P = 0.028), nail  and cardiac tissue  samples. Patients’ age, sex, BMI, hypertension and diabetes status adjusted analysis showed that patients from arsenic-affected areas had significantly higher iAs concentration in cardiac tissue  reflecting higher cardiac tissue injury among them , which in turn allowed the analysis to assume that the iAs exposure have played a vital role in patients’ disease condition. Adjusted analysis showed significant association between urinary iAs concentration with AA (P = 0.012) and AG (P = 0.034) genotypes and cardiac iAs concentration with AA (P = 0.017) genotype of AS3MT rs10748835. The AG genotype of AS3MT rs10748835 , AA genotype of NOS3 rs3918181 , GG genotype of ICAM1 rs281432  and AA genotype of SOD2 rs2758331  were found significantly associated with CVD patients from arsenic-affected areas. Again, adjusted analysis showed significant association of AA genotype of AS3MT rs10748835 with CVD patients from arsenic affected areas. In comparison to the reference genotypes of the selected SNPs, AA of AS3MT 10748835, AG of NOS3 rs3918181 and AC of rs3918188, GG of ICAM1 rs281432, TT of VCAM1 rs3176867, AA of SOD2 rs2758331 and GT of APOE rs405509 significantly increased odds of cardiac tissue injury of CVD patients from arsenic affected areas. The results showed that the selected SNPs played a susceptibility role towards cardiac tissue iAs concentration and injury among CVD patients from iAs affected areas.Inorganic arsenic (iAs) exposure has been reported to have an impact on cardiovascular diseases (CVD). However, there is not much known about the cardiac tissue injury of CVD patients in relation to iAs exposure and potential role of single nucleotide polymorphisms (SNPs) of genes related to iAs metabolism, oxidative stress, endothelial dysfunction and inflammation which may play important roles in such CVD cases. In this dual center cross-sectional study, based on the exclusion and inclusion criteria, we have recruited 50 patients out of 270, who came from known arsenic-affected and- unaffected areas of mainly Chittagong, Dhaka and Rajshahi divisions of Bangladesh and underwent open-heart surgery at the selected centers during July 2017 to June 2018. We found that the patients from arsenic affected areas contained significantly higher average iAs concentrations in their urine (6.72 ± 0.54 ppb,  According to WHO, the acceptable limit of iAs in groundwater is 10 μg/L. However, Due to its geogenic origin, in Bangladesh, the iAs concentration in groundwater is often above 50 μg/L  and sometimes exceeds WHO allowed level at as much as 50 fold higher concentration3. Human exposure to iAs has been found to be related with severe health consequences including several types of malignancies, e.g. skin, bladder, liver and lung; and various non-cancerous diseases including cardiovascular diseases (CVD)6, though heart is not the primary depository organ of iAs in human body. However, iAs exerts its toxicity on multiple organs in the human body and in our study, we focused on the heart, as evidence suggests that iAs has adverse toxic effects on this vital organ7.Arsenic contamination, more specifically the inorganic arsenic (iAs) contamination has been identified as a major health threat to the millions of people worldwide, including Bangldesh8. Recent studies have reported that chronic exposure to iAs (low to moderate level) is widely prevalent for the risk of CVD. However, several other important factors including age, gender and alterations in the iAs biotransformation and oxidative stress response genes can also influence the susceptibility towards iAs related diseases among individuals 12. The hypothesis that iAs induces oxidative stress and vascular inflammation, which play central role in developing atherosclerosis and CVD, has been supported by recent experimental studies14. Moreover, polymorphisms of the genes responsible for iAs biotransformation , oxidative stress , inflammation/endothelial dysfunction ; and inflammation , can be attributed as one of the reasons for the difference in individual susceptibility to iAs-induced CVD18. Some variants of these genes may alter the efficiency of iAs biotransformation and thus may cause difference in the level of susceptibilities of the cardiovascular effects of iAs exposure.Among the non-communicable diseases , CVD is the top ranked cause of death worldwide21. The gene AS3MT  contains 11 exons which encodes the methyltransferase catalysing the biotransformation of trivalent inorganic arsenicals to their respective non-toxic derivatives24. It has been observed that within the same population, individual metabolic responses to different iAs metabolites vary which can be attributed to the polymorphism of AS3MT gene26. Polymorphic variations in AS3MT gene may cause differences in the individual response in such iAs biotransformation process and thus may influence iAs-induced health effects. In addition, the pathophysiological factors including high oxidative stress along with recurrent low-grade inflammation are important for the development of CVD e.g. hypertension and atherosclerosis27. Multiple genetic polymorphisms SOD2 and APOE are likely to be associated with the development of CVD as these genes are related with oxidative stress and chronic inflammation28. NOS3 gene is involved in oxidative stress and two of its reference SNPs (rs3918181 A/G and rs3918188 A/C) have been marked for their association with CVD and coronary artery disease risk and ischemic stroke31. eNOS in humans produces NO from L-arginine and through this metabolic reaction, L-arginine regulates blood pressure and helps to control endothelium-dependent vasodilatation33. Some SNPs of NOS3 gene can affect NOS3 activity, resulting in endothelial dysfunction and might play a vital role in atherosclerotic heart disease formation30. Maintenance of blood pressure homeostasis and vascular integrity are driven by the production of nitric oxide, which is catalysed by the enzyme eNOS. Variation in the expression of eNOS gene and its activity has been linked to primary hypertension. Moreover, a growing body of evidence has reported an association between primary hypertension and polymorphisms of the eNOS gene32. Additionally, the risk factors for atherosclerosis  are also found to be related with the level of adhesion molecules, i.e. ICAM1 and VCAM135. Monocyte recruitment, which regulates the aggregation of atherosclerotic plaques, is facilitated by the endothelial adhesion molecules which include selectins, ICAM-1 and VCAM-137.When iAs enters human body, pentavalent arsenate (AsV) is reduced to arsenite (AsIII) and subsequently converted to methylated forms  as a detoxification process catalyzed by arsenite methyl transferase enzyme (AS3MT)45. Furthermore, we measured the arsenic exposure of the patients by measuring the iAs concentration in their urine and nail samples. Our recruited patients were mostly from the well documented arsenic contaminated areas of Chittagong, Dhaka and Rajshahi divisions of Bangladesh (see Supplementary Dataset).Although genetic constitution responsible for the supposed protection or predilection to the effects of iAs exposure may play a critical role in CVD pathogenesis, to our knowledge, not much research has been done on this association in the Bangladesh perspective. Therefore, to investigate whether there is any association between iAs exposure and CVD incidence in Bangladesh perspective, we designed a cross-sectional study where 50 CVD patients were recruited from a total of 270 patients who underwent open-heart surgery at the Department of Cardiac Surgery, Chittagong Medical College Hospital, Chittagong and National Heart Foundation Hospital & Research Institute, Dhaka in between July 2017 to June 2018. The inclusion criteria include non-congenital CVD patients with the age range 20–70 years, willingness of the patients to participate in our study and adequacy of the samples . We excluded patients with congenital CVDs (see Supplementary Method AS3MT rs10748835), oxidative stress  and inflammation/endothelial dysfunction (ICAM1 rs281432 and VCAM1 rs3176867) within CVD patients. Most of the selected SNPs are within the intronic region of the genes. Unlike the exonic variants, intronic SNPs exhibit difference in association among populations. We wanted to explicate the associations of a selected list of SNPs of intronic origin that were previously found strongly associated with CVD and iAs metabolism in some populations. The intrinsic function of these SNPs are slowly being understood, therefore, the association of these intronic SNPs with diseases in different populations needs to be clarified so that further studies can emphasize on the functional aspect of their disease association. In short, our study aimed to check the existence of any association between single nucleotide polymorphisms (SNPs) of the genes mentioned above and CVD in the patients who were living in the aforementioned areas of Bangladesh and were exposed to varied ranges of iAs.We measured the cardiac tissue iAs concentration of the patients and scored cardiac tissue injury by histopathology. We further evaluated the genetic susceptibility of iAs exposure and subsequent CVD risk, i.e. if the cardiovascular effects of iAs exposure differs due to the polymorphic variations in genes responsible for iAs methylation (P < 0.05) in urine, nail and cardiac tissues than the iAs-unexposed subject group. iAs-exposed subjects also had significantly more cases hypertension (P < 0.05) than the iAs-unexposed subjects. In Table P = 0.046).Table P = 0.012) and AG (P = 0.034) genotypes of rs10748835 of AS3MT were significantly associated with urinary iAs concentration (Table P = 0.017) genotype was significantly associated with cardiac tissue iAs concentration. This genotype also increased the odds of cardiac iAs concentration .Adjusted binary logistic regression revealed that the AA , AA genotype of NOS3 rs3918181 , GG genotype of ICAM1 rs281432  and AA genotype of SOD2 rs2758331  in iAs-exposed CVD patients group. No association was found among iAs-exposed and unexposed groups for the distribution of the other three selected SNPs. Furthermore, we found significant differences (P < 0.05) in the distribution of A allele of NOS3 rs3918181; G allele of ICAM1 rs281432 and A allele of SOD2 rs2758331genes among iAs-exposed and unexposed patient’s groups , NOS3 rs3918181 (P < 0.005), ICAM1 rs281432 (P = 0.003) and VCAM1 rs3176867 (P = 0.035) differed significantly among the patients. The genotypes of NOS3 rs3918181 (P = 0.001) and SOD2 rs2758331 (P = 0.011) were significantly different among the patients in case of nail iAs concentration. Finally, AS3MT rs10748835 (P < 0.005) and NOS3 rs3918181 (P = 0.0001) genotypes differed significantly among the patients in case of cardiac tissue iAs concentration. The other SNP genotypes did not show any significant differences for iAs concentration in urine, nail and cardiac tissue samples of the patients. The significant distribution differences in the genotypes of the aforementioned selected SNPs among the patients for iAs concentration in the samples reflect their possible susceptibility role towards iAs exposure.The difference of genetic polymorphisms with measured iAs concentrations in biological samples  were analyzed by Student’s st Table . In caseAS3MT, NOS3, ICAM1, VCAM1, SOD2 and APOE were analyzed. No significant differences were found , serum bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and random blood glucose (RBS). We found that the level of Serum Creatinine, BUN, AST and ALT increased significantly among the patients from highly iAs-affected areas. The level of serum total protein was found to be significantly decrease among the patients from highly iAs-affected areas Fig. .Figure 1P < 0.05) between ALT levels and polymorphisms of AS3MT rs10748835, NOS3 rs3918181 and rs3918188, AST levels were found to be significantly (P < 0.05) increased in patients with AA genotype of NOS3 rs3918181 (Table P < 0.05) of NOS3 rs3918181 and rs3918188 were also shown higher AST levels. The polymorphisms of APOE rs405509 also marked significant (P < 0.05) association with AST levels. BUN levels were found significantly associated with only VCAM1 rs3176867, while no association were found with other genes. No significant difference was found in serum total protein levels with SNPs of our selected genes. For serum creatinine levels significant association (P < 0.05) was found with AS3MT rs10748835 (AA genotype), APOE rs405509 (GG genotype) and SOD2 rs2758331 (AC genotype). Serum Bilirubin was only found associated (P < 0.05) with NOS3 rs3918181 (GG genotype). RBS levels showed significant difference for the polymorphism of APOE rs405509 (AA genotype)  Table .Table 6RP < 0.05) were observed in the patients from iAs-affected areas than the patients from iAs-unaffected areas   Table . For thi46. It is acknowledged by multiple government and non-government organizations that a large portion of Bangladeshi residents are exposed to iAs on a daily basis ranging from low to higher concentration. However, among these exposed residents, a fraction, due to their genetic differences, may develop CVD as an effect of chronic iAs exposure47. In this cross-sectional study, we reported that patients with CVD from iAs affected areas, found to be associated with higher iAs deposition in the nail, cardiac tissue and lower iAs-methylation capacity, which were indicated by high iAs concentration in the urine. The result also showed that polymorphisms in AS3MT SNP rs10748835, NOS3 SNP rs3918181, ICAM1 SNP rs281432 and SOD2 SNP rs2758331, in concert with arsenic exposure increases the risk to CVD.Recent epidemiological studies have confirmed that among the people living in the same area showed different susceptibility towards iAs exposure due to their genetic variation and such genetic differences alter individual susceptibilities to develop certain diseases, including cardiovascular diseasesAS3MT gene has been reported to be associated with iAs exposure and CVD 50. In their study, Gong and O’Bryant reported that patients with AG genotype at rs10748835 were at elevated risk of coronary heart diseases and hyperlipidemia than patients with AA genotype at the same SNP position49. Inconsistent with their findings, our results showed a significant difference in the distribution of AG genotype  between iAs-exposed and un-exposed patient groups (Table AS3MT rs10748835 in urine (P = 0.047) and cardiac tissue (P < 0.005) iAs concentration among the patients . The AA genotype showed significant association with both urinary and cardiac iAs concentration (P = 0.012 and 0.017 respectively). Some other SNP association studies found no relations to disease susceptibility with this intronic variant, rs10748835 of AS3MT gene in disease susceptibility, however, the outcomes can vary possibly due to differences in populations and races52. Therefore, the findings of our study allow us to suspect that AA and AG genotypes, specifically, the A allele might be the susceptibility factors for iAs exposure, cardiac iAs concentration and subsequent cardiac tissue injury and CVD in the Bangladeshi population.Polymorphic alterations of the reference alleles of this gene are reported to cause reduction in the iAs metabolism efficiency, and in addition, A/G polymorphism at rs10748835 of NOS3 polymorphism and arsenic exposed CVD patients is controversial; one of the reasons might be the differences in populations and races. Among the Chinese Han population, Du et al. (2008) reported GG genotype and G allele of NOS3 rs3918181 (A/G) to be strongly associated with ischemic stroke ; whereas, Yang et al. (2014) showed no association of this SNP with primary hypertension32. In our study subjects, we found AA genotype and A allele of NOS3 rs3918181 (A/G) to be significantly associated with the CVD patients from iAs-affected area (Table P < 0.005), nail (P = 0.001) and cardiac tissue (P = 0.0001) iAs concentrations (Table . (2017) reported that the AA genotype of rs3918188 SNP of NOS3 gene, may cause an individual to be more susceptible to develop T2DM53. Zhao et al. (2016) found no affirmative association between NOS3 rs3918188 with CAD30. Consistently, no significant association was found with a specific genotype of NOS3 rs3918188 with iAs-exposed CVD patients in this study  of the patients indicate the chronic exposure of them to arsenic. The likelihood of cardiac injury due to the presence of AG genotype at NOS3 rs3918181 and AC genotype at rs3918188 was found to be increased in adjusted binary logistic regression analysis, marking the values 2.201, 95%CI 1.167–4.153 (P = 0.015) and 2.567, 95%CI 1.317–5.003 (P = 0.006) respectively.The association of t al. 201 showed nt al. 201 showed n. (2015) reported the association of SOD2 rs2758331 (A/C) with well-water arsenic, and they found no significant association for this SNP with cardiovascular diseases in Bangladeshi population28. But in our study, we found that among iAs-exposed and unexposed patients’ groups, the frequency of AA genotype of SOD2 rs2758331 was significantly different  (Table P = 0.086). The presence of AA genotype increased the odds of cardiac tissue injury significantly . We suspect that AA genotype or A allele of SOD2 rs2758331 (A/C) might be the risk factors for CVD in iAs exposed patients. Though the finding showed dissimilarities with the previous study and there is not much research on this, therefore, further study should consistently explore the role of SOD2 in creating oxidative stress and its contribution to CVD in iAs exposed patients.Wu et alAPOE with CVD has been reported in various studies28. Clark et al. (2009) reported that GG genotype of APOE rs405509 is significantly associated with hyperlipidemia in Caucasian sample population55. Although, in our study, we found no association between APOE rs405509 polymorphism and CVD patients from iAs-affected areas (Tables P = 0.010). The results of rs405509 of APOE gene of our study thus needs to be validated by involving more CVD patients from iAs affected areas of Bangladesh.The association of 36. Moreover, these markers are also found to be positively associated with iAs exposure56. We found that ICAM1 rs281432 is significantly associated with iAs exposure and CVD  in the distribution of GG genotype in iAs-exposed and unexposed patients’ groups (Table P = 0.063). The genotype distribution of ICAM1 rs281432 were also found to be significantly different in case of urinary iAs concentration (P = 0.003) among the patients (Table P = 0.009). Thus, findings of our study support the role of endothelial dysfunction as an underlying mechanism of iAs exposure and subsequent adverse cardiovascular effects and allow us to suspect that GG genotype or G allele might be one of the risk factors for CVD among the people living in iAs affected areas.Circulating sICAM-1 and sVCAM-1 levels can be used for the prediction of CVD risk. (2015) reported a strong association between CC genotype of VCAM1 rs3176867 with iAs exposure and cardiovascular disease28. In our study, we found no significant association with any specific genotypes in iAs-exposed and unexposed patients’ groups  (Table P = 0.035) among the patients. Consequently, such discordance between the association analysis results of previous studies and ours need to further investigated.Although Wu et alps Table . However9) Table . Moreove57. The significantly higher level of liver function enzymes AST, ALT and serum bilirubin, Kidney function markers serum creatinine, and BUN indicated the arsenic intoxication among the iAs-exposed patients' groups59. Arsenic disturbs glucose metabolism by uncoupling of oxidation and phosphorylation, causing excess availability of unutilized glucose60. In the study subjects, an increase was found in the RBS levels in the iAs-exposed patients' group. A significant decrease (P = 0.003) was found in the levels of serum total protein in the iAs-exposed patients' group. This is because increased breakdown (catabolism) of proteins due to possible oxidative stress by reactive oxygen species generated by arsenic toxicity may contribute to decreased protein levels60. Arsenic exposure has been described as a cause of bone marrow depression, causing haematologic abnormalities61. This study also reported a significantly lower average white blood cell (WBC) count and platelet count (P < 0.05), moderately lower average red blood cell (RBC) count and haemoglobin (Hb) level in the iAs-exposed patients.Additionally, biochemical and haematological profiles of the patients also indicated the associated health complications due to iAs exposure. These parameters do not necessarily confirm or rule out iAs exposure, as various factors other than iAs exposure might also cause their fluctuations. More importantly, these parameters are more difficult to explain in humans where exposure is almost entirely natural and both exposure level and outcomes depend on a combination various factors. However, one noteworthy phenomenon here is that the differences in these parameters were almost always more pronounced in patients from > 50 ppb iAs areas, the higher exposed patient category. Chronic iAs exposure causes damages to internal organs such as the liver, the cardiovascular system, the nervous system, kidneys, and lungs31. Therefore, increased oedema, leukocyte infiltration, myocardial fibre swelling, fibre separation, fibrosis and fatty changes are the hallmarks of iAs exposure related cardiac injury62. In this study, we found higher injury score for these parameters among the iAs-exposed patients  in their cardiac tissue samples than the iAs-unexposed patients' group. A strong correlation has also observed between arsenic deposition and genotype. For four genotypes , among the seven tested SNPs, were found significantly (P < 0.05) associated with the arsenic concentrations in the cardiac tissue. Therefore, this finding strengthens the combined role of iAs exposure and the SNP genotypes in possible association with CVDs.In our study, we tried to confirm that iAs-exposure served as one the contributing factors for the CVD among the study subjects and also tried to minimize and exclude other possible contributing factors for the development of CVD among the cardiac surgery patients by involving three criteria. These included, (i) retrieval of National iAs-contamination data of patients’ residential area, (ii) by measuring iAs-concentration of patients’ pre-operative urine and nail; and more importantly, (iii) by measuring iAs-concentration of cardiac tissues collected from the cardiac surgery patients. It may be noted here that confirming iAs-exposure by strictly maintaining these three criteria is relatively difficult than confirming iAs-exposure only by first two of the selected criteria. This is due to the facts that (a) such cardiac tissue collection for research is difficult as patient safety is paramount and (b) cardiac tissue collection from patients who do not meet the first two criteria will go in vain and ultimately jeopardize the actual findings of the study. As such, the number of patients, who meet all the exclusion and inclusion criteria .As this was a study involving human subjects, the detail methods, written informed consent form and data collection questionnaire were submitted to the Ethical Review Committee (ERC) of Chittagong Medical College and obtained prior approval. American College of Cardiology guidelinesNail (fingers and toes), urine and peripheral blood were collected from each patient shortly after their admission at the aforementioned cardiac surgery centers. Usually the surgery took place within a week of the admission. Nail and urine were used for the iAs exposure measurement of each patient. Peripheral blood was used for biochemical and haematological analysis relevant to this study. A very small portion (0.5 cm × 0.5 cm × 1.0 cm) of the cardiac tissue was collected  were processed for total iAs concentration measurement by Hydride Generation Atomic Absorption Spectrophotometry  as described previously67 semi quantitative grading with some modifications showed cardiac injury of a patient as a sum of all parameter scores  was used as preservative and stored at 4°C until transportation to histopathology lab. At the beginning of tissue processing, the tissue samples were sectioned into small longitudinal and transverse pieces. The small gross sections were put in an automatic tissue processor for dehydration by using gradually increased concentration of ethanol (ranging from 50 to 100% v/v). After dehydration xylene treatment was done sequentially three times to remove the ethanol. Paraffin infiltration was done at 65°C and cavity blocks were used for tissue placement and chilled for hardening. A microtome was used to cut ~ 6 μm thick tissue sections from the paraffin blocks. Sections were placed in a 45°C heated water bath and fixed on a slide after a few minutes. The tissue fixed slides were hydrated by placing in gradually decreasing concentration of ethanol (from 100 to 70% v/v), then stained with hematoxylin and eosin, again rehydrated and washed with xylene and then observed with a light microscope . Oedema, leukocyte infiltration, myocardial fibre swelling, fibre separation, fatty changes and fibrosis were examined for pathological grading of the collected samples. Severity for these parameters were graded with scores from 0  to 4 (severe). This previously descrivedComplete blood count  of the pre-operative blood samples was done by using an automatic haematology analyzer . Other blood serum parameters including creatinine level, total protein, BUN, AST, ALT and RBS by using diagnostic kits manufactured by Human GmbH (Germany) with Erba Chem 5v3 Clinical Chemistry Analyzer . The details of the assay protocols are described in Supplementary Method r2 threshold > 0.8 and allele frequency cutoff ≥ 5% 70. Most of the selected SNPs were in the intronic region of the genes. However, many of the intronic SNPs fall within the regulatory region  of the gene and can exert effects splicing and expression. Similar importance is put on the role of these cryptic variations in gene regulation. We used Ensembl variant effect predictor to find out if the selected intronic SNPs of this study can affect transcription , non-sense mediated decay variant  as well as being located upstream and downstream of the protein coding gene (NOS3 rs3918188 and SOD2 rs2758331). All these variations are considered as modifiers with still not known definitive functions.SNPs were selected from the phase III datasets of International HapMap project and further screened with Genome variation Server 138 with the parameters AS3MT rs10748835 and NOS3 rs3918181 were detected by polymerase chain reaction followed by restriction enzyme digestion with ApaLI and RsaI respectively as described previously by Gong and O’Bryant (2012) and Yang et al. (2014)49. For the rest of selected SNPs, the amplicons were sequenced by capillary electrophoresis sequencing . The PCR primers used in this study are given in Supplementary Table 2 , one unit GoTaq Flexi DNA Polymerase , 1X GoTaq Flexi Reaction Buffer  and nuclease free water  in a final reaction volume of 25 μL. The reactions were carried out in a Qantarus Q-Cycler .Genomic DNA from cardiac tissue was isolated by using standard phenol–chloroform-Iso amyl alcohol extraction and ethanol precipitation. SNPs of t test for age, BMI and iAs concentration in urine, nail and cardiac tissues, and by Chi-square test for sex, smoking habit, hypertension and diabetes mellitus. The differences in the allelic and genotypic frequencies for the selected SNPs were analysed by using Chi-square test between two patient groups. Odds ratios were also calculated in each Chi-square test. Significant differences among the genotypic distribution of the SNPs in regard to iAs concentration in urine, nail and cardiac tissue were calculated between the two patient groups by Student’s t test. Single factor ANOVA was done for the analysis of association between biochemical parameters and the SNP genotypes. iAs exposure assessment was done by binary logistic regression between the two patient groups in relation to the iAs concentration measured in urine, nail and cardiac tissue. For toxicokinetic association analysis, binary logistic regression was done among the AS3MT rs10748835 genotypes and iAs concentration measured in urine, nail and cardiac tissue between the two patient groups. Furthermore, binary logistic regression was done for the association between all the selected SNP genotypes and total cardiac tissue injury score. All the logistic regression analyses were adjusted for age, sex, BMI, hypertension and diabetes mellitus status of the patients.All the analyses were done by dividing the patients into two groups: patients from iAs affected areas and patients from iAs unaffected areas. The highest nail iAs concentration of the patients from iAs affected area (276 ppb) was considered as cut off value for the determination of iAs exposed status of the patients; the patients who had higher iAs concentration in nail than 276 ppb was considered iAs exposed, which included all the patients from iAs affected areas. Significant differences were calculated between the two patient groups by Student’s Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Chronic plaque psoriasis and psoriatic arthritis are multifactorial inter-related diseases with strong genetic contributions. Better elucidation of the heritability of psoriatic disease subsets is important for identifying novel genes, risk stratification and potential clinical applications. In this study, we used two mixed-effect modelling methodologies to assess the additive contribution of common single nucleotide polymorphisms from genome-wide association studies to estimate the heritability of cutaneous psoriasis, psoriasis vulgaris and psoriatic arthritis. We found that cutaneous psoriasis and psoriatic arthritis both exhibit considerable heritability, with a greater contribution coming from cutaneous psoriasis. Although the etiology of psoriatic disease is not fully elucidated, it is widely viewed as a multifactorial disease as there is substantive evidence to implicate an interaction between inherited genetic factors and environmental triggers2. PsA can be thought of as a distinctive entity within a broader disease . To investigate the genetics of psoriatic disease, it is prudent to phenotypically segregate this entity into three subsets: psoriasis in the absence of arthritis , inflammatory arthritis with psoriasis  and psoriasis irrespective of inflammatory arthritis . Under this classification, PsA or PsC alone represents a more homogenous group than PsV. Defining such homogeneous phenotypes will facilitate the elucidation of disease-related genes and relevant signalling pathways specific to cutaneous and articular manifestations of psoriatic disease.Chronic plaque psoriasis (henceforth termed ‘psoriasis’) is a complex immune-mediated cutaneous disease. Psoriatic arthritis (PsA) is an inflammatory arthritis that occurs in approximately 20 to 30% of patients with psoriasis3. Broad-sense heritability (H2) refers to the proportion of phenotypic variation due to genetic value including effects due to dominance and epistasis. In contrast, narrow-sense heritability (h2) is the proportion of genetic variation due to additive genetic values, and represents the heritability measure most commonly used in human complex disease studies3.Heritability essentially refers to how much variation in a trait is due to variation in genetic factors. Phenotypes vary within a population due to environmental exposure and genotypes at particular loci. The combined effect of all loci is the genotypic burden or value due to the additive effect of all loci, and possible allelic interactions within a locus (dominance) and between loci (epistasis)4. Population-based genetic epidemiological studies quantitate familial aggregation using prevalence of affected first degree relatives of probands compared with prevalence of affected first degree relatives of unaffected controls5. The recurrence rate of PsA among first degree relatives of PsA probands (λs 30 to 48) is greater than the recurrence of psoriasis among first degree relatives of PsV probands (λs 4 to 10)9. Reliance on the population prevalence of disease is a major limitation of familial aggregation estimates based on population risk. Underestimating the prevalence of PsA in the general population can inflate the familial aggregation estimate when calculating the sibling recurrence risk score (λs). This is of particular relevance in PsA as estimates for population prevalence in the literature vary widely from 0.1% to 0.5%11. Notably, the prevalence in siblings of probands with PsA compared to siblings of probands with PsC was similar in these studies, indicating that the genetic contribution is driven mainly by psoriasis. Genetic contribution to complex disease can also be estimated from twin studies by comparing concordance of monozygotic and dizygotic twins13. Although there are no large PsA twin studies reported in the literature, for PsV the monozygotic twin concordance rate is almost three times higher (62 to 70%) compared with dizygotic twins (21 to 23%) resulting in heritability estimates (h2) between 60% and 90%14.Population-based genetic epidemiological studies, immunogenetics, candidate genes and more recently GWAS studies have demonstrated a strong genetic basis for psoriatic disease as previously reviewed15. The lack of PsA-specific genes identified from GWAS studies combined with the considerable clinical heterogeneity of PsA and the lack of cases in PsA GWAS studies (compared with psoriasis), suggest that additional GWAS on larger PsA cohorts followed by meta-analyses should identify additional PsA-specific variants or that the genetic burden (or variance) for PsA is just not as high as originally thought. Recently, algorithms have been developed to assess heritability of complex disease through unbiased estimates of the variance explained from genome-wide arrays or whole-genome sequencing. The three most widely used single nucleotide polymorphism (SNP)-based methods to estimate heritability are genomic relatedness matrix residual maximum likelihood analysis (GREML) in the genome-wide complex trait analysis package (GCTA)16, linkage disequilibrium (LD) adjusted kinships approach (LDAK)17, and LD score regression18. In this study, we set out to assess the heritability of PsC, PsA and PsV by interrogating SNPs from large-scale genotyping arrays. Better approximation of the heritability of PsC, PsV and PsA will culminate in more efficient genetic profiling of psoriatic disease and facilitate gene identification studies by providing more accurate estimates of sample sizes needed based on the heritability of different subsets of psoriatic disease.Over 60 genetic signals have reached a genome-wide level of significance from genome-wide association scan (GWAS) studies in psoriasis, whereas ~20 genetic signals have achieved the same in PsA cohorts as previously reviewed20. Table 21. The heritability of PsC, PsV and PsA were estimated by interrogating 2938 PsV, 1155 PsC, 715 PsA subjects and 3117 unaffected controls.The data used for this study were obtained from a previously published GWAS study in psoriatic diseaseAll samples were genotyped on a custom Axiom Biobank plus genotyping array with 461,092 autosomal SNPs. Stringent quality control (QC) was performed and genetic markers with a high missing rate (>1%), low minor allele frequency (MAF < 5%), or significant deviation from Hardy-Weinberg equilibrium (p < 0.001) were filtered, with 230k SNPs remaining for heritability estimation. SNPs were imputed based onautosomal reference panel of HapMap Phase 3 (HM3) CEU cohort with 1.5 million sites. After setting imputation quality cut-off as >0.9, 735k high quality SNPs remained. SNPs with low MAF (<5%) and SNPs with significant deviation from Hardy-Weinberg equilibrium (p < 0.001) were excluded, leaving a total of 401k imputed SNPs for the heritability estimation. Since the major histocompatibility complex (MHC) region has a large impact on the SNP heritability estimation for psoriatic disease and related phenotypes, heritability was also estimated after removing SNPs located within the MHC region (chr6:25–35 Mb).2SNP) is defined as the proportion of total phenotypic variation due to additive genetic effects of a given set of typed SNPs, and can be calculated as ratio of the variance of total genetic effects to the total phenotypic variance. The effects of non-additive genetic variation and environment will not confound the h2SNP. These variance components were estimated using genotypic relatedness matrix (GRM) and the restricted maximum likelihood (REML) approach. The heritability of PsC, PsV and PsA in our analysis was estimated using the LDAK and GCTA methods. The LDAK and GCTA methods both rely on the restricted maximum likelihood algorithm (ReML). However, these two approaches have different assumptions for the relationships between the expected SNP heritability and allele frequency, levels of LD and genotype certainty.In this study, the heritability was estimated using the typed SNPs. This SNP heritability : the SNPs were re-weighted to account for LD; SNPs in low LD contribute more than those in high-LD regions; higher-quality SNPs contribute more than lower-quality ones, while in the GCTA method: these effects are independent of LD and strongly inversely proportionate to MAF, also GCTA’s heritability does not vary with genotype certainty. These two methods both have their limitations and are sensitive to assumptions about genetic architecture23. In the heritability estimation, to account the population stratification, we included first five eigenvectors from PCA as covariates. We also included sex as a covariate to control the sex effect. Parallel analyses were performed after removing SNPs from the MHC region. For case–control studies, the heritability estimation can be biased by the prevalence of the disease in the population. Thus, the population prevalence was varied from 0.5% to 4.0%  to assess the robustness of the heritability estimations.The estimation of heritability requires the samples with similar ancestry. The potential population stratification will affect the estimation. We performed principal components analysis(PCA) on our samples with genotype data2 = 0.58) was greater than both PsV (h2 = 0.37) and PsA (h2 = 0.41) (Table 2 = 0.50) was greater than both PsV (h2 = 0.32) and PsA (h2 = 0.43) (Table 2 = 0.29 vs 0.33) was greater than both PsV (h2 = 0.19 vs 0.22) and PsA (h2 = 0.25 vs 0.28) with and without SNP imputation  being contributed from the SNPs located within the MHC region.The heritability assessment for psoriatic disease using both the LDAK and the GCTA methods are presented with and without imputation and including and excluding SNPs located within the MHC region. Although the heritability estimates vary depending on the LDAK and the GCTA methods, there are numerous similarities. Using the LDAK method with 230k SNPs from GWAS data and adjusted with a prevalence as 1%, the heritability of PsC (h2 = 0.42), PsA (h2 = 0.39) and PsV (h2 = 0.26) as compared with the LDAK method (Table 2 = 0.34) was greater than both PsA (h2 = 0.27) and PsV (h2 = 0.20) (Table 2) estimates of the GCTA method were similar irrespective of disease prevalence or imputation  in PsC, PsA and PsV.et al. used the restricted maximum likelihood algorithm (REML), implemented in the GCTA package, to estimate the proportion of variance in phenotypes from the genomic relationship matrix using the mixed-effects model25. This method was applied to common, binned SNPs to capture the amount of heritability caused by possible causal variants. In this model, the SNP effect should conform to a normal distribution without environmental interaction. Yang’s GREML approach is independent of LD; however, it may lead to bias when real causal variants have different distributions of MAF and LD compared with these associated or tagged SNPs24.Yang et al. hypothesized that heritability varies according to local levels of LD17. While the LD varies across the whole-genome, the LDAK model was designed to reduce redundant tagging and estimate the GRM by weighting the SNPs according to the individual LD. Unlike GREML, the LDAK model can correct some degree of overestimation.While the GCTA model assumes that heritability is independent of LD, Speed 26. The GREML methodology mainly captures phenotypic variation explained by SNPs that are correlated with genotyped SNPs in high LD with causative SNPs. Current GWAS genotyping arrays cannot sufficiently cover all genetic variants as rare causal variants with weak effects in low LD are poorly tagged by common SNPs. This can lower heritability estimation using GWAS data compared with pedigree or twin analysis. Second, the GREML methodology assumes the effect sizes are normally distributed. Bias will likely be introduced in the absence of normal distribution of effect sizes and gene/environment interaction. Other factors that could bias the estimation include the SNP MAF and LD. Recent studies28 have demonstrated that existing SNP-heritability estimate methods are sensitive to frequency- and LD-dependent factors. When using these approaches, it is prudent to proceed cautiously. Partitioning SNPs by MAF and LD can help provide a more accurate estimation of heritability22. Hou et al. recently estimated SNP-heritability without partitioning SNPs by MAF and/or LD29.Both methods have limitations for estimating the heritability of complex diseases7. SNP-based heritability estimates suggest a greater or equal heritability for PsC as compared with PsA. The higher heritability estimate for PsA compared with psoriasis  in previous epidemiological studies could be attributed to common environmental factors should be considered to account the strong recurrence rate of PsA over psoriasis among first degree relatives reported. The MHC effects on heritability is notable and stronger for PsC than PsA, leading to higher gene-based heritability estimates for PsC.This study determined the SNP-based heritability of PsC, PsA and PsV through an unbiased estimate of phenotypic variance. Results from this study differ from population-based genetic epidemiological studies that report a much greater heritability for PsA than PsV3. This is partly explained by the much larger number of patients in the PsC or PsV GWAS studies to date compared with PsA. Disease heterogeneity, which may be greater for PsA than PsC, particularly because the ascertainment criteria for PsC have been chronic plaque psoriasis, whereas PsA is more of a polyarticular, oligo-articular and axial disease. This may affect the heritability estimate for PsA. Finally, dominance and epistasis were not accounted for in our narrow-sense heritability (h2) estimate. The interaction between alleles at the various loci may have a significant effect on heritability estimates. Considerably increasing the number of PsA patients in GWAS studies will help clarify the heritability estimate question for PsA. A lower heritability for PsA or greater environmental influence on PsA should also be considered as possible explanations for these findings.If we assume that the heritability estimates based on previous epidemiological studies are accurate, then what is surprising about GWAS findings to date in psoriatic disease is the absence of PsA-specific genes that have reached GWAS significancePopulation- and clinic-based epidemiological studies have consistently documented significant familial aggregation in psoriasis and PsA. This is the first study to quantify the additive heritability of three subsets of psoriatic disease that is attributable to common susceptibility SNPs from large scale genotyping arrays. We noted that PsC, PsV and PsA were heritable. While the contribution of all SNPs resulted in greater heritability, a significant proportion of the heritability was attributed to SNPs located within the MHC region. Further, the heritability of psoriasis was notably greater than PsA in this study.


Neuroendocrine neoplasms comprise a heterogeneous group of tumors, categorized into neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) depending on tumor differentiation. NECs and high-grade NETs (G3) confer a poor prognosis, demanding novel treatment strategies such as immune checkpoint inhibition in tumors with microsatellite instability (MSI). To study any possible intratumoral heterogeneity of MSI, a tissue microarray (TMA) containing 199 NETs and 40 NECs was constructed to screen for MSI using immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. Four cases suspicious for MSI were identified. Validation of MSI by repeated IHC on large sections and polymerase chain reaction (PCR)–based analysis using the “Bethesda Panel” confirmed MSI in 3 cecal NECs. One pancreatic NET G3 with MSI-compatible TMA results was MMR intact on large section IHC and microsatellite stable (MSS). The remaining 235 tumors exhibited intact MMR. Protein loss of MLH1/PMS2 was found in two and MSH6 loss in one cancer with MSI. Large section IHC on all available tumor-containing tissue blocks in NECs with MSI did not identify aberrant tumor areas with intact MMR. Our data indicate that MSI is common in colorectal NECs (3 out of 10) but highly infrequent in neuroendocrine neoplasms from many other sites. The lack of intratumoral heterogeneity of MMR deficiency suggests early development of MSI during tumorigenesis in a subset of colorectal NECs and indicates that microsatellite status obtained from small biopsies may be representative for the entire cancer mass. Neuroendocrine neoplasms occur at various organ sites and comprise a heterogeneous group of epithelial tumors ranging from well-differentiated neuroendocrine tumors (NETs) to poorly differentiated carcinomas, called neuroendocrine carcinomas (NECs). NETs clinically often present as low-grade malignancies, while NECs behave more aggressively with rapid disease progression and poor long-term survival, demanding for novel treatment options.Microsatellite instability (MSI) refers to a distinctive pattern of increased mutational load in certain tumors, typically caused by a defective DNA mismatch repair (MMR) apparatus, and is characterized by accumulated length variations of repetitive DNA sequences (microsatellites) throughout the genome. Protein loss of at least one of the major components of the MMR system—MLH1, PMS2, MSH2, or MSH6, which can easily be analyzed by immunohistochemistry (IHC)—provides strong indirect evidence for MSI. MSI can also be detected via direct analysis of a predefined panel of DNA microsatellite loci with classic polymerase chain reaction (PCR)–based methods or novel high-throughput molecular techniques [Data on MSI in neuroendocrine neoplasms are limited. Recent studies have found tumors with MSI among neuroendocrine carcinomas  of the stomach, small intestine, and colorectum –6. The fTo systematically assess potential intratumoral heterogeneity of MMR protein expression in neuroendocrine neoplasms with MMR deficiency, a cohort of 239 NETs and NECs was screened on a tissue microarray (TMA) format by IHC, followed by a large section evaluation of cancers suspected for MSI by repeated IHC and PCR analysis.A TMA comprising 239 neuroendocrine neoplasms and various control tissues was constructed using one tissue core per tumor from archived material from patients diagnosed with a NET or NEC at the University Medical Center Hamburg-Eppendorf between 2009 and 2018. TMA construction was described earlier . ClinicaTo screen for MSI cancers, freshly taken TMA sections were immunostained on 1 day and in one experiment using an automated immunostainer (Dako/Agilent Autostainer Link 48). Primary antibody specific for MLH1 , PMS2 , MSH2 , and MSH6   was applied for 20 min  or 30 min (PMS2). Bound antibody was visualized using the EnVision Kit  according to the manufacturer’s directions. Tumors were scored as negative (0) when nuclear staining was absent in all captured tumor cells and positive (+) when unequivocal nuclear staining in tumor cells (irrespective of staining intensity or percentage of stained cells) was observed. In spots showing a negative (0) result for the tumor cells, presence (+) or absence (−) of nuclear staining in peritumoral stromal or inflammatory cells was also recorded . For TMA spots with a staining loss of any MMR protein in tumor cells but not in stromal/inflammatory cells (suspected MSI), IHC was repeated on a corresponding large section of the routinely archived tumor material and PCR analysis was performed. In case of non-interpretable TMA spots  tumor cells accompanied by absence of an internal control (−)), staining was repeated on a representative large section.For all cases with suspected MSI based on TMA screening, a fluorescent PCR-based assay was performed , incorporating the five microsatellite loci of the “Bethesda Panel” which includes two mononucleotide repeats  and three dinucleotides repeats  . AnalysiIf MSI was confirmed, all available archived tumor-containing tissue blocks were analyzed to search for intratumoral heterogeneity. All together 20 large sections were analyzed for 3 patients with confirmed MMR deficiency.n = 15)—large section IHC was performed and revealed intact MMR in each cases. Two hundred twenty from the 224 tumors with interpretable results from TMA screening were also MMR intact, resulting in 235 of 239 neuroendocrine neoplasms demonstrating retained expression of all four MMR proteins. The remaining 4 tumors were all high-grade neuroendocrine neoplasms, including one pancreatic NET G3 and 3 colorectal NECs, demonstrating loss of one or two MMR proteins on the TMA accompanied by an adequate internal positive control, and were thus considered suspicious for MSI  revealed consistent MMR protein loss throughout the entire tumor mass. No concurrent tumor areas with intact MMR were found.In the 3 NECs with confirmed MSI, IHC for MLH1, PMS2, MSH2, and MSH6 from all available archived cancer-containing tumor blocks  or 43 tumors exhibiting CD117 overexpression in a cohort of 1654 breast carcinomas (2.6%) . We recen = 15) and the lung (n = 7) exhibited MSI. These data fit with previous studies reporting particularly high rates of MSI in NECs of the colorectum. Sahnane et al. found MSI in 6 of 37 (16%), Olevian et al. in 2 of 29 (7%), Furlan et al. in 2 of 21 (10%), and La Rosa et al. in 3 of 35 (14%) colorectal NECs [Among the 239 analyzed neuroendocrine neoplasms, MSI was confirmed in 3 cases (1.3%), all representing NECs of the cecum, suggesting a considerable prevalence of MSI  in colorectal NECs, although this may not reflect the true MSI rate in colorectal NECs due to the small tumor cohort. None of the other primary NECs from different sites of the gastrointestinal tract (3 of 35 1% colorectal NECs . In contTaken together, the available data suggest that the prevalence of MSI in NECs is site-dependent and closely related to those organ sites where the exocrine neoplastic counterparts of NECs—adenocarcinomas—are also frequently affected by MSI, such as endometrial, colorectal, and gastric adenocarcinomas. All 3 colorectal NECs with MSI in the present study were located in the cecum, corresponding well to the strong association of MSI in sporadic colorectal adenocarcinomas with tumor location in the right colon . A link In contrast to NECs, none of the 199 NETs exhibited MSI in the present study. That MSI is highly infrequent in NETs fits well with data from earlier studies. MSI was not found in previous studies analyzing 56 rectal , 14 smalEvaluation of the MMR status throughout all available cancer-containing tumor blocks revealed homogeneous MMR protein loss in all three NECs with confirmed MSI. This is in agreement with our previous observations of high homogeneity of MSI in prostate , ovarianPCR analysis revealed MSI-high in all 3 cases of MMR deficiency. Although the number of cases is too small to draw major conclusions, the high concordance between IHC and PCR results is not surprising. The “Bethesda Panel,” a selection of 5 specific mono- and dinucleotide repeats, has been developed based on data derived from colorectal cancers . As diffIn summary, the detection of MSI in 3 of 10 colorectal NECs but not in other neuroendocrine neoplasm suggests that MSI affects NECs of the colorectum in a relevant manner, similar to colorectal adenocarcinomas. In contrast, MSI is highly infrequent in NETs irrespective of tumor site. The observed complete homogeneity of MMR deficiency indicates that the use of minor biopsy samples should result in highly representative data and hints towards MSI as an early event in tumorigenesis. Testing for MSI early in the course of disease might open novel treatment options for colorectal NECs.


Whole-genome sequencing of preimplantation human embryos to detect and screen for genetic diseases is a technically challenging extension to preconception screening. Combining preconception genetic screening with preimplantation testing of human embryos facilitates the detection of de novo mutations and self-validates transmitted variant detection in both the reproductive couple and the embryo’s samples. Here we describe a trio testing workflow that involves whole-genome sequencing of amplified DNA from biopsied embryo trophectoderm cells and genomic DNA from both parents. Variant prediction software and annotation databases were used to assess variants of unknown significance and previously not described de novo variants in five single-gene preimplantation genetic testing couples and eleven of their embryos. Pathogenic variation, tandem repeat, copy number and structural variations were examined against variant calls for compound heterozygosity and predicted disease status was ascertained. Multiple trio testing showed complete concordance with known variants ascertained by single-nucleotide polymorphism array and uncovered de novo and transmitted pathogenic variants. This pilot study describes a method of whole-genome sequencing and analysis for embryo selection in high-risk couples to prevent early life fatal genetic conditions that adversely affect the quality of life of the individual and families. In vitro fertilisation (IVF) used in conjunction with monogenic PGT is available for couples to prevent transmission of known hereditary monogenic disorders. PGT for aneuploidy screens embryos for large segmental or whole-chromosome copy number changes and is commonly used for older women (>35 years) who have a history of infertility, miscarriages or chromosomally abnormal conceptions3. The most recent developments in clinical PGT are low-coverage next-generation sequencing and Karyomapping, which uses a highly polymorphic single-nucleotide polymorphism (SNP) microarray to identify disease-causing haplotypes. Next-generation sequencing PGT for aneuploidy  is useful for high-throughput screening at a reasonable cost for detecting chromosomal aneuploidies, structural variations and large copy-number variations (CNVs)6. In addition to pedigree analysis for monogenic disorders, Karyomapping has been reported to identify partial chromosomal aneuploidies as small as 1.8 Mb7.For over two decades, preimplantation genetic testing (PGT) has been available for couples who are aware they carry a genetic condition or have had a child affected by a genetic disease. 8. A mutation or disease-causing variant in one or both copies of approximately 5,000 human genes can cause a syndromic disease or phenotype14. Between 0.5–5% of infants are born with a genetic condition or disorder16. The preconception genetic screening panels that are available to determine a couple’s carrier status for disease-causing genetic variants are limited to a subset of high-risk genes7. Currently, preconception screening and PGT are performed as separate unlinked tests17. An estimated 74 de novo SNP mutations are introduced at embryogenesis, which, when expressed dominantly or as a compound heterozygote, result in severe pathogenic phenotypes20.For couples seeking to ascertain their risk of having an affected child, around 6,000 diseases exist that may be genetically screened for23. We investigated whether whole-genome sequencing of IVF-conceived embryos could screen for hereditary syndromic genetic diseases in addition to identifying the more technically challenging syndromes resulting from de novo mutations15. To date, whole-genome sequencing has been used in a limited number of assisted reproduction cases, principally due to the high cost of high-throughput sequencing21. We hypothesised that whole-genome sequencing of preimplantation embryos combined with sequencing genomic DNA from both parents could address the limitations associated with current PGT techniques. The aim was to use whole-genome sequencing analysis to screen embryos for pathogenic variants that would result in severe childhood-onset diseases24.With the declining cost and increased availability of whole-genome sequencing, we sought to explore the design of combined preconception screening and embryo PGT using whole-genome sequencing to detect disease-causing genetic variants in couples and their embryos in accordance with recommended practice guidelines25. The whole-genome amplified trophectoderm cell biopsy samples and the genomic DNA of the parents’ samples were used as template DNA for library generation for whole-genome sequencing. Each embryo’s resolved genome sequence was triangulated using multiple trio testing of their parents’ sequences for confirmation of variant status and vice versa22. To detect clinically actionable pathogenic variations, multiple trio testing of the parental and the embryo genomes was performed. This was followed by variant annotation using databases to grade variant pathogenicity and the use of pathogenicity prediction algorithms for inherited and de novo mutations and variants of unknown significance26. Detecting disease-causing pathogenic variants necessitated the use of inheritance-mode filtering to exclude false positives caused by sequencing artefacts. For each of the major modes of inheritance, curatable variant filter and classification sets were generated to detect known ClinVar archive pathogenic variants27. Variants of unknown significance were classified using a range of pathogenicity prediction algorithms and functional annotation databases. The threshold for classifying candidate pathogenic variants was based on pathogenic and likely pathogenic ClinVar categories9. For differentiating between type I and type II error calls for de novo mutations, we used the variant allele frequency (VAF) and quality by depth (QD) metric to filter false-positive pathogenic variants in combination. The purpose of this was to detect inherited pathogenic and unacceptably high-risk de novo variations that would be clinically actionable and to guide personalised diagnosis and treatment29. Our study to design and test a framework to determine clinically actionable pathogenic variants is, to our understanding, the first of its kind.For this pilot study, we sequenced the genomes of five IVF couples and 11 of their IVF embryos that had previously undergone clinical PGT for familial diseases with Karyomapping6. Each participant was given the option to have the results of their genomic DNA and their biopsied embryo samples reported or withheld. All participating couples consented to whole-genome sequencing and elected to receive results for themselves and their tested PGT embryos. The study and protocol were approved by the Monash Health Human Research Ethics Committee (Ref: HREC/17/MonH/286) and all experiments were performed in accordance with protocol guidelines and regulations.Couples who had PGT for single gene disorders provided written informed consent to having whole-genome sequencing on themselves and their biopsied embryos included in the study30.Genomic DNA from five couples who had been used as reference templates for PGT using Karyomapping were selected for whole-genome sequencing. The DNA had been extracted from whole blood using a ReliaPrep™ Blood genomic DNA Miniprep System . For the isolation of embryonic DNA, intracytoplasmic sperm injection method created embryos belonging to the five PGT couples underwent trophectoderm biopsy, using laser or mechanical techniques, on day five or six of culture to remove 4–10 trophectoderm cells. Biopsied cells were washed three times in a solution of 1× phosphate-buffered buffer  and 1× polyvinylpyrrolidone  followed by whole-genome amplification by multi-displacement amplification with SureMDA system  as per manufacturer’s instructions. Samples for whole-genome sequencing were selected based on Karyomapping quality control metrics, which indicated a SNP call-rate on the HumanCytoSNP-12 BeadArray of >96% and allele dropout and miscall rates of <1%. A 1 ug sample of parental genomic DNA and embryo whole-genome amplification products were sent to BGI Genomics  for sequencing with the BGI-SEQ500. Briefly, the DNA samples were fragmented to approximately 350 bp with a E220 Covaris  followed by 3′ end-repair, adaptor ligation and amplification by ligation-mediated polymerase chain reaction, single strand separation and cyclisation. DNA nanoballs were produced with rolling-circle amplification, placed in patterned nanoarrays which are 100 bp paired-end reads on a BGI-SEQ50032. Raw reads were mapped to the human reference genome (GRCh37/HG19) with Burrows-Wheeler Aligner34, polymerase chain reaction duplicates were removed using Picard tools35, local realignment was undertaken with Genome Analysis Toolkit37 and variants were called with HaplotypeCaller using the variant quality score recalibration method.Standard raw read processing through to variant call format was performed in accordance with Genome Analysis Toolkit best practices by the BGI Genomics Online portal pipeline40. Clinically actionable variants were defined as those that could be justified in requesting for screening by an accredited medical ethics committee42. Each parental and embryo binary alignment map (BAM) and raw variant call format files were imported into VarSeq . Variant filtering workflows were arranged for the inheritance modes of; dominant heterozygous, recessive homozygous, compound heterozygous, X-linked, de novo and a low-specificity high-sensitivity failsafe filter with a low depth threshold (read depth >1) and was missing the genotype quality filter , atrophin 1 (ATN1), ataxin 2 (ATXN2), ataxin 3 (ATXN3), junctophilin 3 (JPH3), calcium channel, voltage-dependent, P/Q type, alpha 1A subunit (CACNA1A), dystrophia myotonica-protein kinase (DMPK), cystatin B (CSTB), ataxin 10 (ATXN10), ataxin 7 (ATXN7), huntingtin (HTT), protein phosphatase 2, regulatory subunit B beta (PPP2R2B), ataxin 10 (ATXN1), chromosome 9 open reading frame 72 (C9ORF72), frataxin (FXN), androgen receptor (AR) and fragile X mental retardation 1 (FMR1) on all embryo and parental samples.Analysis was guided by the Standards and Guidelines from the American College of Medical Genetics for interpretation of sequence variants51 and structural variations with Breakdancer52 and CREST53. A secondary, overlapping CNV discovery analysis was performed by binning into 10 kb windows, filtering by calling loss of heterozygosity (LoH) in more than 95% of variants in flagged regions55 and annotating using ClinGen Gene Dosage Sensitivity (27-09-2017 release). Structural variations were called and included in the analysis using Breakdancer52. CNVnator and Breakdancer calls were imported into Varseq and then compared with the inherited CNVs from each parent and categorised as having dosage pathogenicity for either haploinsufficiency or triplosensitivity. LoH regions (>100 and 95% of variants) were trio-called compared with the parental LoH regions. Filtering was applied for the haploinsufficiency and triplosensitivity categories of ‘sufficient evidence for dosage pathogenicity’ or ‘gene associated with autosomal recessive phenotype’ and called for pathogenicity using the target copy number state for proband per sample. This was performed by applying a ratio of >2.0 with a Z-score of >0 for duplications and <0.5 with a Z-score of <0, a mean targeted depth >5 and a lack of quality control flags  for detecting true positive CNVs. CNVs with recessive inheritance were cross-checked against the autosomal recessive SNP and indel variants.CNVs were called using CNVnator (v.0.2.7)Sequencing depth was comparable between the amplified trophectoderm-biopsy DNA from embryos and the parents’ from genomic DNA (mean depth of 48.2× versus 46.1×). Embryo reads were equivalent to the couple’s genomic DNA samples for raw and clean reads, bases aligned and transitions to transversion ratios of 2.071 and 2.081  threshold of >12 was added to the non-dbSNP variant subfilters. This QD threshold reduced the number of de novo variants flagged for curation from 285 across all the eleven embryos to 57. QD filtering was not applied to the transmitted variants, but when this stringent filter was applied to the non-dbSNP variants, 8/125 unique and pathogenic transmitted variants were removed from reporting.Variant filters were therefore arranged to classify for each mode of inheritance into two parallel sub-filter sets that all variants would be assessed; one sub-filter of each filter set for annotating variants catalogued in dbSNP and a second for variants not catalogued to date, for which pathogenicity prediction was used Fig. .Three of the five couples had undergone PGT for autosomal dominant conditions, one for an autosomal recessive condition and one for an X-linked condition Table . To confCTFR ΔF508 mutation and resulted in a heterozygote in at least one embryo.For the recessive filter there was an average of 0.82 transmitted pathogenic variants found in dbSNP per embryo . This is compared to an average of 1.27 non-inherited variants per embryo that were predicted pathogenic  for variants which had fewer than 10 reads. An average sum of 2.3 (SD 1.2) pathogenic or predicted pathogenic variants were noted as expected but missing from the embryo sequencing due to low coverage threshold or LoH from all the filters. Pathogenic variants in low-coverage regions were phased using the nearest flanking SNPs of the missing regions to determine the carrier status. A mean of 4.5 (SD 3.7) likely pathogenic or pathogenic variants were found in embryos and a mean of 5.5 (SD 3.4) variants deemed potentially pathogenic and required haplotype curation via LoH to account for dropout of potentially inherited but missing pathogenic variants.ABL1 locus  in a single embryo56.To prevent filtering of true positive de novo mutations, the failsafe filter container was used to capture clinically relevant variants for curation. After elimination of PGT variants, 17 variants were detected in the 11 embryos with review status of 3 stars, of which none were clinically actionable essential or developmental delay genes and were removed following QD filtering. Review status classification revealed that only the failsafe filters had missing calls, with a mean of 2.36 (SD 3.86); none of the variants captured by the failsafe filter resulted in compound heterozygotes derived from transmitted variants. There were no ClinVar review status 1-star (conflicting interpretations) variants found in any of the embryo samples. Similarly, there were no compound heterozygotes, homozygous autosomal recessive or X-linked , or likely pathogenic or pathogenic in American College of Medical Genetics incidental findings variants in embryos or parental genomes. There were 109 unclassified candidate pathogenic de novo mutations across the 11 embryos with nine variants featured repeatedly across multiple embryos, all but two of which occurred in more than one family. There were 10 candidate de novo autosomal dominant variants in four embryos which had a VAF < 0.4 and only one having a VAF > 0.5, indicating the high likelihood of false-positive calls. Addition of the QD minimum threshold to the unclassified filters for QD < 12 reduced the candidate false positive unclassified variant calls to one de novo mutation at the FMR1, ATXN1 and ATXN3.For the 17 loci that Expansion Hunter assessed the tandem repeat number at known disease-causing loci, no parental samples indicated pathogenic repeat numbers. In embryo samples, most of the loci tested provided at least one concordant call in terms of transmission exactness. At three loci, both alleles were discordant: CNVs were assessed by direct transmission and binning reads in 10 kb windows and comparing against inheritance and ClinGen dosage sensitivity scores for pathogenicity. CNVs calls were higher in the embryos compared to parental samples, except for inter-chromosomal structural variants and structural deletions, suggesting a high false-positive rate  in one of the embryos exemplifies the necessity to have specific filter sets for each mode of transmission and variant subtype.De novo variant calling in embryos presented a unique challenge. A custom VAF filter was required to minimise false positives that were likely introduced as a result of multiple displacement amplification from single base substitutions. The VAF soft threshold of <0.35 and quality scores guided the de novo variant calling. This threshold was marginally higher than the reported de novo false-positive threshold of 0.28 to 0.33To avoid pathogenic variants being transmitted in low or missing coverage regions and being undetected, an untransmitted variant filter manually examined uncalled variants flanking haplotypes to confirm the result at each site. The uniform coverage exhibited by multiple displacement amplification of DNA from the embryos suggests that the likelihood of a pathogenic de novo mutation arising in a region with low coverage is remote. These type 2 errors are further mitigated by the failsafe low-coverage assessment filter, although LoH and VAF filtering can guide manual decision-making. We avoided imputation for regions of LoH to focus on what could be ascertained directly from the data.58.For this study we performed pathogenic variant detection of known likely pathogenic and pathogenic variants in accordance with available databases of variants that have high to complete penetrance. Further work is required to stratify the outcomes of compound heterozygotes in which at least one variant is ranked likely pathogenic. Here, we used a non-exhaustive list of essential genes combined with known developmental delay genes. A list of core disease genes for embryo genome screening is necessary to avoid overcallingFor CNV calls, the recommended 10 kb size for the bins represents the lower limit for the annotation software, which coincides with the upper limit for variant call format file indels. For variations exceeding 10 kb, variant calls were inconsistent between the couples and the embryos, and a read-binning approach was required to confidently call CNV and structural variations. CNV detection via analysis of 10 kb bins overcomes the issue of high false-positive CNV calls, as evidenced by the concordance between partner and embryo genomes. The effective 10 kb upper size limit of indels is conveniently bridged by performing binned CNV analysis in 10 kb blocks. This addressed the issue of the limitations of multiple displacement amplification, enabling comprehensive compound CNV detection of inherited variants and de novo mutations. Short tandem repeat loci yielded inconsistent results for parental and embryo genomes, an observation not pursued further. Clinically, it would be beneficial to use preconception short tandem repeat assessment of premutations at loci responsible for short tandem repeat disorders.There are limitations to this pilot study and areas where further work is required. Pathogenic de novo mutations occurring in a region of no or low coverage will be a challenging limitation to overcome. Further work is required to determine the likelihood of one of these highly improbable scenarios occurring. A second limitation is the threshold of VAF, which obfuscates de novo mutation calling. The need to determine the validity of de novo mutation calls meant filtering out variants which were likely polymerase base incorporation errors of the MDA, allele dropout or mis-aligned reads, generating false-positive variants. The advantages to embryo development and implantation rates conferred by the technique of trophectoderm biopsy of 4–8 cells serves as an additional benefit by maximising embryo genome sequencing coverage. Although the VAF suggested that the type of mutation varies in mean VAF, this was not explored in the present study. Minimising amplification and sequencing artefacts through allelic ratio and haplotype scoring effectively minimises the number of candidate de novo mutations to a number that can be, if necessary, curated. An ethnicity-specific penetrance magnitude metric to guide the level of pathogenicity would be highly relevant for IVF-based screening.16.Controversy regarding whole-genome sequencing in IVF is reflected in contemporary questions of the utility of transferring chromosomally mosaic embryos in PGT aneuploidy screening. We provide compelling evidence in favour of using whole-genome sequencing for screening embryos for pathogenic, severe disease-causing and unacceptably high-risk de novo mutations. Offering clinical genome screening of embryos in the IVF clinic, either as a standalone test or after low-coverage PGT, is based on evidence that the major classes of pathogenic variation can be reliably detected. In addition to comprehensive genomic screening, several embryo development-related aneuploidies, that cannot currently be screened for via next-generation sequencing based PGT (i.e. 69XXX and low-level mosaicism), can be directly observed and screened via this protocol because of its unlimited resolution of structural variation. Although low-coverage PGT for aneuploidy is effective for detecting large (>10 Mbp) chromosomal aneuploidies, 1–2% of conceptions carry a de novo CNV or structural aneuploidy of >100 kb, a significant gap in the detection threshold62. The ongoing emotional and psychological burden born by the parents and the monetary cost of support from a healthcare system for caring for an affected individual is vastly greater than the cost of a genome sequencing test63. For IVF patients, undiagnosed reasons for a couple’s subfertility can be diagnosed and factored into the initial screening to produce a viable pregnancy. Additionally, pharmacogenetics guided stimulation regimens for oocyte retrieval and personalised embryo culture media based on metabomic pathway analysis could be ascertained.The concept of applying whole-genome sequencing for PGT is contentious, the main concern being the sensitivity and specificity of a testing system and the ethical questions that ariseCFTR ΔF508 mutation in one of the couples having PGT for an alternative mutation exemplifies the justification, relevance and utility of this study.The method we propose for screening embryos for pathogenic content has provided evidence of the feasibility of whole-genome sequencing to screen biopsied IVF embryos for severe disease-causing pathogenic variants. By including de novo mutations and premutation short tandem repeat disorders in preconception testing, the risk of childhood disease with known genetic aetiologies can be significantly reduced, should any couple choose to. The discovery of the This study is the first to demonstrate the validity of using whole-genome sequencing in the IVF clinic. Further research is required for stratifying variant penetrance across ethnicities and expanding the variant data to include variants of unknown significance and idiopathic disorders with polygenic risk is warranted.Supplementary Information.


Perturbations in the intrauterine environment can result in lifelong consequences for metabolic health during postnatal life. Intrahepatic cholestasis of pregnancy (ICP) can predispose offspring to metabolic disease in adulthood, likely due to a combination of the effects of increased bile acids, maternal dyslipidemia and deranged maternal and fetal lipid homeostasis. Whereas ursodeoxycholic acid (UDCA) is a commonly used treatment for ICP, no studies have yet addressed whether it can also prevent the metabolic effects of ICP in the offspring and fetoplacental unit. We therefore analyzed the lipid profile of fetal serum from untreated ICP, UDCA-treated ICP and uncomplicated pregnancies and found that UDCA ameliorates ICP-associated fetal dyslipidemia. We then investigated the effects of UDCA in a mouse model of hypercholanemic pregnancy and showed that it induces hepatoprotective mechanisms in the fetal liver, reduces hepatic fatty acid synthase (Fas) expression and improves glucose tolerance in the adult offspring. Finally, we showed that ICP leads to epigenetic changes in pathways of relevance to the offspring phenotype. We therefore conclude that UDCA can be used as an intervention in pregnancy to reduce features of metabolic disease in the offspring of hypercholanemic mothers. Increasing evidence links gestational, perinatal and early infancy factors to susceptibility of metabolic disease in adulthood2. Type 2 diabetes is more prevalent among people whose mothers experienced malnutrition during the Dutch famine in 1944-453, and a substantive literature suggests that maternal obesity and gestational diabetes mellitus (GDM) are associated with elevated metabolic syndrome markers in childhood and later life4. These observational studies have been strengthened by studies in animal models, which have shown that the maternal nutritional and metabolic status can profoundly influence cardiovascular and metabolic function in young and adult offspring7.The prevalence of non-communicable diseases such as obesity and type 2 diabetes has grown rapidly over the last three decades8. Its etiology is complex, with genetic and endocrine components. A subgroup of affected women possess genetic variants in biliary transporters (ABCB4/MDR3 and ABCB11/BSEP) and nuclear receptors (NR1H4/FXR) that perturb bile acid (BA) homeostasis10. The phenotype of ICP is unmasked by pathologically elevated levels of reproductive hormone metabolites in genetically predisposed women14. Women with ICP present with pruritus, hepatobiliary injury and hypercholanemia (elevated serum BA concentrations), as well as dyslipidemia15. Increased rates of GDM have been reported16. Maternal BA concentrations ≥40 μmol/L are associated with adverse fetal outcomes, including spontaneous preterm labor, fetal hypoxia and meconium-stained amniotic fluid19. Fetal BA levels are raised in umbilical cord blood in ICP pregnancies20.Intrahepatic cholestasis of pregnancy (ICP) affects 0.5% of pregnancies in UK and up to 2% globallyin utero as a result of ICP predisposes the adolescent offspring to metabolic disease. Analysis of the Northern Finland Birth cohort database (1986) revealed that 16-year old children from cholestatic pregnancies had increased body mass index and dyslipidemia, and the males had raised fasting insulin compared to age matched adolescents of uncomplicated pregnancies21. In a mouse model of 0.5% cholic acid (CA) feeding that replicates the hypercholanemia of cholestatic pregnancy, female offspring exposed to increased BAs in utero developed hepatic steatosis, glucose intolerance and insulin resistance when challenged with a Western diet (WD) for six weeks. Human samples and in vivo experiments showed that increased transplacental cholesterol transport and greater fat storage in the fetoplacental unit contribute to this phenotype21. Furthermore, maternal hypercholanemia resulted in altered DNA methylation patterns in the agouti Avy yellow mouse offspring, indicating epigenetic alterations in the ICP-associated offspring phenotype21.Exposure of the fetus to elevated BA 23.Administration of ursodeoxycholic acid (UDCA) is a treatment for ICP that reduces BA levels and improves symptoms and biochemical abnormalities in some mothers. However, it is not known whether UDCA improves fetal abnormalities and susceptibility of the offspring to metabolic disease later in life21 and we investigated the impact on the metabolic phenotype of the adult offspring. Based on our previous findings in the agouti Avy yellow mouse models and the emerging evidence that epigenetic processes in early life stages are key factors that influence metabolism25, we also explored modifications in the methylation patterns of umbilical cord leukocytes and placentas from human ICP using an epigenome-wide association study (EWAS) and global methylation studies.We hypothesized that treatment with UDCA in cholestatic pregnancy can reverse the fetal metabolic phenotype, as well as placental dyslipidemia. This may also prevent features of metabolic syndrome in the adult offspring. To address our hypothesis, we first compared the fetal and maternal biochemical profile in UDCA-treated ICP pregnancies, untreated ICP and uncomplicated pregnancies. Based on these findings, we then administered UDCA in a mouse model of maternal hypercholanemia to mimic UDCA treatment in ICP20.Women with untreated ICP (n = 16), UDCA-treated ICP (n = 17) and controls (n = 18) were recruited from Queen Charlotte’s and Chelsea and St Thomas’ Hospitals, London. ICP diagnosis was based on presentation with gestational pruritus and serum BA over 10 µmol/L and no additional identifiable cause of liver dysfunction. Exclusion criteria included other causes of hepatic dysfunction  syndrome, acute fatty liver of pregnancy, primary biliary cholangitis, active viral hepatitis, and any ultrasound abnormality that may result in biliary obstruction) and multi-fetal pregnancy. Controls were women with uncomplicated pregnancies. The decision to treat with UDCA was made by the individual specialist clinician looking after each woman with ICP. Maternal serum and umbilical cord blood were collected and prepared as previously described21. A week before mating for the second litter, females were randomly assigned to one of the three dietary groups: RM3 control diet , RM3 diet supplemented with 0.5% CA  or RM3 diet supplemented with 0.5% CA plus 0.5% UDCA . All diets were purchased from LBS Serving Biotechnology Ltd, Horley, UK. To address the effects of UDCA in gestational cholestasis, pregnant mothers were sacrificed on day 18 of pregnancy after a 4-hour fast. Maternal and fetal liver and blood as well as placentas were collected. Maternal body weight, liver weight, litter number, fetal weight and placental weight were monitored.Age-matched (6–8-week-old) female and male C57BL/6 inbred mice were purchased from Harlan Laboratories  and maintained in a 24-hour light-dark cycle (12 h/12 h) with free access to normal chow diet  and water. Mice were allowed to acclimatize for one week before any experiment. For all studies, the second litters were used as previously described21. At 12 weeks of age, one female mouse from each litter was challenged with a western diet for six weeks (LBS Serving Biotechnology Ltd), whereas female littermates continued on a NC diet(n = 4–6 per dietary regimen from each different diet mothers). A glucose tolerance test was performed two days before sacrifice of the animals at 18 weeks of life as previously described 21.To investigate the effects of UDCA on long-term health of the offspring, a different set of mice were placed on an identical maternal dietary regime and were allowed to give birth to offspring (n = 4–6 mothers per dietary regimen). Litters were reduced to four mice per mother on post-natal day 2; 2 females and 2 males from each mother were kept when possible. Only female offspring were analyzed in this study, as it was previously shown that females from this dietary regime develop a more extreme phenotype21. BA measurements were performed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with the use of internal standards for extraction and quantification as previously reported11. The same techniques were used for both human and animal studies.Serum and tissue biochemical parameters  and FFA) were measured using an LX20 autoanalyzer (Beckman Coulter) as previously describedhttp://www.cmmt.ubc.ca).Venous umbilical cord samples were collected from pregnancies affected by ICP  and uncomplicated pregnancies (n = 17). Samples were collected in plain vacutainers and centrifuged at 3500 rpm for 10 minutes. The serum was stored at −80 °C until use. EDTA blood collection tubes were used to store whole blood at −80 °C. DNA was extracted using the Qiagen QIAamp Blood mini kit, according to the manufacturers instructions . Following elution of DNA into AE buffer, samples were returned to storage at −80 °C until use. 1 µg of the genomic DNA was treated with Sodium Bisulfite using Zymo EZ DNA Methylation-Gold kit  and processing of the HumanMethylationEPIC  platform was carried out by the Centre for Molecular Medicine and Therapeutics (CMMT) (26. We identified probes that were detected (P > 0.01) in one or more samples  and were not at the sites of single-nucleotide polymorphisms (SNPs) 26  or cross-reactive (identified for the EPIC dataset27). After excluding probes that did not meet all these criteria, a total of 792,068 probes, including those on sex chromosomes, were maintained for subsequent analyses.Raw image array files were processed using the R statistical software (R version 3.5.0). A total of 866,836 probes for 60 samples were analysed with minfi (version 1.26) and probe signals normalised using the stratified quantile normalisation algorithm28. Differentially methylated CpGs (dmCpGs) were assigned using Limma (version 3.36.1)29 and adjusted for status, gender and ethnicity as confounders as well as treatment, gender and ethnicity in the second model. A 5% Benjamini-Hochberg False Discovery Rate (FDR)30 correction for multiple testing was applied. All dmCpGs were assigned to their corresponding genomic context and location using the R/Bioconductor package IlluminaHumaMethylationEPICanno.ilm10b2.hg19 (version 0.6.0)31.Statistical analyses for the methylation data were performed using the logit transformed M-values for methylation to avoid bias that would have incurred if beta values were selected32, including 18 passing FDR at 5%.Top 1000 dmCpGs, with a p value <1.74E-04 and FDR < 13%, were selected from the disease (treated and untreated) vs control probe-wise differential methylation analyses to perform IPA pathway analyses . The increase in FDR was adjusted to maintain a significant number of CpGs33. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed with R/Bioconductor package missMethyl (version 1.14.0) using the gometh function34 and the MethylationEPIC genes as background. Further pathway analyses were completed with Ingenuity IPA  utilising the Ingenuity knowledge base reference gene set. An IPA search of individual genes was also run to establish their biological significance.Differentially methylated regions were identified using the DMRcate package (version 1.16.0)DNA extraction of placentas was carried out with a DNeasy Blood and Tissue Kit (Qiagen) as per supplier’s instructions. DNA was eluted in buffer AE, which guarantees optimal DNA stability, and stored at −20 °C for further use. Quantification of DNA methylation was performed using the Epigentek MethylFlash Methylated DNA Kit , according to manufacturer’s instructions. 200 ng of sample DNA as well as positive and negative controls were included. The light absorbance was quantified in a microplate reader at 450 nm . Final amounts were calculated as percentages based on the controls’ standard curve as instructed by the manufacturer.21. Primer sequences  are given in Supplementary Table 1 μg of total RNA  from mouse tissues and human placentas was processed as previously describedpost hoc testing with the GraphPad Prism 7.00 software . The significance cutoff was set at p ≤ 0.05.Data are presented as means ± sem unless otherwise stated. Statistical analysis for multiple comparisons was performed by repeated measures of ANOVA and Newman–Keuls Written informed consent was received from participants prior to inclusion in the study, which conformed to the 1975 Declaration of Helsinki guidelines. Biochemical studies were approved by the local ethics committee of Hammersmith Hospital NHS Trust (11/L0/0396). Epigenome-wide studies were also approved by the ethics committees of Hammersmith Hospitals NHS Trust, London (97/5197 and 08/H0707/21), and King’s College Hospitals NHS Trust, London (03WH06).All experimental procedures were approved by the ethical committee for animal welfare at King’s College London, and all animal studies were compliant with the UK Animals (Scientific Procedures) Act of 1986 and the guidelines from the biological sciences unit at King’s College London.21. Analysis of umbilical cord blood lipid profiles in fetal cord blood from ICP + UDCA treatment showed an increase in cholesterol and FFA in females exposed to ICP, whereas FFA and TG were increased in exposed males. These features were reversed by UDCA treatment  diet fed to pregnant mice mimicked the hypercholanemia of ICP, as previously described21. We first investigated whether dietary supplementation with UDCA is sufficient to reverse the CA diet-induced increase in maternal and fetal BA concentrations (CA + UDCA group). UDCA prevented the elevation of taurocholic acid (TCA) and taurodeoxycholic acid (TDCA) in maternal serum and liver  and fatty acid synthase (Fas) mRNA. No effects were observed in the hepatic Hmgcr mRNA either in the mother or fetus in response to UDCA Fig. . In contin utero develop glucose intolerance when fed a WD for six weeks21. We therefore tested whether exposure of the offspring to UDCA in pregnancy can ameliorate this phenotype. Although UDCA did not affect the liver to body weight ratio  when all cases of ICP  were compared to controls  and associated impairment of metabolic health is therefore plausible, and might be attributable to the restoration of a healthy FFA metabolism.This concurs with previous studies in rodents that have successfully improved obesity-induced hepatosteatosis with UDCA administrationde novo biosynthesis gene Hmgcr, suggesting that UDCA mediates these effects via alternative mechanisms.UDCA reduced hepatic TG in hypercholanemic mothers, as well as in the fetal serum and placenta. It also reversed the hypercholesterolemic phenotype of the mother and placentas, and the CA-induced reduction in HDL levels in the fetal serum. Curiously, these results were not accompanied by a reduction in mRNA expression of maternal and fetal hepatic cholesterol Avy mouse model, when born to mothers fed a CA diet, have reduced methylation of the IAP promoter21, suggesting that maternal hypercholanemia can trigger epigenetic changes in the offspring. By performing an EWAS in fetal umbilical cord blood, we found that ICP also induces methylation changes in pathways that could contribute to the offspring phenotype. When ICP cases (both treated and untreated) were compared with controls (uncomplicated pregnancies), a total of eighteen differentially methylated CpGs were found. IPA pathway analysis showed that these methylated CpGs are involved in fatty acid oxidation pathways, as well as pathways potentially related to glucose metabolism such as glucose-6-phosphate dehydrogenase activity and glucose 1-dehydrogenase [NAD(P)] activity. Interestingly, such pathways are involved in diseases related to endocrine system, cardiovascular disease, lipid metabolism, carbohydrate metabolism, and metabolic disease, all of which are consistent with our previous findings.DNA methylation is a typical chromatin modification that can occur when the mother is exposed to an environmental stimulus. We previously established that female offspring of the The control versus untreated and the treated versus untreated comparisons yielded limited results and did not identify any loci directly relevant to the phenotype studied. The relatively small number of samples precluded detailed investigation of whether beneficial effects of UDCA stem from epigenetic changes or not. Another limitation of the analysis is that the samples from UDCA-treated and untreated ICP cases were not matched for disease severity as they would be in a randomised, placebo-controlled trial.36, suggesting that placental epigenetic marks may be of relevance to metabolic homeostasis in later life.While the use of surrogate tissues for human EWAS offers valuable insights into the epigenome, it also presents a challenge for interpretation due to possible tissue-specific epigenetic alterations. However, alterations in DNA methylation in the placenta have been previously reportedTo gain further insight into the epigenetic component of hypercholenemic pregnancy, we also performed a global DNA methylation analysis of human placentas. As suggested by the EWAS results, ICP altered the human placental epigenome.UDCA treatment improves ICP-associated fetal dyslipidaemia. This finding is consistent in mouse hypercholanemic pregnancy. In addition, maternal UDCA administration improved the lipid profile of mothers and placentas and triggered hepatoprotective mechanisms in the fetal liver in mouse gestational hypercholanemia. In adult offspring, UDCA prevented the development of glucose intolerance. Our data support an epigenetic component to the impact of ICP upon offspring metabolic phenotypes. Larger sample sizes will enable more detailed assessment of the effects of UDCA on the epigenome, and investigation of sex-specific alterations. Human cohort studies will be necessary to investigate whether such UDCA effects can persist through adulthood. These data indicate that UDCA may be used as an effective intervention in ICP to prevent susceptibility of the adult offspring to metabolic disease.Supplementary information.


The arachidonate 5-lipoxygenase (ALOX5) pathway has been investigated in diverse chronic inflammatory diseases including metabolic disorders. Recently, the ALOX5 polymorphism rs4987105 was identified to confer susceptibility to type 2 diabetes mellitus (T2DM), implicating its role in regulating glucose homeostasis. Gestational diabetes mellitus (GDM) shares similar pathogenic mechanism with T2DM. Thus, we aimed to evaluate the association between rs4987105 and gestational glucose metabolism in Chinese pregnant women.P = 0.011). In addition, we observed a significant association between rs4987105 and FPG levels after adjusting confounding variables in the linear regression analysis using dominant genetic model . The present study for the first time reported that the rs4987105 of 5-lipoxygenase (ALOX5) gene was associated with gestational glucose metabolism in Chinese pregnant women.A total of 380 unrelated Chinese pregnant women including 241 GDM patients and 139 controls were included in this study. The genotypes of rs4987105 were examined by the Agena MassARRAY iPLEX platform, the association between rs4987105 and fasting plasma glucose (FPG) levels at 24–28 gestational weeks was evaluated using different statistical methods. We found that carriers of rs4987105 CT/TT genotypes exhibited significantly lower FPG levels ( Gestational diabetes mellitus (GDM) is defined as abnormal glucose intolerance with onset or first recognition during pregnancy. It is a prevalent and clinically significant disease threatening both the mother and the offspring –4. The iNormal pregnancy is associated with an altered inflammatory profile compared to the non-pregnant state. Tightly controlled balance between pro- and anti-inflammatory cytokines is necessary for normal implantation, trophoblast invasion and placentation –12. DysrALOX5 encodes the central enzyme in proinflammatory leukotriene biosynthesis   and multivariate linear regression  showed that the T allele of rs4987105 was significantly associated with lower FPG levels in the studied subjects, demonstrating that this allele may prevent the development of hyperglycemia during pregnancy.In a recent study conducted in a German cohort of 533 T2DM patients and 473 controls, Nejatian et al. reported that the minor T allele of rs4987105 was significantly less frequent in T2DM patients than in controls [OR (95% CI) = 0.7; otective . In agreALOX5 is a synonymous SNP close to the promoter region of this gene. Klotsman et al. showed that rs4987105 had functional impacts on the clinical responses to montelukast in asthma patients [The polymorphism rs4987105 residing in the exon 1 of patients . It is cThe minor allele frequency (MAF) of rs4987105 displays ethnic variations. According to the 1000 genome data, it was lower in American (13%) and African (16%) populations, and higher in European (18%) and Asian (19%) populations. In the present study, it was 21%, which was close to the MAF in Southern Han Chinese (23%) and the MAF for the control groups reported in previous study such as Cai’s study  in China2 and above, whereas in Asian populations such as in our study subjects, lower BMI of only 22–23 kg/m2 could also confer risk of the disease. Therefore for Asian pregnant women, BMI by itself should not be considered as a reliable indicator for GDM risk prediction. Aside from obesity, it has been shown that dietary patterns and physical activity significantly influence GDM risk [It is worth noticing that BMI thresholds for increased GDM risk displays racial/ethnic distinction , 36. AmoGDM risk –40. LifeALOX5 and gestational glucose metabolism.As far as we know, this is the first report of the association between rs4987105 of The main drawback of this study is the limited statistical power in only 241 patients and 139 controls. Besides, several confounding factors were not included and analyzed in this study, such as gestational weight gain, dietary and physical activity habits. Future multi-centered studies in different ethnic groups with larger sample sizes are needed to validate this finding.