Homozygous Variant Carriers Research Articles

A novel germline Pregnane X Receptor (PXR) variant predisposing to Hodgkin lymphoma in two siblings

Hodgkin's lymphoma (HL) is the most common cancer in adolescents and young adults. A family history of HL increases the risk of developing HL in other family members. Identification of genetic predisposition variants in HL is important for understanding disease aetiology, prognosis, and response to treatment. Aberrant activation of the NF-κB pathway is a hallmark feature of HL, contributing to the survival and proliferation of the malignant cells' characteristic of HL.The family with multiple consanguineous marriages with siblings of diagnosed HL was examined by whole-exome sequencing. We found a germline homozygous variation in the PXR ligand binding domain (NM_003889.3:c.811G>A, p.(Asp271Asn)), which was classified as pathogenic by prediction tools and segregated in HL cases. Increased PXR expression was found in homozygous variant carriers compared to heterozygous carriers by quantitative real time PCR (qRT-PCR) and immunofluorescence staining of patients' formalin-fixed paraffin-embedded tissues showed upregulation of PXR, particularly in Hodgkin Reed/Sternberg (HRS) cells.Patients with homozygous PXR variant showed significantly high expression compared to PXR wild-type HL, heterozygous and controls (p = 0.0001, p = 0.0004 and p = 0.0001, respectively). PXR homozygous HRS cells had significantly higher PXR expression compared to PXR wild-type HRS cells (p < 0.0001, 3.27-fold change). Albeit PXR's prominent expression in cytoplasm of HRS cells, homozygous PXR HRS cells showed increased PXR expression in nucleus (p < 0.001).PXR is a member of the nuclear receptor superfamily and previous studies have demonstrated a pleiotropic effect of PXR on malignant transformation. Expression analysis showed that cell proliferation, apoptosis and inflammation related genes were deregulated, in homozygous PXR HL cases. This study provided clinical evidence to previously reported Sxr−/− mice model that develop multifocal lymphomas, had an aberrantly increased NF-κB expression and consistent inflammation. Further functional studies are needed to elucidate the exact mechanisms of action of PXR in HL pathogenesis.

Prevalence and Clinical Characteristics of Heterozygous RNF213 p.Arg4810Lys Variant Carriers Diagnosed With Chronic Thromboembolic Pulmonary Hypertension.

Ring finger protein 213 (RNF213) p.Arg4810Lys is a susceptibility gene for moyamoya disease, peripheral pulmonary artery stenosis (PPS), and other vascular diseases and thrombosis. We investigated the prevalence and clinical characteristics of RNF213 variant carriers diagnosed with chronic thromboembolic pulmonary hypertension (CTEPH). We retrospectively analyzed the prevalence of the RNF213 p.Arg4810Lys variant in patients diagnosed with CTEPH (n=112) and PPS (n=10). Clinical and angiographic characteristics were evaluated between RNF213 variant carriers diagnosed with CTEPH and noncarriers with CTEPH and homozygous variant carriers with PPS. Eight heterozygous RNF213 p.Arg4810Lys variant carriers (7.1%) were identified among patients diagnosed with CTEPH, while 5 patients with PPS (50%) carried the homozygous variant. The clinical characteristics of heterozygous variant carriers with CTEPH were not remarkably different from those of noncarriers with CTEPH. All heterozygous variant carriers with CTEPH showed webs/bands lesions at the segmental/subsegmental level, with 75% showing distal tortuous vessels. None of the heterozygous variant carriers with CTEPH exhibited the string-of-beads pattern or elongated stenosis. Homozygous variant carriers with PPS showed the string-of-beads pattern, elongated stenosis, and distal tortuous vessels without webs/bands lesions. A subset of patients diagnosed with CTEPH (7.1%) carried the heterozygous RNF213 p.Arg4810Lys variant. Clinical and angiographic characteristics of heterozygous variant carriers were not remarkably different from those of noncarriers of CTEPH. However, both heterozygous variant carriers with CTEPH and homozygous variant carriers with PPS showed tortuous vessels on angiography.

A bipolar disorder-associated missense variant alters adenylyl cyclase 2 activity and promotes mania-like behavior.

The single nucleotide polymorphism rs13166360, causing a substitution of valine (Val) 147 to leucine (Leu) in the adenylyl cyclase 2 (ADCY2), has previously been associated with bipolar disorder (BD). Here we show that the disease-associated ADCY2 missense mutation diminishes the enzyme´s capacity to generate the second messenger 3',5'-cylicadenosine monophosphate (cAMP) by altering its subcellular localization. We established mice specifically carrying the Val to Leu substitution using CRISPR/Cas9-based gene editing. Mice homozygous for the Leu variant display symptoms of a mania-like state accompanied by cognitive impairments. Mutant animals show additional characteristic signs of rodent mania models, i.e., they are hypersensitive to amphetamine, the observed mania-like behaviors are responsive to lithium treatment and the Val to Leu substitution results in a shifted excitatory/inhibitory synaptic balance towards more excitation. Exposure to chronic social defeat stress switches homozygous Leu variant carriers from a mania- to a depressive-like state, a transition which is reminiscent of the alternations characterizing the symptomatology in BD patients. Single-cell RNA-seq (scRNA-seq) revealed widespread Adcy2 mRNA expression in numerous hippocampal cell types. Differentially expressed genes particularly identified from glutamatergic CA1 neurons point towards ADCY2 variant-dependent alterations in multiple biological processes including cAMP-related signaling pathways. These results validate ADCY2 as a BD risk gene, provide insights into underlying disease mechanisms, and potentially open novel avenues for therapeutic intervention strategies.

Potential association of SULT2A1 and ABCG2 variant alleles with increased risk for palbociclib toxicity.

Aim: This study evaluated associations between CYP3A4*22 and variants in other pharmacogenes (CYP3A5, SULT2A1, ABCB1, ABCG2, ERCC1) and the risk for palbociclib-associated toxicities.Materials & methods: Two hundred cancer patients who received standard-of-care palbociclib were genotyped and associations with toxicity were evaluated retrospectively.Results: No significant associations were found for CYP3A4*22, CYP3A5*3, ABCB1_rs1045642, ABCG2_rs2231142, ERCC1_rs3212986and ERCC1_rs11615. Homozygous variant carriers of SULT2A1_rs182420 had higher incidence of dose modifications due to palbociclib toxicity (odds ratio [OR]: 4.334, 95% CI: 1.057-17.767, p=0.042). ABCG2_rs2231137 variant carriers had borderline higher incidence of grade 3-4 neutropenia (OR: 4.14, 95% CI: 0.99-17.37, p=0.052).Conclusion: Once validated, SULT2A1 and ABCG2 variants may be useful to individualize palbociclib dosing to minimize toxicities and improve treatment outcomes.

The clinical significance of transthyretin gene variant homozygosity in variant ATTR-CM

Abstract Background Transthyretin Amyloid Cardiomyopathy (ATTR-CM) is a progressive condition characterised by the infiltration of the myocardial extracellular space resulting in progressive heart failure and mortality. Owing to advances in imaging and diagnostics, ATTR-CM is an increasingly recognised form of heart failure worldwide. ATTR-CM can be hereditary (hATTR) and associated with a pathogenic TTR gene variant which exhibits an autosomal dominant pattern of inheritance. Studies have shown that homozygosity in Mendelian diseases confers a more severe clinical phenotype. This has yet to be determined for hATTR-CM. Purpose To determine the clinical significance of TTR gene variant homozygosity in hATTR-CM. Methods All patients whom were diagnosed with hATTR-CM at a single UK national referral centre for amyloidosis were identified retrospectively. TTR gene zygosity, clinical, biochemical and echocardiographic parameters were obtained from patient clinical records. Results Only patients that were homozygous for the V122I TTR gene variant were identified in sufficient numbers for meaningful analysis. Twenty three patients with hATTR-CM whom were homozygous for the V122I variant were identified. Three hundred and ninety heterozygous V122I hATTR-CM patients were identified as a control group. There were no statistically significant differences in echocardiographic parameters (heterozygous vs homozygous means were as follows: IVSd 16.9mm v 16.6mm, LVEF 43.8% v 44.8%, stroke volume 30.7ml v 34.4ml, TAPSE 14.4mm v 15.3mm and GLS -9.7% vs -9.0%). Median NT-proBNP was lower in the homozygotes compared to heterozygotes (2480ng/L v 3179ng/L, p&amp;lt;0.05). Median survival from baseline was similar between the two groups (Figure 1), with homozygous and heterozygous patients having median survivals of 28.7 months and 24.5 months, respectively. However, homozygous patients were significantly younger at baseline diagnosis (mean age 66.4 years v 76.3 years, p&amp;lt;0.01) and therefore died at a much younger age (mean age at death 72.5 years v 79.9 years, p&amp;lt;0.01). Conclusions To our knowledge, this is the first study reporting outcomes in hATTR-CM patients whom are homozygous TTR gene variant carriers. Whilst the cardiac phenotype, natural history and length of survival were not substantially different between heterozygotes and homozygotes, the mean age at first diagnosis was substantially younger. Given the natural course of the disease was relatively similar between the groups, this resulted in a significantly younger age at death in hATTR patients whom are homozygous TTR variant carriers. Larger studies are required to confirm if this is applicable across all TTR variants. Nonetheless our study suggests that hATTR patients whom are homozygous carriers of TTR variants should be afforded closer clinical surveillance from a younger age than their heterozygous counterparts.Figure 1

Germline founder variant c.1998delinsTTCT in the RET oncogene: a cohort study in 15 Belgian families.

The c.1998delinsTTCT variant in the RET gene (codon 666) is linked to medullary thyroid carcinoma in Belgium. We aimed to study the clinical phenotype and the age-dependent penetrance in predictive variant carriers. Retrospective study of index patients and predictive variant carriers, identified through familial cascade testing between 2001 and 2020. The total cohort comprised 119 patients: 15 index patients, 102 heterozygous, and 2 homozygous predictive variant carriers. Among heterozygous carriers, high suspicion of clinical disease was present in 25 patients at initial evaluation and in 3 patients during follow-up. No high suspicion of clinical disease was observed during surveillance in 56 patients, and 18 patients did not proceed to screening for clinical disease. Compared to index patients, surgically treated heterozygous predictive variant carriers had a lower presurgical basal calcitonin, a lower disease stage, less need for adjuvant therapy, and higher chances of remission. In heterozygous carriers, median age at developing high suspicion of disease is 52 years (range 7-75), with a predicted penetrance of 62% (9% SE) at the age of 70 years. Two patients were identified with pheochromocytoma and 1 patient with primary hyperparathyroidism. The 2 homozygous predictive variant carriers presented with higher disease severity at first clinical evaluation. The c.1998delinsTTCT variant in the RET gene is pathogenic and associated with a moderate risk for medullary thyroid carcinoma and rarely with other multiple endocrine neoplasia type 2A (MEN2A) manifestations. Active surveillance is a possible option in heterozygous gene carriers with a negative first clinical evaluation.

Molybdenum Cofactor Catabolism Unravels the Physiological Role of the Drug Metabolizing Enzyme Thiopurine S-Methyltransferase.

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt-/- ) mice as well as human liver cytosol. Urothione levels were quantified by liquid-chromatography tandem mass spectrometry in the kidneys and urine of mice. TPMT-genotype/phenotype and excretion levels of urothione were investigated in human samples and validated in an independent population-based study. As Moco provides a physiological substrate (thiopterin) of TPMT, thiopterin-methylating activity was associated with TPMT activity determined with its drug substrate (6-thioguanin) in mice and humans. Urothione concentration was extremely low in the kidneys and urine of Tpmt-/- mice. Urinary urothione concentration in TPMT-deficient patients depends on common TPMT polymorphisms, with extremely low levels in homozygous variant carriers (TPMT*3A/*3A) but normal levels in compound heterozygous carriers (TPMT*3A/*3C) as validated in the population-based study. Our work newly identified an endogenous substrate for TPMT and shows an unprecedented link between Moco catabolism and drug metabolism. Moreover, the TPMT example indicates that phenotypic consequences of genetic polymorphisms may differ between drug- and endogenous substrates.

A polymorphism in histidine-rich calcium binding protein as second hit in phospholamban cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Netherlands Cardio Vascular Research Initiative (CVON): the Dutch Heart Foundation, Dutch Federation of University Medical Center, the Netherlands Organization for Health Re-search and Development and the Royal Netherlands Academy of Sciences Introduction Sudden cardiac death (SCD) is one of the severe manifestations in carriers with a phospholamban (PLN p.Arg14del) cardiomyopathy. Prediction whether a patient with this pathogenic variant will be at risk for SCD is difficult. PLN has an important role in cardiac calcium homeostasis as regulator of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). It has been shown that the p.Arg14del pathogenic variant leads to Ca2+ overload in cardiomyocytes. Recently, it was found that dilated cardiomyopathy (DCM) patients who have a polymorphism in histidine-rich calcium binding protein (HRC Ser96Ala, rs3745297), displayed an increased risk for malignant arrhythmias and SCD. HRC resides within the SR, where it acts as a regulator of Ca2+ homeostasis. This Ser96Ala gene variant is widespread, as 60% of the general population bears at least one copy of this allele. Objective To explore the effect of the HRC Ser96Ala polymorphism on ventricular arrhythmias and disease expression in PLN p.Arg14del pathogenic variant carriers. Methods 337 p.Arg14del patients were included into the study; divided into wildtype (WT) (n=134, 24 index patients), heterozygous for Ser96Ala variant (n=142, 30 index patients) and homozygous for Ser96Ala variant (n=61, 11 index patients). The study was conducted according to the Declaration of Helsinki. Blood samples were genotyped on the Infinium® Global Screening Array-24 v3.0. Clinical data were subtracted from health records. Results In total 23% of PLN variant carriers were diagnosed with DCM while 11% of the variant carriers were diagnosed with arrhythmogenic cardiomyopathy. A significant difference in age of presentation (p=0.019) of p.Arg14del patients diagnosed with a DCM phenotype was found in homozygous HRC variant carriers (median 43 years, [36-47.3], n=8) compared to WT (median 49 years, [41-56.8], n=24) and heterozygous variant carriers (median 58.5 years, [51-66.5], n=22). No significant differences between the 3 groups were detected in manifestations of premature ventricular contractions (n=188, p=0.203), non-sustained ventricular tachycardia (n=248, p=0.314) and appropriate ICD shocks (n=308, p=0.901). Conclusion Although a significant difference in disease onset was found in PLN p.Arg14del patients with DCM who were homozygous for the HRC polymorphism, no correlations with arrhythmogenic parameters were found between patients with and without the HRC polymorphism. Therefore, we conclude that presence of the HRC polymorphism is not a discriminative predictor for arrhythmogenic events in PLN p.Arg14del.

DNAJC30 defect: a frequent cause of recessive Leber hereditary optic neuropathy and Leigh syndrome.

The recent description of biallelic DNAJC30 variants in Leber hereditary optic neuropathy (LHON) and Leigh syndrome challenged the longstanding assumption for LHON to be exclusively maternally inherited and broadened the genetic spectrum of Leigh syndrome, the most frequent paediatric mitochondrial disease. Herein, we characterize 28 so far unreported individuals from 26 families carrying a homozygous DNAJC30 p.Tyr51Cys founder variant, 24 manifesting with LHON, two manifesting with Leigh syndrome, and two remaining asymptomatic. This collection of unreported variant carriers confirms sex-dependent incomplete penetrance of the homozygous variant given a significant male predominance of disease and the report of asymptomatic homozygous variant carriers. The autosomal recessive LHON patients demonstrate an earlier age of disease onset and a higher rate of idebenone-treated and spontaneous recovery of vision in comparison to reported figures for maternally inherited disease. Moreover, the report of two additional patients with childhood- or adult-onset Leigh syndrome further evidences the association of DNAJC30 with Leigh syndrome, previously only reported in a single childhood-onset case.

UGT1A1 genotype-guided dosing of irinotecan: A prospective safety and cost analysis in poor metaboliser patients

AimTo determine the safety, feasibility, pharmacokinetics, and cost of UGT1A1 genotype-guided dosing of irinotecan. Patients and methodsIn this prospective, multicentre, non-randomised study, patients intended for treatment with irinotecan were pre-therapeutically genotyped for UGT1A1∗28 and UGT1A1∗93. Homozygous variant carriers (UGT1A1 poor metabolisers; PMs) received an initial 30% dose reduction. The primary endpoint was incidence of febrile neutropenia in the first two cycles of treatment. Toxicity in UGT1A1 PMs was compared to a historical cohort of UGT1A1 PMs treated with full dose therapy, and to UGT1A1 non-PMs treated with full dose therapy in the current study. Secondary endpoints were pharmacokinetics, feasibility, and costs. ResultsOf the 350 evaluable patients, 31 (8.9%) patients were UGT1A1 PM and received a median 30% dose reduction. The incidence of febrile neutropenia in this group was 6.5% compared to 24% in historical UGT1A1 PMs (P = 0.04) and was comparable to the incidence in UGT1A1 non-PMs treated with full dose therapy. Systemic exposure of SN-38 of reduced dosing in UGT1A1 PMs was still slightly higher compared to a standard-dosed irinotecan patient cohort (difference: +32%). Cost analysis showed that genotype-guided dosing was cost-saving with a cost reduction of €183 per patient. ConclusionUGT1A1 genotype-guided dosing significantly reduces the incidence of febrile neutropenia in UGT1A1 PM patients treated with irinotecan, results in a therapeutically effective systemic drug exposure, and is cost-saving. Therefore, UGT1A1 genotype-guided dosing of irinotecan should be considered standard of care in order to improve individual patient safety.

Hardy-Weinberg Equilibrium in the Large Scale Genomic Sequencing Era.

Hardy-Weinberg Equilibrium (HWE) is used to estimate the number of homozygous and heterozygous variant carriers based on its allele frequency in populations that are not evolving. Deviations from HWE in large population databases have been used to detect genotyping errors, which can result in extreme heterozygote excess (HetExc). However, HetExc might also be a sign of natural selection since recessive disease causing variants should occur less frequently in a homozygous state in the population, but may reach high allele frequency in a heterozygous state, especially if they are advantageous. We developed a filtering strategy to detect these variants and applied it on genome data from 137,842 individuals. The main limitations of this approach were quality of genotype calls and insufficient population sizes, whereas population structure and inbreeding can reduce sensitivity, but not precision, in certain populations. Nevertheless, we identified 161 HetExc variants in 149 genes, most of which were specific to African/African American populations (∼79.5%). Although the majority of them were not associated with known diseases, or were classified as clinically “benign,” they were enriched in genes associated with autosomal recessive diseases. The resulting dataset also contained two known recessive disease causing variants with evidence of heterozygote advantage in the sickle-cell anemia (HBB) and cystic fibrosis (CFTR). Finally, we provide supporting in silico evidence of a novel heterozygote advantageous variant in the chromodomain helicase DNA binding protein 6 gene (CHD6; involved in influenza virus replication). We anticipate that our approach will aid the detection of rare recessive disease causing variants in the future.

Influence of GRK5 gene polymorphisms on ritodrine efficacy and adverse drug events in preterm labor treatment

The present prospective follow-up study aimed to evaluate the effects of GRK5 polymorphisms on ritodrine efficacy and adverse drug events (ADEs) in pregnant women undergoing preterm labor. A total of 162 women undergoing preterm labor were included in the study. Seven single nucleotide polymorphisms (SNPs) in the GRK5 gene (rs915120, rs2230345, rs2230349, rs7923896, rs1020672, rs4752308, and rs4752292) were assessed. Homozygous variant carriers of rs4752292 and rs1020672 had 0.6 times the hazard of delivery compared to wild-type allele carriers (95% confidence interval [CI], 0.41~0.99 and 0.38~0.99, respectively). In addition, homozygous variant carriers of rs4752292 and rs1020672 had 2.4-fold more (95% CI, 1.10~4.98) and 2.3-fold more (95% CI, 1.04~5.06) ADEs compared to those with the wild-type homozygotes, respectively. Among demographic variables, gestational age and modified Bishop score were significant factors associated with time to delivery, while body weight and maximum ritodrine infusion rate were significant factors associated with ADEs. In silico analysis showed that both rs4752292 and rs1020672 had the potential to affect mRNA splicing by alteration of splicing motifs. The present study shows that ritodrine efficacy and ADEs are associated with GRK5 gene polymorphisms in pregnant women undergoing preterm labor.

A founder homozygous DSG2 variant in East Asia results in ARVC with full penetrance and heart failure phenotype

BackgroundVariants in the desmoglein-2 (DSG2) gene account for a significant proportion of patients with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). The aim of this study was to evaluate the genetic epidemiology of DSG2 and the impact of a frequent homozygous DSG2 variant in East Asia. MethodsGenetic screening of 14 ARVC related genes was performed in 118 unrelated index patients using next-generation sequencing. Following that, family screening, clinical evaluation and haplotype analysis were performed among eight probands who carry the same homozygous DSG2 variant. We also examined the histopathology and protein expression using immunofluorescence staining on the myocardial tissue of two probands undergoing heart transplant. ResultsEighteen (15.2%) patients bear rare putatively deleterious variants in DSG2, among which 8 patients shared the homozygous DSG2 p.Phe531Cys variant. Family screening demonstrated that only homozygous variant carriers exhibited definite ARVC phenotype with 100% penetrance, while heterozygous variant carriers were either unaffected or only presented mild ARVC related symptoms in 25% relatives. Left ventricular involvement and bi-ventricular failure were common among homozygous p. Phe531Cys variant patients even at early age. Haplotype analysis demonstrated p. Phe531Cys was a founder variant in East Asia population with an allele frequency of 0.12%. ConclusionsWe identified, for the first time, a homozygous founder variant of DSG2 in East Asia, which was at surprisingly high frequency of 8.47% among Chinese ARVC patients with a full penetrance. This result suggested an urgent demand of genetic counseling for the probands and their relatives with heterozygous variant.

Genotypic and Phenotypic Factors Influencing Drug Response in Mexican Patients With Type 2 Diabetes Mellitus.

The treatment of Type 2 Diabetes Mellitus (T2DM) consists primarily of oral antidiabetic drugs (OADs) that stimulate insulin secretion, such as sulfonylureas (SUs) and reduce hepatic glucose production (e.g., biguanides), among others. The marked inter-individual differences among T2DM patients’ response to these drugs have become an issue on prescribing and dosing efficiently. In this study, fourteen polymorphisms selected from Genome-wide association studies (GWAS) were screened in 495 T2DM Mexican patients previously treated with OADs to find the relationship between the presence of these polymorphisms and response to the OADs. Then, a novel association screening method, based on global probabilities, was used to globally characterize important relationships between the drug response to OADs and genetic and clinical parameters, including polymorphisms, patient information, and type of treatment. Two polymorphisms, ABCC8-Ala1369Ser and KCNJ11-Glu23Lys, showed a significant impact on response to SUs. Heterozygous ABCC8-Ala1369Ser variant (A/C) carriers exhibited a higher response to SUs compared to homozygous ABCC8-Ala1369Ser variant (A/A) carriers (p-value = 0.029) and to homozygous wild-type genotypes (C/C) (p-value = 0.012). The homozygous KCNJ11-Glu23Lys variant (C/C) and wild-type (T/T) genotypes had a lower response to SUs compared to heterozygous (C/T) carriers (p-value = 0.039). The screening of OADs response related genetic and clinical factors could help improve the prescribing and dosing of OADs for T2DM patients and thus contribute to the design of personalized treatments.

T300A variant of AT16L1 gene in a cohort of Algerian Crohn disease patients

The T300A variant is among the most Crohn's disease (CD) associated genetic variants. The aim of our study is to bring a first insight about the contribution of the T300A variant in a cohort of Algerian CD. In a case/control design, 118 Algerian CD patients and 161 unrelated healthy subjects were genotyped for the T300A variant using the allelic discrimination test by Applied Biosystems Taqman® genotyping technology. A serological analysis was carried out using Biosystems™ ELISA kit for the assessment of the anti-Saccharomyces cerevisiae antibodies and immunofluorimetry via Luminex® technology for the evaluation of cytokine levels (TNFα, IFNγ, IL-6 and IL-17). The comparison between allelic and genotypic frequencies was performed using the χ2 test and the exact Fischer test. The odds ratio (OR) was noted adopting confidence interval of 95%. The comparison between the averages was carried out by the Mann-Whitney and Kruskal-Wallis tests. A factorial discriminant analysis and a binary logistic regression were performed as further analyses. The T300A variant showed an increased risk of CD within homozygous variant carriers (P=0.027). Moreover, the carriage of the G allele was associated with the early onset of CD (P=0.01) and a severe CD impairment (P=0.045). We were not able to comfort the association of the T300A variant and ASCA IgA, ASCA IgG and IFNγ levels detected at the univariate analysis. Our results suggest a possible association between the T300A variant and CD in this cohort of Algerian CD patients. Moreover, this variant might be incriminated in the early onset of CD and a severe disease impairment. At the serological study, the univariate and the multivariate analyses yielded contradictory results. Further investigations of larger cohorts of Algerian CD are needed to better assess the suggested associations at the present study.

The HNF1A mutant Ala180Val: Clinical challenges in determining causality of a rare HNF1A variant in familial diabetes.

Heterozygous mutations in hepatocyte nuclear factor-1A (HNF1A) cause maturity-onset diabetes of the young type 3 (MODY3). Our aim was to compare two families with suspected dominantly inherited diabetes and a new HNF1A variant of unknown clinical significance. The HNF1A gene was sequenced in two independently recruited families from the Norwegian MODY Registry. Both familes were phenotyped clinically and biochemically. Microsatellite markers around and within the HNF1A locus were used for haplotyping. Chromosomal linkage analysis was performed in one family, and whole-exome sequencing was undertaken in two affected family members from each family. Transactivation activity, DNA binding and nuclear localization of wild type and mutant HNF-1A were assessed. The novel HNF1A variant c.539C>T (p.Ala180Val) was found in both families. The variant fully co-segregated with diabetes in one family. In the other family, two subjects with diabetes mellitus and one with normal glucose levels were homozygous variant carriers. Chromosomal linkage of diabetes to the HNF1A locus or to other genomic regions could not be established. The protein functional studies did not reveal significant differences between wild type and variant HNF-1A. In each family, whole-exome sequencing failed to identify any other variant that could explain the disease. The HNF1A variant p.Ala180Val does not seem to cause MODY3, although it may confer risk for type 2 diabetes mellitus. Our data demonstrate challenges in causality evaluation of rare variants detected in known diabetes genes.

Short Report: TRPV1-polymorphism 1911 A>G alters capsaicin-induced sensory changes in healthy subjects.

BackgroundC-fibers express transient receptor potential (TRP) channels. These high-voltage gated channels function as integrators of different physical stresses (e.g. heat, protons, ATP). Additionally channel activation can be induced by capsaicin. Topically applied, capsaicin elicits burning pain, heat and mechanical hyperalgesia and serves as a human surrogate model for pain. It was suggested that the TRPV1-variant rs8065080 (1911A>G) plays a pivotal role in patients with neuropathic pain syndromes. We investigated the effect of this TRPV1-SNP on thermal sensitivity and superficial skin perfusion in 25 healthy subjects.Methods and findingsNine subjects being homozygous TRPV1 wild type (AA), 8 heterozygous (AG) and 8 homozygous variant (GG) carriers were selected out of a pool of genotyped healthy individuals. Under physiological conditions (no capsaicin application), there was no statistical significant difference in thermal thresholds or skin perfusion between carriers of different TRPV1 1199A>G genotypes. However, intra-individual calculations (Δ% pre vs. post capsaicin) revealed (1) less warm-detection in AA/AG (-82.1%) compared to GG (-13.1%) and (2) a gain of heat pain sensitivity in AA/AG (+22.2%) compared to GG carriers (+15.6%) after adjustment for perfusion measurements ((1)p = 0.009, (2)p = 0.021).ConclusionPresence of homozygous variant TRPV1 genotype (GG) demonstrated less capsaicin-induced warm hypoesthesia in warm-detection and less capsaicin-induced heat pain sensitivity suggesting an altered channel function. This demonstrates not only the functional influence of TRPV1 rs8065080 polymorphism itself; it further more underpins the relevance of genotyping-based approaches in both patients and surrogate models of neuropathic pain in healthy volunteers.

Limb girdle myasthenia with digenic RAPSN and a novel disease gene AK9 mutations

Though dysfunction of neuromuscular junction (NMJ) is associated with congenital myasthenic syndrome (CMS), the proteins involved in neuromuscular transmission have not been completely identified. In this study, we aimed to identify a novel CMS gene in a consanguineous family with limb-girdle type CMS. Homozygosity mapping of the novel CMS gene was performed using high-density single-nucleotide polymorphism microarrays. The variants in CMS gene were identified by whole-exome sequencing (WES) and Sanger sequencing. A 20 MB-region of homozygosity (ROH) was mapped on chromosome 6q15-21. This was the only ROH that present in all clinically affected siblings and absent in all clinically unaffected siblings. WES showed a novel variant of AK9 gene located in this ROH. This variant was a start-gain mutation and introduced a cryptic 5'-UTR signal in intron 5 of the AK9 gene. The normal splicing signal would be interfered by the cryptic translation signal leading to defective splicing. Another 25 MB-ROH was found on chromosome 11p13-q12 in all siblings. WES showed a homozygous RAPSN pathogenic variant in this ROH. Since RAPSN-associated limb-girdle type CMS was only manifested in AK9 homozygous variant carriers, the disease phenotype was of digenic inheritance, and was determined by the novel disease modifier AK9 which provides NTPs for N-glycosylation. This is the first time that this specific genotype-phenotype correlation is reported. Importantly, the AK9-associated nucleotide deficiency may replete by dietary supplements. Since AK9 is a disease modifier, enhancing N-glycosylation by increasing dietary nucleotides may be a new therapeutic option for CMS patients.

Genetic Variations in Pattern Recognition Receptor Loci Are Associated with Anti-TNF Response in Patients with Rheumatoid Arthritis.

ObjectivesTo determine whether genetic variation within genes related to the Toll-like receptor, inflammasome and interferon-γ pathways contributes to the differences in treatment response to tumour necrosis factor inhibitors (anti-TNF) in patients with rheumatoid arthritis (RA).MethodsIn a retrospective case-case study, we assessed 23 functional single nucleotide polymorphisms (SNPs) in 15 genes. We included 538 anti-TNF naïve Danish RA patients from the nationwide DANBIO database. Multivariable logistic regression analyses were performed to detect associations (p-value<0.05) between genotypes and European League Against Rheumatism (EULAR) treatment responses. False Discovery Rate corrections for multiple testing (q-value) and stratified analyses were performed to investigate association with individual therapies and IgM-rheumatoid factor (RF) status.ResultsSix of twenty successfully genotyped polymorphisms were nominally associated with EULAR treatment response. Three of these were in weak to moderate linkage disequilibrium with polymorphisms previously reported associated with anti-TNF treatment response. TLR5(rs5744174) variant allele carriers (odds ratio(OR) = 1.7(1.1–2.5),p = 0.010,q = 0.46) and TLR1(rs4833095) homozygous variant carriers (OR = 2.8(1.1–7.4),p = 0.037,q = 0.46) had higher odds for a positive treatment response. NLRP3(rs10754558) variant allele carriers (odds ratio(OR) = 0.6(0.4–1.0),p = 0.045,q = 0.46) were more likely to have a negative treatment response.The association in TLR5(rs5744174) remained significant after correction for multiple comparisons among patients negative for RF (OR = 6.2(2.4–16.3),p = 0.0002,q = 0.024). No other association withstood correction for multiple testing. Post hoc analyses showed that change in Patient Global score on a visual analogue scale (VAS) and change in pain VAS were the main factors responsible for the association.ConclusionsWe reproduced previously reported associations between genetic variation in the TLR10/1/6 gene cluster, TLR5, and NLRP3 loci and response to anti-TNF treatment in RA. Changes in VAS pain and patient global scores were the main contributors to the association found for TLR5. Furthermore, we identified other candidate genes that require replication in independent cohorts.

Organophosphate Pesticide Exposures, Nitric Oxide Synthase Gene Variants, and Gene-Pesticide Interactions in a Case-Control Study of Parkinson's Disease, California (USA).

Background:Nitric oxide synthase (NOS) genes are candidates for Parkinson’s disease (PD) because NOS enzymes produce nitric oxide (NO), a pro-oxidant that can damage neurons. Widely used organophosphate (OP) pesticides can induce oxidative stress and are reported to increase PD risk. Additionally, two single nucleotide polymorphisms (SNPs) from the PON1 (paraoxonase 1) gene influence the ability to metabolize OPs.Objective:Here, we investigated contributions of NOS genes and OP pesticides to PD risk, controlling for PON1 status.Methods:In 357 incident PD cases and 495 population controls, we investigated eight NOS SNPs and interactions with both household and ambient agricultural OP exposures assessed with geographic information system (GIS).Results:In comparing PD in homozygous variant carriers of NOS2A rs1060826 versus homozygous wild-type or heterozygotes, we estimate an adjusted odds ratio (OR) of 1.51 (95% CI: 0.95, 2.41). When considering interactions between NOS1 rs2682826 and OP exposure from household use, the OR for frequent OP use alone was 1.30 (95% CI: 0.72, 2.34) and for the CT+TT genotype alone was 0.89 (95% CI: 0.58, 1.39), and for frequent OP use combined with the CT+TT genotype the OR was 2.84 (95% CI: 1.49, 5.40) (interaction p-value 0.04). Similar results were seen for ambient OP exposure. Interactions between OP exposure and three other NOS1 SNPs and a genetic risk score combining all NOS1 SNPs reached statistical significance.Conclusions:We found that OP pesticides were more strongly associated with PD among participants with variant genotypes in NOS1, consistent with the importance of oxidative stress-inducing mechanisms. Our data provide evidence for NOS1 modifying PD risk in OP exposed populations.Citation:Paul KC, Sinsheimer JS, Rhodes SL, Cockburn M, Bronstein J, Ritz B. 2016. Organophosphate pesticide exposures, nitric oxide synthase gene variants, and gene–pesticide interactions in a case–control study of Parkinson’s disease, California (USA). Environ Health Perspect 124:570–577; http://dx.doi.org/10.1289/ehp.1408976