. All SNP-based methods were found to be independently associated with PCa （all P 〈 0.05; hence their clinical validity）. The mean GRSs in men with or without PCa using GRS-RAC were 55.15 and 53.46, respectively, using GRS-wRAC were 7.42 and 6.97, respectively, and using GRS-PS were 1.12 and 0.84, respectively （all P 〈 0.05 for differences between patients with or without PCa）. All three SNP-based methods performed similarly in discriminating PCa from non-PCa based on AUC and in predicting PCa risk based on PPV （all P 〉 0.05 for comparisons between the three methods）, and all three SNP-based methods had a significantly higher AUC than family history （all P 〈 0.05）. Results from this study suggest that while the three most commonly used SNP-based methods performed similarly in discriminating PCa from non-PCa at the population level, GRS-PS is the method of choice for risk assessment at the individual level because its value （where 1.0 represents average population risk） can be easily interpreted regardless of the number of risk-associated SNPs used in the calculation.

Machine Learning Models for Genetic Risk Assessment of Infants with Non-syndromic Orofacial Cleft

The isolated type of orofacial cleft, termed non-syndromic cleft lip with or without cleft palate(NSCL/P), is the second most common birth defect in China, with Asians having the highest incidence in the world. NSCL/P involves multiple genes and complex interactions between genetic and environmental factors, imposing difficulty for the genetic assessment of the unborn fetus carrying multiple NSCL/P-susceptible variants. Although genome-wide association studies(GWAS)have uncovered dozens of single nucleotide polymorphism(SNP) loci in different ethnic populations, the genetic diagnostic effectiveness of these SNPs requires further experimental validation in Chinese populations before a diagnostic panel or a predictive model covering multiple SNPs can be built. In this study, we collected blood samples from control and NSCL/P infants inHan and Uyghur Chinese populations to validate the diagnostic effectiveness of 43 candidate SNPs previously detected using GWAS. We then built predictive models with the validated SNPs using different machine learning algorithms and evaluated their prediction performance. Our results showed that logistic regression had the best performance for risk assessment according to the area under curve. Notably, defective variants in MTHFR and RBP4, two genes involved in folic acid and vitamin A biosynthesis, were found to have high contributions to NSCL/P incidence based on feature importance evaluation with logistic regression. This is consistent with the notion that folic acid and vitamin A are both essential nutritional supplements for pregnant women to reduce the risk of conceiving an NSCL/P baby. Moreover, we observed a lower predictive power in Uyghur than in Han cases, likely due to differences in genetic background between these two ethnic populations.Thus, our study highlights the urgency to generate the HapMap for Uyghur population and perform resequencing-based screening of Uyghur-specific NSCL/P markers.

Race-specific genetic risk score is more accurate than nonrace-specific genetic risk score for predicting prostate cancer and high-grade diseases

Genetic risk score （GRS） based on disease risk-associated single nucleotide polymorphisms （SNPs） is an informative tool that can be used to provide inherited information for specific diseases in addition to family history, However, it is still unknown whether only SNPs that are implicated in a specific racial group should be used when calculating GRSs. The objective of this study is to compare the performance of race-specific GRS and nonrace-specitic GRS for predicting prostate cancer （PCa） among 1338 patients underwent prostate biopsy in Shanghai, China. A race-specific GRS was calculated with seven PCa risk-associated SNPs implicated in East Asians （GRS7）, and a nonrace-specific GRS was calculated based on 76 PCa risk-associated SNPs implicated in at least one racial group （GRS76）. The means of GRS7 and GRS76 were 1.19 and 1.85, respectively, in the study population. Higher GRS7 and GRS76 were independent predictors for PCa and high-grade PCa in univariate and multivariate analyses. GRS7 had a better area under the receiver-operating curve （AUC） than GRS76 for discriminating PCa （0.602 vs 0.573） and high-grade PCa （0.603 vs 0.575） but did not reach statistical significance. GRS7 had a better （up to 13% at different cutoffs） positive predictive value （PPV） than GRS76. In conclusion, a race-specific GRS is more robust and has a better performance when predicting PCa in East Asian men than a GRS calculated using SNPs that are not shown to be associated with East Asians.

A comparison of genetic risk score with family history for estimating prostate cancer risk

Prostate cancer （PCa） testing is recommended by most authoritative groups for high-risk men including those with a family history of the disease. However, family history information is often limited by patient knowledge and clinician intake, and thus, many men are incorrectly assigned to different risk groups. Alternate methods to assess PCa risk are required. In this review, we discuss how genetic variants, referred to as PCa-risk single-nucleotide polymorphisms, can be used to calculate a genetic risk score （GRS）. GRS assigns a relatively unique value to all men based on the number of PCa-risk SNPs that an individual carries. This GRS value can provide a more precise estimate of a man＇s PCa risk. This is particularly relevant in situations when an individual is unaware of his family history. In addition, GRS has utility and can provide a more precise estimate of risk even among men with a positive family history. It can even distinguish risk among relatives with the same degree of family relationships. Taken together, this review serves to provide support for the clinical utility of GRS as an independent test to provide supplemental information to family history. As such, GRS can serve as a platform to help guide-shared decision-making processes regarding the timing and frequency of PCa testing and biopsies.

Clinical validity and utility of genetic risk scores in prostate cancer

Current issues related to prostate cancer （PCa） clinical care （e.g., over-screening, over-diagnosis, and over-treatment of nonaggressive PCa） call for risk assessment tools that can be combined with family history （FH） to stratify disease risk among men in the general population. Since 2007, genome-wide association studies （GWASs） have identified more than 100 SNPs associated with PCa susceptibility. In this review, we discuss （1） the validity of these PCa risk-associated SNPs, individually and collectively; （2） the various methods used for measuring the cumulative effect of multiple SNPs, including genetic risk score （GRS）; （3） the adequate number of SNPs needed for risk assessment; （4） reclassification of risk based on evolving numbers of SNPs used to calculate genetic risk, （5） risk assessment for men from various racial groups, and （6） the clinical utility of genetic risk assessment. In conclusion, data available to date support the clinical validity of PCa risk-associated SNPs and GRS in risk assessment among men with or without FH. PCa risk-associated SNPs are not intended for diagnostic use; rather, they should be used the same way as FH. Combining GRS and FH can significantly improve the performance of risk assessment. Improved risk assessment may have important clinical utility in targeted PCa testing. However, clinical trials are urgently needed to evaluate this clinical utility as well as the acceptance of GRS by patients and physicians.

A Genetic Susceptibility Study of Lung Cancer Risk Potentially Associated with Polycyclic Aromatic Hydrocarbon Inhalation Exposure

For lifetime non-smokers, lung cancer risk is mainly associated with inhalation exposure to air pollution. For the Chinese population, indoor air pollution due to solid fuel combustion has been the primary source of inhalation exposure for decades. Polycyclic aromatic hydrocarbons （PAHs） are the by-products of incomplete combustion.

Genetic and phenotypic heterogeneity in tropical calcific pancreatitis

Tropical calcific pancreatitis(TCP)is a form of chronic non-alcoholic pancreatitis initially reported in the developing parts of the tropical world.The clinical phenotype of TCP has undergone marked changes since its first description in 1968.The disease is now seen in relatively older people with less severe symptoms.In addition,there are varying reports on the proportion of cases presenting with imaging abnormalities like calcification,ductal dilation,and glandular atrophy.Significant progress has also been made in understanding the etiopathology of TCP.The role of malnutrition and cassava toxicity in its pathogenesis is disproven and few studies have focused on the role of micronutrient deficiency and oxidative stress in the etiopathogenesis of TCP.Emerging evidence support an important role for genetic risk factors in TCP.Several studies have shown that,rather than mutations in trypsinogens,variants in serine protease inhibitor kazal type 1,cathepsin B,chymotrypsin C,cystic fibrosis transmembrane regulator,and carboxypeptidase A1,predict risk of TCP.These studies also provided evidence of mutational heterogeneity between TCP and chronic pancreatitis in Western populations.The current review summarizes recent advances that have implications in the understanding of the pathophysiology and thus,heterogeneity in genotype-phenotype correlations in TCP.

Diabetes Mellitus Type 2 Prevalence, Risk Factors and Complications in the Region of Kardzhali, Bulgaria

The present study was conducted within the CINDI program and covers 1,600 individuals, divided into four age groups. The aim of this study is to investigate the key biomedical and lifestyle factors for the development of type 2 diabetes as well as the cardiovascular diabetes complications in the population aged 25-64 from Kardzhali region, Bulgaria. The results of the study show the existence of behavioral and biological risk factors of health, as well as family history in the studied individuals who suffer from diabetes. The connection between diabetes and certain cardiovascular diseases such as ischemic heart disease, myocardial infarction and cardiovascular disease is confirmed. This requires the development and implementation of intervention activities among the population of Kardzhali, aimed at reducing the level of diabetes risk factors, early identification of risk groups and timely treatment of the diseased.

Pancreatic cancer risk variant ABO rs505922 in patients with cholangiocarcinoma

The aim of this study was to investigate an association between the development of cholangiocarcinoma(CCA)and the ABO variant rs505922(known to increase pan-creatic cancer risk)in a large cohort of European individuals with CCA.In total,180 individuals with CCA and 350 CCA-free controls were included.The ABO variant rs505922 was genotyped using a polymerase chain reaction-based assay.Association between this single nucleotide polymorphism(SNP)and CCA was tested in contingency tables.Neither allele distributions nor association tests and regression analysis provided evidence for an increased risk of CCA among carriers of the ABO variant(all P > 0.05).Nevertheless,we documented a deviation from Hardy-Weinberg equilibrium in the entire CCA cohort(P = 0.028)and for patients with intrahe-patic(P = 0.037)but not extrahepatic tumor localization(P > 0.05).The association tests did not provide evidence for a prominent role of the investigated SNP in the genetic risk of CCA.However,Hardy-Weinberg disequilibrium in the entire cohort and the intrahepatic CCA subgroup warrants future studies investigating a potential CCA risk modulation by individual blood groups.

Progress and challenges in genome-wide studies to understand the genetics of diabetic retinopathy

There are many advantages to understanding the genetics of human disease.Genetic markers can be used to calculate the risk of developing a disease,and elucidation of genetic risk factors can pinpoint the molecular aetiology of disease,which can facilitate the development of targeted therapies.Diabetic retinopathy(DR)is a common complication of diabetes that has a significant impact on quality of life.It has a clear genetic component,but determination of the genetic risk factors has proven difficult.To date,genome-wide studies for DR have been conducted on relatively small patient cohorts compared to other complex eye diseases and replication of genetic findings has been limited.The disease is highly heterogeneous,confounding attempts to classify patients into appropriate groups for genetic analysis and making direct comparisons between studies challenging.Future studies to determine the genetic causes of DR will need to focus on larger sample sizes,detailed phenotyping and appropriate classification of patients.Global co-operation and meta-analyses combining data from multiple studies will be critical to the discovery of genetic risk loci for DR.

The First Pilot Epigenetic Type Improvement of Neuropsychiatric Symptoms in a Polymorphic Dopamine D2 (-DRD2/ANKK (Taq1A)), OPRM1 (A/G), DRD3 (C/T), and MAOA (4R) Compromised Preadolescence Male with Putative PANDAS/CANS: Positive Clinical Outcome with Precision-Guided DNA Testing and Pro-Dopamine Regulation (KB220) and Antibacterial Therapies

Pediatric autoimmune neuropsychiatric disorders associated with or without streptococcal and other bacterial infections (PANDAS/CANS) are emerging as a featured pediatric disorder. Although there is some controversy regarding treatment approaches, especially related to the behavioral sequelae, we have hypothesized in other published work that it is characterized by the rapid onset of Reward Deficiency Syndrome (RDS) in children. We propose utilizing a multi-systems biological approach involving the coupling of genetic addiction risk testing and pro-dopamine regulation (KB220/POLYGEN®) to help induce “dopamine homeostasis” in patients with PANDAS, especially those with known DNA-induced hypodopaminergia. This case study examines a 12-year-old Caucasian male with no prior psychiatric issues who presented with a sudden onset of severe anxiety, depression, emotional liability, and suicidal ideation. The patient underwent genotyping and the genetic addiction risk score (GARS) testing, which revealed risk polymorphisms in the dopamine D2 (-DRD2/ANKK (Taq1A), OPRM1 (A/G), DRD3 (C/T), and MAOA (4R) genes. These polymorphisms have been linked to hypodopaminergia. The patient was subsequently placed on research ID-KB220ZPBMPOLY (POLYGEN®), and albeit the possibility of bias, based upon self and parental assessment, a marked rapid improvement in psychiatric symptoms was observed. In the second phase of treatment (102 days utilizing KB220), the patient received standard antibody testing, which was positive for Lyme. Antibacterial therapy started immediately, and KB220z was discontinued to provide a wash-out period. A monotonic trend analysis was performed on each outcome measure, and a consistently decreasing trend was observed utilizing antibacterial therapy. Our recommendation, albeit only one case, is to utilize and further research a combined therapeutic approach, involving precision-guided DNA testing and pro-dopamine regulation along with antibacterial therapy, as well as glutathione to address offensive enhanced cytokines, in patients with suspected PANDAS/CANS.

The power of a healthy lifestyle for cancer prevention:the example of colorectal cancer

Objective:We aimed to directly compare the estimated effects of adherence to a healthy lifestyle with those of risk predisposition according to known genetic variants affecting colorectal cancer(CRC)risk,to support effective risk communication for cancer prevention.Methods:A healthy lifestyle score(HLS)was derived from 5 lifestyle factors:smoking,alcohol consumption,diet,physical activity,and body adiposity.The association of lifestyle and polygenic risk score(PRS)(based on 140 CRC-associated risk loci)with CRC risk was assessed with multiple logistic regression and compared through the genetic risk equivalent(GRE),a novel approach providing an estimate of the effects of adherence to a healthy lifestyle in terms of percentile differences in PRS.Results:A higher HLS was associated with lower CRC risk(4,844 cases,3,964 controls).Those adhering to all 5 healthy lifestyle factors had a 62%(95%CI 54%-68%)lower CRC risk than those adhering to≤2 healthy lifestyle factors.The estimated effect of adherence to all 5 compared with≤2 healthy lifestyle factors was as strong as the effect of having a 79 percentile(GRE 79,95%CI 61-97)lower PRS.The association between a healthy lifestyle and CRC risk was independent of PRS level but was particularly pronounced among those with a family history of CRC in≥1 first-degree relative(P-interaction=0.0013).Conclusions:A healthy lifestyle was strongly inversely associated with CRC risk.The large GRE indicated that CRC risk determined by polygenic risk may be offset to a substantial extent by adherence to a healthy lifestyle.

A common variant in the precursor miR-146a sequence does not predispose to cholangiocarcinoma in a large European cohort

BACKGROUND:Micro-RNAs(miRNAs) are small,non-coding RNA species considered to fine-tune basic cellular functions by modulating target gene translation and/or mRNA stability.A common G/C polymorphism(rs2910164) in the precursor(pre-) miR-146a gene engaged in NF-κB signaling and apoptosis pathways has been reported to modulate the genetic risk of hepatocellular carcinoma by increased G-allelic production of mature miR-146a.We investigated rs2910164 in a large Europeanbased cholangiocarcinoma(CCA) cohort.METHODS:We recruited 182 CCA patients and 350 controls in three academic medical centers.Genotyping for rs2910164 was performed by PCR-based assays with 5’-nuclease and fluorescence detection.Genotype frequencies were tested for consistency with the Hardy-Weinberg equilibrium using an exact test;allelic and genotypic differences between the patients and controls were assessed by the Chi-square test and Armitage’s trend test.Exploratory subgroup analyses included gender,tumor localization(extra-versus intrahepatic CCA) and early-onset CCA.RESULTS:Genotype distributions were consistent with the Hardy-Weinberg equilibrium.No significant differences in either allele or genotype distributions were detected between the CCA and control groups or the respective subgroups investigated.However,there was a trend for a protective effect of the heterozygous single-nucleotide polymorphism state GC,as indicated by an underrepresentation in the CCA group in general(29% vs 35%;P=0.18) and,in particular,for extrahepatic tumor sites(26% vs 35%;OR=0.67;95% CI,0.43-1.02;P=0.065).CONCLUSIONS:Our data do not support a prominent contribution of the pre-miR-146a sequence variant in the genetic predisposition to CCA.However,current studies functionally characterizing rs2910164 have proposed that distinct repertoires of target genes are addressed by genotype-specific mature miR146a species.Given the detected trend towards a potentially protective role of GC heterozygosity,a subtle modulation of genetic CCA risk by the pre-miR-146a GC genotype may exist and should be evaluated further.