Genome-Wide Association Study-Guided Exome Rare Variant Burden Analysis Identifies IL1R1 and CD3E as Potential Autoimmunity Risk Genes for Celiac Disease.

Celiac disease (CeD) is a multifactorial autoimmune enteropathy characterized by the overactivation of the immune system in response to dietary gluten. The molecular etiology of CeD is still not well-understood. Therefore, this study aims to identify potential candidate genes involved in CeD pathogenesis by applying multilayered system biology approaches. Initially, we identified rare coding variants shared between the affected siblings in two rare Arab CeD families by whole-exome sequencing (WES). Then we used the STRING database to construct a protein network of rare variants and genome-wide association study (GWAS) loci to explore their molecular interactions in CeD. Furthermore, the hub genes identified based on network topology parameters were subjected to a series of computational validation analyses like pathway enrichment, gene expression, knockout mouse model, and variant pathogenicity predictions. Our findings have shown the absence of rare variants showing classical Mendelian inheritance in both families. However, interactome analysis of rare WES variants and GWAS loci has identified a total of 11 hub genes. The multidimensional computational analysis of hub genes has prioritized IL1R1 for family A and CD3E for family B as potential genes. These genes were connected to CeD pathogenesis pathways of T-cell selection, cytokine signaling, and adaptive immune response. Future multi-omics studies may uncover the roles of IL1R1 and CD3E in gluten sensitivity. The present investigation lays forth a novel approach integrating next-generation sequencing (NGS) of familial cases, GWAS, and computational analysis for solving the complex genetic architecture of CeD.

Novel MYO1D Missense Variant Identified Through Whole Exome Sequencing and Computational Biology Analysis Expands the Spectrum of Causal Genes of Laterality Defects.

Laterality defects (LDs) or asymmetrically positioned organs are a group of rare developmental disorders caused by environmental and/or genetic factors. However, the exact molecular pathophysiology of LD is not yet fully characterised. In this context, studying Arab population presents an ideal opportunity to discover the novel molecular basis of diseases owing to the high rate of consanguinity and genetic disorders. Therefore, in the present study, we studied the molecular basis of LD in Arab patients, using next-generation sequencing method. We discovered an extremely rare novel missense variant in MYO1D gene (Pro765Ser) presenting with visceral heterotaxy and left isomerism with polysplenia syndrome. The proband in this index family has inherited this homozygous variant from her heterozygous parents following the autosomal recessive pattern. This is the first report to show MYO1D genetic variant causing left-right axis defects in humans, besides previous known evidence from zebrafish, frog and Drosophila models. Moreover, our multilevel bioinformatics-based structural (protein variant structural modelling, divergence, and stability) analysis has suggested that Ser765 causes minor structural drifts and stability changes, potentially affecting the biophysical and functional properties of MYO1D protein like calmodulin binding and microfilament motor activities. Functional bioinformatics analysis has shown that MYO1D is ubiquitously expressed across several human tissues and is reported to induce severe phenotypes in knockout mouse models. In conclusion, our findings show the expanded genetic spectrum of LD, which could potentially pave way for the novel drug target identification and development of personalised medicine for high-risk families.

TagSNP approach for HLA risk allele genotyping of Saudi celiac disease patients: effectiveness and pitfalls.

BACKGROUND: Celiac disease (CD) is a genetically complex autoimmune disease which is triggered by dietary gluten. Human leukocyte antigen (HLA) class II genes are known to act as high-risk markers for CD, where >95% of CD patients carry (HLA), DQ2 and/or DQ8 alleles. Therefore, the present study was conducted to investigate the distribution of HLA haplotypes among Saudi CD patients and healthy controls by using the tag single nucleotide polymorphisms (SNP). METHODS: HLA-tag SNPs showing strong linkage value (r2>0.99) were used to predict the HLA DQ2 and DQ8 genotypes in 101 Saudi CD patients and in 103 healthy controls by using real-time polymerase chain reaction technique. Genotype calls were further validated by Sanger sequencing method. RESULTS: A total of 63.7% of CD cases and of 60.2% of controls were predicted to carry HLA-DQ2 and DQ8 heterodimers, either in the homozygous or heterozygous states. The prevalence of DQ8 in our CD patients was predicted to be higher than the patients from other ethnic populations (35.6%). More than 32% of the CD patients were found to be non-carriers of HLA risk haplotypes as predicted by the tag SNPs. CONCLUSION: The present study highlights that the Caucasian specific HLA-tag SNPs would be of limited value to accurately predict CD specific HLA haplotypes in Saudi population, when compared with the Caucasian groups. Prediction of risk haplotypes by tag SNPs in ethnic groups is a good alternate approach as long as the tag SNPs were identified from the local population genetic variant databases.

Exploring celiac disease candidate pathways by global gene expression profiling and gene network cluster analysis.

Celiac disease (CeD) is a gastrointestinal autoimmune disorder, whose specific molecular basis is not yet fully interpreted. Therefore, in this study, we compared the global gene expression profile of duodenum tissues from CeD patients, both at the time of disease diagnosis and after two years of the gluten-free diet. A series of advanced systems biology approaches like differential gene expression, protein-protein interactions, gene network-cluster analysis were deployed to annotate the candidate pathways relevant to CeD pathogenesis. The duodenum tissues from CeD patients revealed the differential expression of 106 up- and 193 down-regulated genes. The pathway enrichment of differentially expressed genes (DEGs) highlights the involvement of biological pathways related to loss of cell division regulation (cell cycle, p53 signalling pathway), immune system processes (NOD-like receptor signalling pathway, Th1, and Th2 cell differentiation, IL-17 signalling pathway) and impaired metabolism and absorption (mineral and vitamin absorptions and drug metabolism) in celiac disease. The molecular dysfunctions of these 3 biological events tend to increase the number of intraepithelial lymphocytes (IELs) and villous atrophy of the duodenal mucosa promoting the development of CeD. For the first time, this study highlights the involvement of aberrant cell division, immune system, absorption, and metabolism pathways in CeD pathophysiology and presents potential novel therapeutic opportunities.

Whole exome sequencing of a Saudi family and systems biology analysis identifies CPED1 as a putative causative gene to Celiac Disease.

Celiac disease (CD) is a gastrointestinal disorder whose genetic basis is not fully understood. Therefore, we studied a Saudi family with two CD affected siblings to discover the causal genetic defect. Through whole exome sequencing (WES), we identified that both siblings have inherited an extremely rare and deleterious CPED1 genetic variant (c.241 A > G; p.Thr81Ala) segregating as autosomal recessive mutation, suggesting its putative causal role in the CD. Saudi population specific minor allele frequency (MAF) analysis has confirmed its extremely rare prevalence in homozygous condition (MAF is 0.0004). The Sanger sequencing analysis confirmed the absence of this homozygous variant in 100 sporadic Saudi CD cases. Genotype-Tissue Expression (GTEx) data has revealed that CPED1 is abundantly expressed in gastrointestinal mucosa. By using a combination of systems biology approaches like protein 3D modeling, stability analysis and nucleotide sequence conservation analysis, we have further established that this variant is deleterious to the structural and functional aspects of CPED1 protein. To the best of our knowledge, this variant has not been previously reported in CD or any other gastrointestinal disease. The cell culture and animal model studies could provide further insight into the exact role of CPED1 p.Thr81Ala variant in the pathophysiology of CD. In conclusion, by using WES and systems biology analysis, present study for the first-time reports CPED1 as a potential causative gene for CD in a Saudi family with potential implications to both disease diagnosis and genetic counseling.

A mutation in the major autophagy gene, WIPI2, associated with global developmental abnormalities.

We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377-5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5-12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux.

Exome Analysis Identifies a Novel Compound Heterozygous Alteration in TGM1 Gene Leading to Lamellar Ichthyosis in a Child From Saudi Arabia: Case Presentation.

Background: Lamellar ichthyosis is an autosomal recessive type of rare skin disorders characterized with defective epidermis leading hyperkeratosis with brownish-gray scales over the body. These patients are born as collodion babies and may also exhibit additional features like erythema, ectropion, and eclabium. This disease is mainly caused by homozygous and compound heterozygous alterations in transglutaminase 1 encoding gene (TGM1), which is located on 14q12. Case presentation: This study reports the genetic analysis of a 4-year Saudi girl presenting lamellar ichthyosis. She was the first child of unrelated parents. The family had no previous history of the disease phenotype. She was born as a collodion baby without any prenatal complications. At the time of this study she had developed rough scaly skin on her legs, arms and trunk regions with thick palms and soles. Whole exome sequencing (WES) followed by Sanger sequence validation identified a novel compound heterozygous variant in TGM1 gene. The paternal variant was a missense transition (c.1141G>A; p.Ala381Thr) present at exon 7, while maternal variant (c.758-1G>C) was present at the intron4-exon5 boundary. To the best of our knowledge these variants had not been reported before in TGM1 gene. Conclusion: In isolated and inbred populations, homozygous variants are identified more frequently; however, our results suggest that compound heterozygous variants should also be considered especially when the marriages are not consanguineous.

A missense mutation in TRAPPC6A leads to build-up of the protein, in patients with a neurodevelopmental syndrome and dysmorphic features.

Childhood onset clinical syndromes involving intellectual disability and dysmorphic features, such as polydactyly, suggest common developmental pathways link seemingly unrelated phenotypes. We identified a consanguineous family of Saudi origin with varying complex features including intellectual disability, speech delay, facial dysmorphism and polydactyly. Combining, microarray based comparative genomic hybridisation (CGH) to identify regions of homozygosity, with exome sequencing, led to the identification of homozygous mutations in five candidate genes (RSPH6A, ANKK1, AMOTL1, ALKBH8, TRAPPC6A), all of which appear to be pathogenic as predicted by Proven, SIFT and PolyPhen2 and segregate perfectly with the disease phenotype. We therefore looked for differences in expression levels of each protein in HEK293 cells, expressing either the wild-type or mutant full-length cDNA construct. Unexpectedly, wild-type TRAPPC6A appeared to be unstable, but addition of the proteasome inhibitor MG132 stabilised its expression. Mutations have previously been reported in several members of the TRAPP complex of proteins, including TRAPPC2, TRAPPC9 and TRAPPC11, resulting in disorders involving skeletal abnormalities, intellectual disability, speech impairment and developmental delay. TRAPPC6A joins a growing list of proteins belonging to the TRAPP complex, implicated in clinical syndromes with neurodevelopmental abnormalities.

Whole exome sequencing of a consanguineous family identifies the possible modifying effect of a globally rare AK5 allelic variant in celiac disease development among Saudi patients.

Celiac disease (CD), a multi-factorial auto-inflammatory disease of the small intestine, is known to occur in both sporadic and familial forms. Together HLA and Non-HLA genes can explain up to 50% of CD's heritability. In order to discover the missing heritability due to rare variants, we have exome sequenced a consanguineous Saudi family presenting CD in an autosomal recessive (AR) pattern. We have identified a rare homozygous insertion c.1683_1684insATT, in the conserved coding region of AK5 gene that showed classical AR model segregation in this family. Sequence validation of 200 chromosomes each of sporadic CD cases and controls, revealed that this extremely rare (EXac MAF 0.000008) mutation is highly penetrant among general Saudi populations (MAF is 0.62). Genotype and allelic distribution analysis have indicated that this AK5 (c.1683_1684insATT) mutation is negatively selected among patient groups and positively selected in the control group, in whom it may modify the risk against CD development [p<0.002]. Our observation gains additional support from computational analysis which predicted that Iso561 insertion shifts the existing H-bonds between 400th and 556th amino acid residues lying near the functional domain of adenylate kinase. This shuffling of amino acids and their H-bond interactions is likely to disturb the secondary structure orientation of the polypeptide and induces the gain-of-function in nucleoside phosphate kinase activity of AK5, which may eventually down-regulates the reactivity potential of CD4+ T-cells against gluten antigens. Our study underlines the need to have population-specific genome databases to avoid false leads and to identify true candidate causal genes for the familial form of celiac disease.

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism.

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

Shotgun Metagenomics of 250 Adult Twins Reveals Genetic and Environmental Impacts on the Gut Microbiome.

The gut microbiota has been typically viewed as an environmental factor for human health. Twins are well suited for investigating the concordance of their gut microbiomes and decomposing genetic and environmental influences. However, existing twin studies utilizing metagenomic shotgun sequencing have included only a few samples. Here, we sequenced fecal samples from 250 adult twins in the TwinsUK registry and constructed a comprehensive gut microbial reference gene catalog. We demonstrate heritability of many microbial taxa and functional modules in the gut microbiome, including those associated with diseases. Moreover, we identified 8 million SNPs in the gut microbiome and observe a high similarity in microbiome SNPs between twins that slowly decreases after decades of living apart. The results shed new light on the genetic and environmental influences on the composition and function of the gut microbiome that could relate to risk of complex diseases.

Whole-exome sequencing reveals a recurrent mutation in the cathepsin C gene that causes Papillon-Lefevre syndrome in a Saudi family.

Papillon-Lefevre syndrome (PALS) is a rare, autosomal recessive disorder characterized by periodontitis and hyperkeratosis over the palms and soles. Mutations in the cathepsin C gene (CTSC) have been recognized as the cause of PALS since the late 1990s. More than 75 mutations in CTSC have been identified, and phenotypic variability between different mutations has been described. Next generation sequencing is widely used for efficient molecular diagnostics in various clinical practices. Here we investigated a large consanguineous Saudi family with four affected and four unaffected individuals. All of the affected individuals suffered from hyperkeratosis over the palms and soles and had anomalies of both primary and secondary dentition. For molecular diagnostics, we combined whole-exome sequencing and genome-wide homozygosity mapping procedures, and identified a recurrent homozygous missense mutation (c.899G>A; p.Gly300Asp) in exon 7 of CTSC. Validation of all eight family members by Sanger sequencing confirmed co-segregation of the pathogenic variant (c.899G>A) with the disease phenotype. This is the first report of whole-exome sequencing performed for molecular diagnosis of PALS in Saudi Arabia. Our findings provide further insights into the genotype-phenotype correlation of CTSC pathogenicity in PALS.

A novel homozygous PTH1R variant identified through whole-exome sequencing further expands the clinical spectrum of primary failure of tooth eruption in a consanguineous Saudi family.

OBJECTIVES: The present study aimed to identify the genetic cause of non-syndromic primary failure of tooth eruption in a five-generation consanguineous Saudi family using whole-exome sequencing (WES) analysis. DESIGN: The family pedigree and phenotype were obtained from patient medical records. WES of all four affected family members was performed using the 51 Mb SureSelect V4 library kit and then sequenced using the Illumina HiSeq2000 sequencing system. Sequence alignment, variant calling, and the annotation of single nucleotide polymorphisms and indels were performed using standard bioinformatics pipelines. The genotype of candidate variants was confirmed in all available family members by Sanger sequencing. RESULTS: Pedigree analysis suggested that the inheritance was autosomal recessive. WES of all affected individuals identified a novel homozygous variant in exon 8 of the parathyroid hormone 1 receptor gene (PTH1R) (NM_000316: c.611T>A: p.Val204Glu). CONCLUSION: To the best of our knowledge, this is the first report of primary failure of eruption caused by a homozygous mutation in PTH1R. Our findings prove the application of WES as an efficient molecular diagnostics tool for this rare phenotype and further broaden the clinical spectrum of PTH1R pathogenicity.

The alkylglycerol monooxygenase (AGMO) gene previously involved in autism also causes a novel syndromic form of primary microcephaly in a consanguineous Saudi family.

Autosomal recessive primary microcephaly (MCPH) refers to a genetically heterogeneous group of neurodevelopmental disorders in which patients exhibit a marked decrease in occipitofrontal head circumference at birth and a variable degree of intellectual disability. To date, 18 genes have been reported for MCPH worldwide. We enrolled a consanguineous family from Saudi Arabia presenting with primary microcephaly, developmental delay, short stature and intellectual disability. Whole exome sequencing (WES) with 100× coverage was performed on two affected siblings after defining common regions of homozygosity through genome-wide single nucleotide polymorphism (SNP) microarray genotyping. WES data analysis, confirmed by subsequent Sanger sequence validation, identified a novel homozygous deletion mutation (c.967delA; p.Glu324Lysfs12*) in exon 10 of the alkylglycerol monooxygenase (AGMO) gene on chromosome 7p21.2. Population screening of 178 ethnically matched control chromosomes and consultation of the Exome Aggregation Consortium database, containing 60,706 individuals' exomes worldwide, confirmed that this mutation was not present outside the family. To the best of our knowledge, this is the first evidence of an AGMO mutation underlying primary microcephaly and intellectual disability in humans. Our findings further expand the genetic heterogeneity of MCPH in familial cases.

The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment.

We carried out metagenomic shotgun sequencing and a metagenome-wide association study (MGWAS) of fecal, dental and salivary samples from a cohort of individuals with rheumatoid arthritis (RA) and healthy controls. Concordance was observed between the gut and oral microbiomes, suggesting overlap in the abundance and function of species at different body sites. Dysbiosis was detected in the gut and oral microbiomes of RA patients, but it was partially resolved after RA treatment. Alterations in the gut, dental or saliva microbiome distinguished individuals with RA from healthy controls, were correlated with clinical measures and could be used to stratify individuals on the basis of their response to therapy. In particular, Haemophilus spp. were depleted in individuals with RA at all three sites and negatively correlated with levels of serum autoantibodies, whereas Lactobacillus salivarius was over-represented in individuals with RA at all three sites and was present in increased amounts in cases of very active RA. Functionally, the redox environment, transport and metabolism of iron, sulfur, zinc and arginine were altered in the microbiota of individuals with RA. Molecular mimicry of human antigens related to RA was also detectable. Our results establish specific alterations in the gut and oral microbiomes in individuals with RA and suggest potential ways of using microbiome composition for prognosis and diagnosis.

BRAF gene: From human cancers to developmental syndromes.

The BRAF gene encodes for a serine/threonine protein kinase that participates in the MAPK/ERK signalling pathway and plays a vital role in cancers and developmental syndromes (RASopathies). The current review discusses the clinical significance of the BRAF gene and other members of RAS/RAF cascade in human cancers and RAS/MAPK syndromes, and focuses the molecular basis and clinical genetics of BRAF to better understand its parallel involvement in both tumourigenesis and RAS/MAPK syndromes-Noonan syndrome, cardio-facio-cutaneous syndrome and LEOPARD syndrome.

Truncating mutation in intracellular phospholipase A1 gene (DDHD2) in hereditary spastic paraplegia with intellectual disability (SPG54).

BACKGROUND: Hereditary spastic paraplegias (HSP), a group of genetically heterogeneous neurological disorders with more than 56 documented loci (SPG1-56), are described either as uncomplicated (or pure), or complicated where in addition to spasticity and weakness of lower extremeties, additional neurological symptoms are present, including dementia, loss of vision, epilepsy, mental retardation and ichthyosis. We identified a large consanguineous family of Indian descent with four affected members with childhood onset HSP (SPG54), presenting with upper and lower limb spasticity, mental retardation and agenesis of the corpus callosum. RESULTS: A common region of homozygosity on chromosome 8 spanning seven megabases (Mb) was identified in the affected individuals using the Illumina human cytoSNP-12 DNA Analysis BeadChip Kit. Exome sequencing identified a homozygous stop gain mutation (pR287X) in the phospholipase A1 gene DDHD2, in the affected individuals, resulting in a premature stop codon and a severely truncated protein lacking the SAM and DDHD domains crucial for phosphoinositide binding and phospholipase activity. CONCLUSION: This mutation adds to the knowledge of HSP, suggests a possible founder effect for the pR287X mutation, and adds to the list of genes involved in lipid metabolism with a role in HSP and other neurodegenerative disorders.

Exome analysis identified a novel missense mutation in the CLPP gene in a consanguineous Saudi family expanding the clinical spectrum of Perrault Syndrome type-3.

Perrault syndrome (PRLTS) is a clinically and genetically heterogeneous disorder. Both male and female patients suffer from sensory neuronal hearing loss in early childhood, and female patients are characterized by premature ovarian failure and infertility after puberty. Clinical diagnosis may not be possible in early life, because key features of PRLTS, for example infertility and premature ovarian failure, do not appear before puberty. Limb spasticity, muscle weakness, and intellectual disability have also been observed in PRLTS patients. Mutations in five genes, HSD17B4, HARS2, CLPP, LARS2, and C10orf2, have been reported in five subtypes of PRLTS. We discovered a consanguineous Saudi family with the PRLTS3 phenotype showing an autosomal recessive mode of inheritance. The patients had developed profound hearing loss, brain atrophy, and lower limb spasticity in early childhood. For molecular diagnosis, we complimented genome-wide homozygosity mapping with whole exome sequencing analyses and identified a novel homozygous mutation in exon 6 of CLPP at chromosome 19p13.3. To our knowledge, early onset with regression is a unique feature of these PRLTS patients that has not been reported so far. This study broadens the clinical spectrum of PRLTS3.

Gut microbiome development along the colorectal adenoma-carcinoma sequence.

Colorectal cancer, a commonly diagnosed cancer in the elderly, often develops slowly from benign polyps called adenoma. The gut microbiota is believed to be directly involved in colorectal carcinogenesis. The identity and functional capacity of the adenoma- or carcinoma-related gut microbe(s), however, have not been surveyed in a comprehensive manner. Here we perform a metagenome-wide association study (MGWAS) on stools from advanced adenoma and carcinoma patients and from healthy subjects, revealing microbial genes, strains and functions enriched in each group. An analysis of potential risk factors indicates that high intake of red meat relative to fruits and vegetables appears to associate with outgrowth of bacteria that might contribute to a more hostile gut environment. These findings suggest that faecal microbiome-based strategies may be useful for early diagnosis and treatment of colorectal adenoma or carcinoma.