Interaction between genetic and epigenetic variation defines gene expression patterns at the asthma-associated locus 17q12-q21 in lymphoblastoid cell lines

Phenotypic variation results from variation in gene expression, which is modulated by genetic and/or epigenetic factors. To understand the molecular basis of human disease, interaction between genetic and epigenetic factors needs to be taken into account. The asthma-associated region 17q12-q21 harbors three genes, the zona pellucida binding protein 2 (ZPBP2), gasdermin B (GSDMB) and ORM1-like 3 (ORMDL3), that show allele-specific differences in expression levels in lymphoblastoid cell lines (LCLs) and CD4+ T cells. Here, we report a molecular dissection of allele-specific transcriptional regulation of the genes within the chromosomal region 17q12-q21 combining in vitro transfection, formaldehyde-assisted isolation of regulatory elements, chromatin immunoprecipitation and DNA methylation assays in LCLs. We found that a single nucleotide polymorphism rs4795397 influences the activity of ZPBP2 promoter in vitro in an allele-dependent fashion, and also leads to nucleosome repositioning on the asthma-associated allele. However, variable methylation of exon 1 of ZPBP2 masks the strong genetic effect on ZPBP2 promoter activity in LCLs. In contrast, the ORMDL3 promoter is fully unmethylated, which allows detection of genetic effects on its transcription. We conclude that the cis-regulatory effects on 17q12-q21 gene expression result from interaction between several regulatory polymorphisms and epigenetic factors within the cis-regulatory haplotype region

Role of DNA methylation in common disease: analysis of two asthma-associated regions

Two chromosomal regions, 5q31 and 17q21, are both among the best replicated asthma-associated regions from genome-wide association studies (GWAS) and share certain characteristics.They are among the highest genome-wide significance allele-specific expression differences: in the 17q region, the childhood asthma-associated allele shows increased expression of gasdermin B (GSDMB) and ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) genes and in the 5q31 region the asthma-associated allele shows increased expression of the solute carrier family 22 (organic 3 cation/carnitine transporter) member 5 (SLC22A5) genes. This suggests that cis-regulatory mechanisms may influence disease development in these regions. Among children, the prevalence of asthma is higher in boys than girls, 5q31 and 17q21 regions show sex specificity of genetic association: the association with asthma is stronger in males than females, and both are associated with several autoimmune disorders such as asthma and Crohn’s disease, among different populations. We hypothesize that variation in DNA methylation at regulatory elements within asthma-associated genomic regions 5q31 and 17q21 may acts as a modifier of the effect of genotype on phenotype. To better understand the relationship between genetic variation in these regions, DNA methylation, and predisposition to asthma, we established DNA methylation profiles of 13 genes. We used sodium bisulfite sequencing methylation assay to examine the effects that genotype has on DNA methylation in the peripheral blood cells from individuals in the Saguenay-Lac-Saint-Jean (SLSJ) asthma familial collection and lymphoblastoid cell lines (LCLs). We found a single regulatory region, the zona pellucida binding protein 2 (ZPBP2) promoter, which showed a statistically significantly higher methylation level in females compared to males. The local genotype influenced methylation levels of SLC22A5, ZPBP2 and gasdermin A (GSDMA) promoter regions. The genotype had a dominant effect on SLC22A5, ZPBP2 and GSDMA methylation, with lower methylation levels in individuals that carry the asthma-predisposing alleles. To understand whether the moderate change in DNA methylation leads to a change in gene expression levels in 5q31 and 17q21, the effect of treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) on expression and promoter methylation of genes in 5q31 and 17q21 in the human airway epithelium cell line, NuLi-1 and embryonic kidney epithelium cell line, HEK293T/17 was assessed. We found that SLC22A5 and GSDMA were upregulated after 5-aza-dC treatment in both cell lines. ZPBP2 expression was increased in NuLi-1 but remained silent in HEK293T/17, while ORMDL3 showed upregulation in HEK293T/17 but not NuLi-1. Increased2expression of ZPBP2 and GSDMA was accompanied by a reduction in promoter methylation, whereas no change in SLC22A5 promoter methylation levels was observed. Furthermore, we noticed modification in allelic expression of ZPBP2 and ORMDL3, suggesting that each region may react in uniquely to 5-aza-dC.We hypothesized that sex-specific differences in DNA methylation levels at the ZPBP2 promoter resulted from the dosage of the sex chromosomes. Thus, ZPBP2 DNA methylation was analyzed using the sodium bisulfite sequencing assay in DNA samples from fibroblast cell lines derived from individuals with different sex phenotypes and sex chromosome dosages. No significant influence of the sex phenotype was detected. However, a significant positive correlation was found between ZPBP2 methylation levels and the number of X chromosomes. Our results suggest that the presence of more than one X in the absence of the sex-determining region Y (SRY) gene is associated with higher methylation levels.Deux régions chromosomiques, 5q31 et 17q21, font partie des régions associées à l’asthme les mieux répliquées des études d’association pangénomique (GWAS) et partagent certaines caractéristiques. Ces régions ont les plus grandes différences significatives d’expression génique associée aux allèles au niveau du génome : dans la région 17q, l’allèle associé à l’asthme chez l’enfant montre une augmentation de l’expression des gènes gasdermin B (GSDMB) et «Régulateur de biosynthèse des sphingolipides ORMDL 3» (ORMDL3); alors que c’est le gène «famille de support de soluté 22 (transporteur de 3 cation / carnitine organique) membre 5» (SLC22A5) qui est augmenté dans la région 5q31. Ceci suggère que les mécanismes régulés en cis peuvent influencer le développement des maladies dans ces régions. Chez les enfants, la prévalence de l’asthme est plus élevée chez les garçons que chez les filles. Les régions 5q31 et 17q21 montrent une association génétique spécifique liée au sexe: l’association avec l’asthme est plus forte chez les males que chez les femelles, et les deux sont associés à de nombreux troubles auto-immunes, tels que l’asthme et la maladie de Crohn, dans les différentes populations. Nous posons l’hypothèse que la variation de la méthylation de l’ADN des éléments régulateurs dans les régions génomiques associées à l’asthme, 5q31 et 17q21, peut agir comme modificateur des effets du génotype sur le phénotype. Afin de mieux comprendre la relation entre la variation génétique de ces régions, la méthylation de l’ADN et la prédisposition à l’asthme, nous avons établi les profils de méthylation d’ADN de 13 gènes. Nous avons utilisé des essais de méthylation au bisulfite de sodium et de séquençage pour examiner les effets que le génotype a sur la méthylation de l’ADN dans les cellules du sang périphérique provenant d’individus de la collection familiale asthmatique du Saguenay-Lac-Saint-Jean (SLSJ) et les lignées de cellules lymphoblastoïdes (LCLs). Nous avons identifié une seule région régulatrice, le promoteur de «zona pellucida binding protein 2» (ZPBP2), lequel a montré un plus haut niveau statistiquement significatif de la méthylation chez les femelles comparativement aux males. Le génotype local a influencé les niveaux de méthylation des promoteurs de SLC22A5, ZPBP2 et gasdermin A (GSDMA). Le génotype avait un effet dominant sur la méthylation de SLC22A5, ZPBP2 et GSDMA, avec des niveaux de méthylation plus bas chez les individus porteurs des allèles de prédisposition à l’asthme. Pour comprendre comment le changement modéré dans la méthylation d’ADN mène à un changement dans les niveaux de l’expression des gènes dans les régions 5q31 et 17q21, nous avons examiné les effets d’un traitement à l’inhibiteur de la méthyltransférase de l’ADN 5-aza-2’-4déoxycitidine (5-aza-dC), lequel induit la déméthylation de l’ADN, sur l’expression et la méthylation des promoteurs des gènes situés aux chromosomes 5q31 et 17q21 dans les lignées de cellules humaines épithéliales des voies aériennes (NuLi-1) et du rein embryonnaire (HEK293T/17). Nous avons trouvé que SLC22A5 et GSDMA étaient régulés positivement suite à un traitement au 5-aza-dC dans les deux lignées cellulaires. L’expression de ZPBP2 était augmentée dans les cellules NuLi-1 mais restait silencieuse dans les cellules HEK293T/17, tandis qu’ORMDL3 montrait une régulation positive dans les HEK293T/17 mais pas dans les NuLi-1. Les expressions augmentées de ZPBP2 et de GSDMA étaient accompagnées par une diminution de la méthylation du promoteur, alors qu’aucun changement des niveaux de méthylation du promoteur de SLC22A5 n’a été observé. De plus, nous avons remarqué une modification dans l’expression allélique de ZPBP2 et ORMDL3, suggérant que chaque région peut réagir de façon unique au 5-aza-dC.Nous avons émis l’hypothèse que les différences liées au sexe dans les niveaux de méthylation de l’ADN du promoteur de ZPBP2 étaient dues au dosage des chromosomes sexuels

A novel homozygous splice site variant in the CLCN7 causes osteopetrosis

Objectives: Osteopetrosis is a monogenic disorder represented by disturbed osteoclast resorption or osteoclastogenesis differentiation. Clinical symptoms are intensive and brittle bones, recurrent fractures, thrombocytopenia, impaired immune function, optic nerve compression, and anemia. Several osteopetrosis-causing genes have been identified and reported. Methods: The present study describes two consanguineous Saudi families segregating a severe autosomal recessive osteopetrosis disease. A single proband (II-2) in family A and two probands (II-2; II-4) in family B exhibited increased bone density, multiple fractures, teeth abnormalities, bilateral optic atrophy with nystagmus, and progressive blindness. DNA of the affected individuals was exposed to whole-exome sequencing (WES) and Sanger sequencing. Further, reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting analysis were done to investigate the impact of the identified mutation. Results: WES revealed a novel homozygous splice site variant (c.739-18G > A) in the CLCN7 gene on chromosome 16p13.3, segregating perfectly with the disorder phenotype. RT-PCR resulted in the retention of a 50 bp sequence of intron 8 in the mutated sequence. As a result, this variant resulted in a large size exon 9 compared to the wild type. In addition, the western blot revealed the heteromeric form of ClC-7 disappeared in the patient’s fibroblasts versus the control, indicating identified variant pathogenicity. Conclusion: The present research provides certain proof that homozygous variants in the CLCN7 gene are responsible for intense osteopetrosis disorders with diverse phenotypes. These findings have significant implications for decisions regarding the clinical therapeutic regimen, prognosis assessment, and antenatal diagnosis

Next-Generation Sequencing-Based Pre-Implantation Genetic Testing for Aneuploidy (PGT-A): First Report from Saudi Arabia

Recently, high-throughput next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidies techniques came into use. This technique is essential for successful embryo transfer and accomplishing pregnancy, thus reducing the time and cost of additional cycles. In this study, we describe our first experience in introducing an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) service using next-generation sequencing in King Abdulaziz Medical City located in Riyadh, Saudi Arabia. Our main goal was to report the successful implementation of this new technology in clinical practice and highlight the factors that may affect the results. In total, 200 blastomere biopsies were obtained from 36 in vitro fertilization (IVF) cycles from Saudi couples suffering from prolonged infertility or recurrent embryo transfer failure. NGS-based PGT-A was performed in all embryos. The results were analyzed in five age groups, showing that aneuploidy rates increased with maternal age. Moreover, the results also showed that complex abnormal embryos with (2–5) aneuploidy are the most common type of embryos. Additionally, our data showed that chromosome 16-related abnormality was the most frequent abnormality detected among all reported abnormalities. In conclusion, our study suggests that NGS-based PGT-A is an applicable and reliable technique for routine-based embryo screening, especially for couples suffering from recurrent miscarriages or multiple embryo transfer failures

Role of DNA methylation in expression control of the IKZF3-GSDMA region in human epithelial cells.

Chromosomal region 17q12-q21 is associated with asthma and harbors regulatory polymorphisms that influence expression levels of all five protein-coding genes in the region: IKAROS family zinc finger 3 (Aiolos) (IKZF3), zona pellucida binding protein 2 (ZPBP2), ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), and gasdermins A and B (GSDMA, GSDMB). Furthermore, DNA methylation in this region has been implicated as a potential modifier of the genetic risk of asthma development. To further characterize the effect of DNA methylation, we examined the impact of treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) that causes DNA demethylation, on expression and promoter methylation of the five 17q12-q21 genes in the human airway epithelium cell line NuLi-1, embryonic kidney epithelium cell line 293T and human adenocarcinoma cell line MCF-7. 5-aza-dC treatment led to upregulation of expression of GSDMA in all three cell lines. ZPBP2 was upregulated in NuLi-1, but remained repressed in 293T and MCF-7 cells, whereas ORMDL3 was upregulated in 293T and MCF-7 cells, but not NuLi-1. Upregulation of ZPBP2 and GSDMA was accompanied by a decrease in promoter methylation. Moreover, 5-aza-dC treatment modified allelic expression of ZPBP2 and ORMDL3 suggesting that different alleles may respond differently to treatment. We also identified a polymorphic CTCF-binding site in intron 1 of ORMDL3 carrying a CG SNP rs4065275 and determined its methylation level. The site's methylation was unaffected by 5-aza-dC treatment in NuLi-1 cells. We conclude that modest changes (8-13%) in promoter methylation levels of ZPBP2 and GSDMA may cause substantial changes in RNA levels and that allelic expression of ZPBP2 and ORMDL3 is mediated by DNA methylation

Pancytopenia, Recurrent Infection, Poor Wound Healing, Heterotopia of the Brain Probably Associated with A Candidate Novel de Novo CDC42 Gene Defect: Expanding the Molecular and Phenotypic Spectrum

CDC42 (cell division cycle protein 42) belongs to the Rho GTPase family that is known to control the signaling axis that regulates several cellular functions, including cell cycle progression, migration, and proliferation. However, the functional characterization of the CDC42 gene in mammalian physiology remains largely unclear. Here, we report the genetic and functional characterization of a non-consanguineous Saudi family with a single affected individual. Clinical examinations revealed poor wound healing, heterotopia of the brain, pancytopenia, and recurrent infections. Whole exome sequencing revealed a de novo missense variant (c.101C > A, p.Pro34Gln) in the CDC42 gene. The functional assays revealed a substantial reduction in the growth and motility of the patient cells as compared to the normal cells control. Homology three-dimensional (3-D) modeling of CDC42 revealed that the Pro34 is important for the proper protein secondary structure. In conclusion, we report a candidate disease-causing variant, which requires further confirmation for the etiology of CDC42 pathogenesis. This represents the first case from the Saudi population. The current study adds to the spectrum of mutations in the CDC42 gene that might help in genetic counseling and contributes to the CDC42-related genetic and functional characterization. However, further studies into the molecular mechanisms that are involved are needed in order to determine the role of the CDC42 gene associated with aberrant cell migration and immune response

Identification of novel interacts partners of ADAR1 enzyme mediating the oncogenic process in aggressive breast cancer

Abstract Triple-negative breast cancer (TNBC) subtype is characterized by aggressive clinical behavior and poor prognosis patient outcomes. Here, we show that ADAR1 is more abundantly expressed in infiltrating breast cancer (BC) tumors than in benign tumors. Further, ADAR1 protein expression is higher in aggressive BC cells (MDA-MB-231). Moreover, we identify a novel interacting partners proteins list with ADAR1 in MDA-MB-231, using immunoprecipitation assay and mass spectrometry. Using iLoop, a protein–protein interaction prediction server based on structural features, five proteins with high iloop scores were discovered: Histone H2A.V, Kynureninase (KYNU), 40S ribosomal protein SA, Complement C4-A, and Nebulin (ranged between 0.6 and 0.8). In silico analysis showed that invasive ductal carcinomas had the highest level of KYNU gene expression than the other classifications (p < 0.0001). Moreover, KYNU mRNA expression was shown to be considerably higher in TNBC patients (p < 0.0001) and associated with poor patient outcomes with a high-risk value. Importantly, we found an interaction between ADAR1 and KYNU in the more aggressive BC cells. Altogether, these results propose a new ADAR-KYNU interaction as potential therapeutic targeted therapy in aggressive BC

X chromosome dosage and presence of SRY shape sex-specific differences in DNA methylation at an autosomal region in human cells

Abstract Background Sexual dimorphism in DNA methylation levels is a recurrent epigenetic feature in different human cell types and has been implicated in predisposition to disease, such as psychiatric and autoimmune disorders. To elucidate the genetic origins of sex-specific DNA methylation, we examined DNA methylation levels in fibroblast cell lines and blood cells from individuals with different combinations of sex chromosome complements and sex phenotypes focusing on a single autosomal region––the differentially methylated region (DMR) in the promoter of the zona pellucida binding protein 2 (ZPBP2) as a reporter. Results Our data show that the presence of the sex determining region Y (SRY) was associated with lower methylation levels, whereas higher X chromosome dosage in the absence of SRY led to an increase in DNA methylation levels at the ZPBP2 DMR. We mapped the X-linked modifier of DNA methylation to the long arm of chromosome X (Xq13-q21) and tested the impact of mutations in the ATRX and RLIM genes, located in this region, on methylation levels. Neither ATRX nor RLIM mutations influenced ZPBP2 methylation in female carriers. Conclusions We conclude that sex-specific methylation differences at the autosomal locus result from interaction between a Y-linked factor SRY and at least one X-linked factor that acts in a dose-dependent manner

Polymorphic CTCF binding site rs4065275 (C9b) shows genotype dependent CTCF-binding, FAIRE enrichment and variable methylation levels.

<p>(A) Effect of genotype on CTCF-enrichment at the rs4065275 SNP region. Cell lines that carry the rs4065275-G allele show CTCF enrichment, whereas cell lines that carry the rs4065275-A allele do not. (B) Allelic bias in FAIRE enrichment in LCLs. In 3 out of 4 cell lines the rs4065275-G (C in the diagram) is enriched in the nucleosome-free fraction. (C) Methylation levels of the two putative CBS within the C9 CTCF-enriched region, rs4065275 CG (C9b) and the adjacent non-polymorphic CG (C9a), in human epithelial cells were determined using a pyrosequencing methylation assay. Name of cell line and rs4065275 genotype are shown below the x-axis.</p