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Arthrogryposis is a clinical feature defined by congenital joint contractures in two or more different body areas which occurs in between 1/3000 and 1/5000 live births. Variants in multiple genes have been associated with distal arthrogryposis syndromes. Heterozygous variants in MYH3 have been identified to cause the dominantly-inherited distal arthrogryposis conditions, Freeman-Sheldon syndrome, Sheldon-Hall syndrome, and multiple pterygium syndrome. In contrast, MYH3 variants underlie both dominantly and recessively inherited Contractures, Pterygia, and Spondylocarpotarsal Fusion syndromes (CPSFS) which are characterized by extensive bony abnormalities in addition to congenital contractures. Here we report two affected sibs with distal arthrogryposis born to unaffected, distantly related parents. Sequencing revealed that both sibs were homozygous for two ultra-rare MYH3 variants, c.3445G>A (p.Glu1149Lys) and c.4760T>C (p.Leu1587Pro). Sequencing and deletion/duplication analysis of 169 other arthrogryposis genes yielded no other compelling candidate variants. This is the first report of biallelic variants in MYH3 being implicated in a distal arthrogryposis phenotype without the additional features of CPSFS. Thus, akin to CPSFS, both dominant and recessively inherited distal arthrogryposis can be caused by variants in MYH3., (© 2024 The Author(s). Clinical Genetics published by John Wiley & Sons Ltd.)

Ephb6 gene knockout causes hypertension in castrated mice. EPHB6 controls catecholamine secretion by adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent way. Nicotinic acetylcholine receptor (nAChR) is a ligand-gated Ca 2+ /Na + channel, and its opening is the first signaling event leading to catecholamine secretion by AGCCs. There is a possibility that nAChR might be involved in EPHB6 signaling, and thus sequence variants of its subunit genes are associated with hypertension risks. CHRNA3 is the major subunit of nAChR used in human and mouse AGCCs. We conducted a human genetic study to assess the association of CHRNA3 variants with hypertension risks in hypogonadic males. The study cohort included 1,500 hypogonadic Chinese males with (750 patients) or without (750 patients) hypertension. The result revealed that SNV rs3743076 in the fourth intron of CHRNA3 was significantly associated with hypertension risks in the hypogonadic males. We further showed that EPHB6 physically interacted with CHRNA3 in AGCCs, providing a molecular basis for nAChR being in the EPHB6 signaling pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Wu, Wang, Shi, Zhang, Raelson, Yao, Wu, Xu, Marois-Blanchet, Ledoux, Blunck, Sheng, Hu, Luo and Wu.)

Objective: We investigated the association of genetic variants of EPHA4, a receptor tyrosine kinase, with hypertension, and its role in vascular smooth muscle cell (VSMC) contractility., Methods: Data from two human genetic studies, ADVANCE and HCHS/SOL, were analyzed for association of EPHA4 single nucleotide variants (SNVs) with hypertension risks. The effect of EPHA4 signalling on mouse VSMC contractility was assessed., Results: We identified a SNV (rs75843691 hg19 chr2:g.222395371 C>G), located in the third intron of EPHA4 gene, being significantly associated with hypertension in human female patients (P value = 8.3 × 10, below the Bonferroni-corrected critical P value) but not male patients with type 2 diabetes from the ADVANCE clinical trial. We found that EPHA4 was expressed in VSMCs and its stimulation by anti-EPHA4 antibody led to reduced VSMC contractility. Estrogen enhanced the contractility-lowering effect of EPHA4 stimulation. Conversely, siRNA knockdown of Epha4 expression in VSMCs resulted in increased contractility of VSMCs from female mice but not from male mice., Conclusion: EPHA4 appears to be a sex-specific hypertension risk gene in type 2 diabetic patients. Forward EPHA4 signalling reduces VSMC contractility, and estrogen is a modifier of this effect. The effect of EPHA4 on VSMCs contractility explains the association of EPHA4 gene with hypertension risks in female patients.

Several members of the EPH kinase family and their ligands are involved in blood pressure regulation, and such regulation is often sex- or sex hormone-dependent, based on animal and human genetic studies. EPHB6 gene knockout (KO) in mice leads to hypertension in castrated males but not in un-manipulated KO males or females. To assess whether this finding in mice is relevant to human hypertension, we conducted a human genetic study for the association of EPHB6 and its two ligands, EFNB1 and EFNB3, with hypertension in hypogonadic patients. Seven hundred and fifty hypertensive and 750 normotensive Han Chinese patients, all of whom were hypogonadic, were genotyped for single nucleotide polymorphisms (SNPs) within the regions of the genes, plus an additional 50 kb 5' of the genes for EPHB6, EFNB1 and EFNB3. An imputed insertion/deletion polymorphism, rs35530071, was found to be associated with hypertension at p-values below the Bonferroni-corrected significance level of 0.0024. This marker is located 5' upstream of the EFNB3 gene start site. Previous animal studies showed that while male EFNB3 gene knockout mice were normotensive, castration of these mice resulted in hypertension, corroborating the results of the human genetic study. Considering the significant associations of EFNB3 SNPs with hypertension in hypogonadic males and supporting evidence from castrated EFNB3 KO mice, we conclude that loss-of-function variants of molecules in the EPHB6 signaling pathway in the presence of testosterone are protective against hypertension in humans.

Background: The prevalence of diabetic nephropathy varies according to ethnicity. Environmental as well as genetic factors contribute to the heterogeneity in the presentation of diabetic nephropathy. Our objective was to evaluate this heterogeneity within the Caucasian population., Methods: The geo-ethnic origin of the 3409 genotyped Caucasian type 2 diabetes (T2D) patients of Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation was determined using principal component analysis. Genome-wide association studies analyses of age of onset of T2D were performed for geo-ethnic groups separately and combined., Results: The first principal component separated the Caucasian study participants into Slavic and Celtic ethnic origins. Age of onset of diabetes was significantly lower in Slavic patients (P = 7.3 × 10), whereas the prevalence of hypertension (P = 4.9 × 10) and albuminuria (5.1 × 10) were significantly higher. Age of onset of T2D and albuminuria appear to have an important genetic component as the values of these traits were also different between Slavic and Celtic individuals living in the same countries. Common and geo-ethnic-specific loci were found to be associated to age of onset of diabetes. Among the latter, the PROX1/PROX1-AS1 genes (rs340841) had the highest impact. Single-nucleotide polymorphism rs340841 CC genotype was associated with a 4.4 year earlier onset of T2D in Slavic patients living or not in countries with predominant Slavic populations., Conclusion: These results reveal the presence of distinct genetic architectures between Caucasian ethnic groups that likely have clinical relevance, among them PROX1 gene is a strong candidate of early onset of diabetes with variations depending on ethnicity.

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions. We recently reported that Efnb3 gene deletion results in hypertension in female but not male mice. These data suggest that EFNB3 regulates blood pressure in a sex- and sex hormone-dependent way. In the present study, we conducted a human genetic study to assess the association of EFNB3 single nucleotide polymorphisms with human hypertension risks, using 3,448 patients with type 2 diabetes from the ADVANCE study (Action in Diabetes and Vascular Disease: Peterax and Diamicron MR Controlled Evaluation). We have observed significant association between 2 SNPs in the 3' untranslated region or within the adjacent region just 3' of the EFNB3 gene with hypertension, corroborating our findings from the mouse model. Thus, our investigation has shown that EFNB3 is a hypertension risk gene in certain individuals.

Several erythropoietin-producing hepatocellular receptor B family (EPHB) and their ligands, ephrinBs (EFNBs), are involved in blood pressure regulation in animal models. We selected 528 single nucleotide polymorphisms (SNPs) within the genes of EPHB6, EFNB2, EFNB3 and GRIP1 in the EPH/EFN signalling system to query the International Blood Pressure Consortium dataset. A SNP within the glutamate receptor interacting protein 1 (GRIP1) gene presented a p-value of 0.000389, approaching the critical p-value of 0.000302, for association with diastolic blood pressure of 60,396 individuals. According to echocardiography, we found that Efnb3 gene knockout mice showed enhanced constriction in the carotid arteries. In vitro studies revealed that in mouse vascular smooth muscle cells, siRNA knockdown of GRIP1, which is in the EFNB3 reverse signalling pathway, resulted in increased contractility of these cells. These data suggest that molecules in the EPHB/EFNB signalling pathways, specifically EFNB3 and GRIP1, are involved blood pressure regulation.

Objective: Single-nucleotide polymorphisms (SNPs) associated with the response to recombinant human growth hormone (r-hGH) have previously been identified in growth hormone deficiency (GHD) and Turner syndrome (TS) children in the PREDICT long-term follow-up (LTFU) study (Nbib699855). Here, we describe the PREDICT validation (VAL) study (Nbib1419249), which aimed to confirm these genetic associations., Design and Methods: Children with GHD (n = 293) or TS (n = 132) were recruited retrospectively from 29 sites in nine countries. All children had completed 1 year of r-hGH therapy. 48 SNPs previously identified as associated with first year growth response to r-hGH were genotyped. Regression analysis was used to assess the association between genotype and growth response using clinical/auxological variables as covariates. Further analysis was undertaken using random forest classification., Results: The children were younger, and the growth response was higher in VAL study. Direct genotype analysis did not replicate what was found in the LTFU study. However, using exploratory regression models with covariates, a consistent relationship with growth response in both VAL and LTFU was shown for four genes - SOS1 and INPPL1 in GHD and ESR1 and PTPN1 in TS. The random forest analysis demonstrated that only clinical covariates were important in the prediction of growth response in mild GHD (>4 to <10 μg/L on GH stimulation test), however, in severe GHD (≤4 μg/L) several SNPs contributed (in IGF2, GRB10, FOS, IGFBP3 and GHRHR)., Conclusions: The PREDICT validation study supports, in an independent cohort, the association of four of 48 genetic markers with growth response to r-hGH treatment in both pre-pubertal GHD and TS children after controlling for clinical/auxological covariates. However, the contribution of these SNPs in a prediction model of first-year response is not sufficient for routine clinical use., (© 2016 European Society of Endocrinology.)

Ephrin B2 (EFNB2) is a ligand for erythropoietin-producing hepatocellular kinases (EPH), the largest family of receptor tyrosine kinases. It has critical functions in many biological systems, but is not known to regulate blood pressure. We generated mice with a smooth muscle cell (SMC)-specific deletion of EFNB2 and investigated its roles in blood pressure regulation and vascular SMC (VSMC) contractility. Male Efnb2 knockout (KO) mice presented reduced blood pressure, whereas female KO mice had no such reduction. Both forward signaling from EFNB2 to EPHs and reverse signaling from EPHs to EFNB2 were involved in regulating VSMC contractility, with EPHB4 serving as a critical molecule for forward signaling, based on crosslinking studies. We also found that a region from aa 313 to aa 331 in the intracellular tail of EFNB2 was essential for reverse signaling regulating VSMC contractility, based on deletion mutation studies. In a human genetic study, we identified five SNPs in the 3' region of the EFNB2 gene, which were in linkage disequilibrium and were significantly associated with hypertension for male but not female subjects, consistent with our findings in mice. The coding (minor) alleles of these five SNPs were protective in males. We have thus discovered a previously unknown blood pressure-lowering mechanism mediated by EFNB2 and identified EFNB2 as a gene associated with hypertension risk in humans.

Transforming growth factor beta-induced (TGFBI/βIG-H3), also known as βig-H3, is a protein inducible by TGFβ1 and secreted by many cell types. It binds to collagen, forms part of the extracellular matrix and interacts with integrins on the cell surface. Recombinant TGFBI and transgenic TGFBI overexpression can promote both islet survival and function. In this study, we generated TGFBI KO mice and further assessed TGFBI function and signaling pathways in islets. Islets from KO mice were of normal size and quantity, and these animals were normoglycemic. However, KO islet survival and function was compromised in vitro. In vivo, KO donor islets became inferior to wild-type donor islets in achieving normoglycemia when transplanted into KO diabetic recipients. TGFBI KO mice were more prone to straptozotocin-induced diabetes than the wild-type counterpart. Phosphoprotein array analysis established that AKT1S1, a molecule linking the AKT and mTORC1 signaling pathways, was modulated by TGFBI in islets. Phosphorylation of four molecules in the AKT and mTORC1 signaling pathway, i.e. AKT, AKT1S1, RPS6 and EIF4EBP1, was upregulated in islets upon TGFBI stimulation. Suppression of AKT activity by a chemical inhibitor, or knockdown of AKT1S1, RPS6 and EIF4EBP1 expression by small interfering RNA, modulated islet survival, proving the relevance of these molecules in TGFBI-triggered signaling. Human genetic studies revealed that in the TGFBI gene and its vicinity, three single-nucleotide polymorphisms were significantly associated with type 1 diabetes risks, and one with type 2 diabetes risks. Our study suggests that TGFBI is a potential risk gene for human diabetes., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Epigenetic inactivation of chromatin plays an important role in determining cell phenotype in both normal and cancer cells, but our knowledge is still incomplete with respect to any potential monoallelic nature of the phenomenon. We have genotyped DNA isolated from chromatin of two colorectal cancer-derived lines and a culture of normal human intestinal epithelial cells (HIEC), which was immunoprecipitated with antibodies to acetylated vs. methylated histone H3K9, and presented the data as B allele frequency differences over multiple single-nucleotide polymorphism (SNP) moving window averages. [B allele is an arbitrary term defined as one of the two alleles at any given SNP, named A and B]. Three different validation tests confirmed that peaks exhibiting differences represented monoallelic domains. These complementary tests confirmed the following: 1) genes in the regions of high B allele frequency difference were expressed monoallelically; 2) in normal cells all five imprinting control regions which carried heterozygous SNPs were characterized by B allele difference peaks; and 3) the haplotypes in the B allele difference peaks were faithfully maintained in the chromatin immunoprecipitated with the respective antibodies. In both samples most of the monoallelic domains were found at the boundaries between regions of open and closed chromatin. With respect to the cancer line, this supports the established concept of conformation spreading, but the results from the normal cells were unexpected. Since these cells were polyclonal, the monoallelic structures were probably not determined by random choice as occurs in X-inactivation, so we propose that epigenetic inactivation in some domains may be heritable and polymorphic in normal human cells.

Essential tremor (ET) is a common neurodegenerative disorder that is characterized by a postural or motion tremor. Despite a strong genetic basis, a gene with rare pathogenic mutations that cause ET has not yet been reported. We used exome sequencing to implement a simple approach to control for misdiagnosis of ET, as well as phenocopies involving sporadic and senile ET cases. We studied a large ET-affected family and identified a FUS p.Gln290(∗) mutation as the cause of ET in this family. Further screening of 270 ET cases identified two additional rare missense FUS variants. Functional considerations suggest that the pathogenic effects of ET-specific FUS mutations are different from the effects observed when FUS is mutated in amyotrophic lateral sclerosis cases; we have shown that the ET FUS nonsense mutation is degraded by the nonsense-mediated-decay pathway, whereas amyotrophic lateral sclerosis FUS mutant transcripts are not., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Essential tremor (ET) is a complex genetic disorder for which no causative gene has been found. Recently, a genome-wide association study reported that two variants in the LINGO1 locus were associated to this disease. The aim of the present study was to test if this specific association could be replicated using a French-Canadian cohort of 259 ET patients and 479 ethnically matched controls. Our genotyping results lead us to conclude that no association exists between the key variant rs9652490 and ET (P(corr) = 1.00).

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.

Multiple sclerosis (MS) is a complex disease that is partly genetic in origin. Although an association of MS with specific human leukocyte antigen (HLA) types has been known for almost 30 years, the nature of this relationship has remained unclear. Furthermore, genetic resolution sufficient to implicate a specific gene in the HLA region has not been achieved. Many loci in the HLA region have been found to be significantly associated with MS, which is largely explained by the extended haplotype sharing and varying marker informativity of the region. We have determined 248 haplotypes of MS patients from the population of the northern Netherlands and 226 haplotypes of their relatives as controls using a set of 22 microsatellite markers covering the HLA region. The data were analyzed using standard association methods and a new statistical method, haplotype sharing statistics (HSS). Haplotype sharing statistics determines the extent of haplotype sharing for all pairs of haplotypes of patients and of controls and calculates the difference in mean haplotype sharing between patients and controls. Haplotype sharing was found to be significantly greater among patients than among controls in a region of 1.1 Mb between markers G511525 and TNFalpha. The involvement of this region is also supported by association analysis and the transmission/disequilibrium test (TDT). Within this region, HSS, which is largely independent of association and TDT, indicated the interval of 51 kb between G511525 and D6S1666 as that most likely to contain a susceptibility gene for MS. As DQB1 is the sole gene known in this interval at present, the results of our analysis suggest that this gene plays a role in the pathogenesis of MS.

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.

All-trans retinoic acid (tRA), a naturally occurring ligand of the nuclear retinoic acid receptors (RARs), induces differentiation of leukemic cells and clinical complete remission in patients with acute promyelocytic leukemia (APL). This differentiation effect can also be seen in vitro in both fresh leukemic cells and in the unique permanent APL cell line, NB4. However, APL cells become resistant to RA-induced differentiation both in vitro and in patients. Although pharmacodynamic mechanisms of resistance have been reported, there is growing evidence that resistance both in patients, as well as in vitro, can be mediated by changes in the sensitivity of leukemic cells to retinoids. To investigate possible mechanisms of retinoid resistance, we established subclones of NB4 that are stably resistant to both tRA and 9-cisRA. Unlike the previously reported NB4.306 retinoid-resistant cells, these subclones expressed PML/RAR-alpha RNA and protein, but demonstrated altered ligand binding patterns of PML/RAR-alpha and differed in retinoid-induced gene expression. They were significantly less able to stimulate transcription of an RARE driven CAT-reporter gene on induction by tRA and showed altered DNA binding activity on a RARE. These data suggest that NB4 cells selected for resistance to retinoids demonstrate abnormal ligand binding to PML/RAR-alpha that lead to altered transcriptional activation by retinoids.

Cystinuria, a hereditary disorder of cystine and dibasic amino acid reabsorption, has been classified into three subtypes on the basis of urinary excretion in obligate heterozygous parents. Thirteen cystinuric patients, identified primarily through the Quebec newborn urinary screening program, were investigated by phenotypic classification and by mutational analysis of the D2H (rBAT) gene. Mutations were identified on 7 of 25 alleles; all of these 7 mutant alleles were associated with Type I cystinuria. Four of the mutations (a large deletion, a 5'splice site mutation, a 2 bp deletion, and a nonsense mutation) have not been previously reported. These findings suggest that abnormalities in the D2H gene may account for only one subtype (Type I) of cystinuria, and that this subtype can be caused by a wide variety of population-specific mutations.