A loss-of-function AGTR1 variant in a critically-ill infant with renal tubular dysgenesis: case presentation and literature review.

BACKGROUND: Renal tubular dysgenesis (RTD) is a severe disorder with poor prognosis significantly impacting the proximal tubules of the kidney while maintaining an anatomically normal gross structure. The genetic origin of RTD, involving variants in the ACE, REN, AGT, and AGTR1 genes, affects various enzymes or receptors within the Renin angiotensin system (RAS). This condition manifests prenatally with oligohydramninos and postnatally with persistent anuria, severe refractory hypotension, and defects in skull ossification. CASE PRESENTATION: In this report, we describe a case of a female patient who, despite receiving multi vasopressor treatment, experienced persistent hypotension, ultimately resulting in early death at five days of age. While there was a history of parental consanguinity, no reported family history of renal disease existed. Blood samples from the parents and the remaining DNA sample of the patient underwent Whole Genome Sequencing (WGS). The genetic analysis revealed a rare homozygous loss of function variant (NM_000685.5; c.415C > T; p.Arg139*) in the Angiotensin II Receptor Type 1 (AGTR1) gene. CONCLUSION: This case highlights the consequence of loss-of-function variants in AGTR1 gene leading to RTD, which is characterized by high mortality rate at birth or during the neonatal period. Furthermore, we provide a comprehensive review of previously reported variants in the AGTR1 gene, which is the least encountered genetic cause of RTD, along with their associated clinical features.

Burden of Mendelian disorders in a large Middle Eastern biobank.

BACKGROUND: Genome sequencing of large biobanks from under-represented ancestries provides a valuable resource for the interrogation of Mendelian disease burden at world population level, complementing small-scale familial studies. METHODS: Here, we interrogate 6045 whole genomes from Qatar-a Middle Eastern population with high consanguinity and understudied mutational burden-enrolled at the national Biobank and phenotyped for 58 clinically-relevant quantitative traits. We examine a curated set of 2648 Mendelian genes from 20 panels, annotating known and novel pathogenic variants and assessing their penetrance and impact on the measured traits. RESULTS: We find that 62.5% of participants are carriers of at least 1 known pathogenic variant relating to recessive conditions, with homozygosity observed in 1 in 150 subjects (0.6%) for which Peninsular Arabs are particularly enriched versus other ancestries (5.8-fold). On average, 52.3 loss-of-function variants were found per genome, 6.5 of which affect a known Mendelian gene. Several variants annotated in ClinVar/HGMD as pathogenic appeared at intermediate frequencies in this cohort (1-3%), highlighting Arab founder effect, while others have exceedingly high frequencies (> 5%) prompting reconsideration as benign. Furthermore, cumulative gene burden analysis revealed 56 genes having gene carrier frequency > 1/50, including 5 ACMG Tier 3 panel genes which would be candidates for adding to newborn screening in the country. Additionally, leveraging 58 biobank traits, we systematically assess the impact of novel/rare variants on phenotypes and discover 39 candidate large-effect variants associating with extreme quantitative traits. Furthermore, through rare variant burden testing, we discover 13 genes with high mutational load, including 5 with impact on traits relevant to disease conditions, including metabolic disorder and type 2 diabetes, consistent with the high prevalence of these conditions in the region. CONCLUSIONS: This study on the first phase of the growing Qatar Genome Program cohort provides a comprehensive resource from a Middle Eastern population to understand the global mutational burden in Mendelian genes and their impact on traits in seemingly healthy individuals in high consanguinity settings.

Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

Integrating Genome Sequencing and Untargeted Metabolomics in Monozygotic Twins with a Rare Complex Neurological Disorder.

Multi-omics approaches, which integrate genomics, transcriptomics, proteomics, and metabolomics, have emerged as powerful tools in the diagnosis of rare diseases. We used untargeted metabolomics and whole-genome sequencing (WGS) to gain a more comprehensive understanding of a rare disease with a complex presentation affecting female twins from a consanguineous family. The sisters presented with polymicrogyria, a Dandy-Walker malformation, respiratory distress, and multiorgan dysfunctions. Through WGS, we identified two rare homozygous variants in both subjects, a pathogenic variant in ADGRG1(p.Arg565Trp) and a novel variant in CNTNAP1(p.Glu910Val). These genes have been previously associated with autosomal recessive polymicrogyria and hypomyelinating neuropathy with/without contractures, respectively. The twins exhibited symptoms that overlapped with both of these conditions. The results of the untargeted metabolomics analysis revealed significant metabolic perturbations relating to neurodevelopmental abnormalities, kidney dysfunction, and microbiome. The significant metabolites belong to essential pathways such as lipids and amino acid metabolism. The identification of variants in two genes, combined with the support of metabolic perturbation, demonstrates the rarity and complexity of this phenotype and provides valuable insights into its underlying mechanisms.

A variant in sperm-specific glycolytic enzyme enolase 4 (ENO4) causes human male infertility.

BACKGROUND: Although defects in sperm morphology and physiology lead to male infertility, in many instances, the exact disruption of molecular pathways in a given patient is often unknown. The glycolytic pathway is an essential process to supply energy in sperm cell motility. Enolase 4 (ENO4) is crucial for the glycolytic process, which provides the energy for sperm cells in motility. ENO4 is located in the sperm principal piece and is essential for the motility and organization of the sperm flagellum. In the present study, we characterized a family with asthenozoospermia and abnormal sperm morphology as a result of a variant in the enolase 4 (ENO4) gene. METHODS: Computer-assisted semen analysis, papanicolaou smear staining and scanning electron microscopy were used to examine sperm motility and morphology for semen analysis in patients. For genetic analysis, whole-exome sequencing followed by Sanger sequencing was performed. RESULTS: Two brothers in a consanguineous family were being clinically investigated for sperm motility and morphology issues. Genetic analysis by whole-exome sequencing revealed a homozygous variant [c.293A>G, p.(Lys98Arg)] in the ENO4 gene that segregated with infertility in the family, shared by affected but not controls. CONCLUSIONS: In view of the association of asthenozoospermia and abnormal sperm morphology in Eno4 knockout mice, we consider this to be the first report describing the involvement of ENO4 gene in human male infertility. We also explore the possible involvement of another variant in explaining other phenotypic features in this family.

Validation of plasma protein glycation and oxidation biomarkers for the diagnosis of autism.

Autism Spectrum Disorder (ASD) is a common neurodevelopmental disorder in children. It is currently diagnosed by behaviour-based assessments made by observation and interview. In 2018 we reported a discovery study of a blood biomarker diagnostic test for ASD based on a combination of four plasma protein glycation and oxidation adducts. The test had 88% accuracy in children 5-12 years old. Herein, we present an international multicenter clinical validation study (N = 478) with application of similar biomarkers to a wider age range of 1.5-12 years old children. Three hundred and eleven children with ASD (247 male, 64 female; age 5.2 ± 3.0 years) and 167 children with typical development (94 male, 73 female; 4.9 ± 2.4 years) were recruited for this study at Sidra Medicine and Hamad Medical Corporation hospitals, Qatar, and Hospital Regional Universitario de Málaga, Spain. For subjects 5-12 years old, the diagnostic algorithm with features, advanced glycation endproducts (AGEs)-Nε-carboxymethyl-lysine (CML), Nω-carboxymethylarginine (CMA) and 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and oxidative damage marker, o,o'-dityrosine (DT), age and gender had accuracy 83% (CI 79 - 89%), sensitivity 94% (CI 90-98%), specificity 67% (CI 57-76%) and area-under-the-curve of receiver operating characteristic plot (AUROC) 0.87 (CI 0.84-0.90). Inclusion of additional plasma protein glycation and oxidation adducts increased the specificity to 74%. An algorithm with 12 plasma protein glycation and oxidation adduct features was optimum for children of 1.5-12 years old: accuracy 74% (CI 70-79%), sensitivity 75% (CI 63-87%), specificity 74% (CI 58-90%) and AUROC 0.79 (CI 0.74-0.84). We conclude that ASD diagnosis may be supported using an algorithm with features of plasma protein CML, CMA, 3DG-H and DT in 5-12 years-old children, and an algorithm with additional features applicable for ASD screening in younger children. ASD severity, as assessed by ADOS-2 score, correlated positively with plasma protein glycation adducts derived from methylglyoxal, hydroimidazolone MG-H1 and Nε(1-carboxyethyl)lysine (CEL). The successful validation herein may indicate that the algorithm modifiable features are mechanistic risk markers linking ASD to increased lipid peroxidation, neuronal plasticity and proteotoxic stress.

Equity, diversity, and inclusion at the Global Alliance for Genomics and Health.

A lack of diversity in genomics for health continues to hinder equitable leadership and access to precision medicine approaches for underrepresented populations. To avoid perpetuating biases within the genomics workforce and genomic data collection practices, equity, diversity, and inclusion (EDI) must be addressed. This paper documents the journey taken by the Global Alliance for Genomics and Health (a genomics-based standard-setting and policy-framing organization) to create a more equitable, diverse, and inclusive environment for its standards and members. Initial steps include the creation of two groups: the Equity, Diversity, and Inclusion Advisory Group and the Regulatory and Ethics Diversity Group. Following a framework that we call "Reflected in our Teams, Reflected in our Standards," both groups address EDI at different stages in their policy development process.

Genomic architecture of autism spectrum disorder in Qatar: The BARAKA-Qatar Study.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impaired social and communication skills, restricted interests, and repetitive behaviors. The prevalence of ASD among children in Qatar was recently estimated to be 1.1%, though the genetic architecture underlying ASD both in Qatar and the greater Middle East has been largely unexplored. Here, we describe the first genomic data release from the BARAKA-Qatar Study-a nationwide program building a broadly consented biorepository of individuals with ASD and their families available for sample and data sharing and multi-omics research. METHODS: In this first release, we present a comprehensive analysis of whole-genome sequencing (WGS) data of the first 100 families (372 individuals), investigating the genetic architecture, including single-nucleotide variants (SNVs), copy number variants (CNVs), tandem repeat expansions (TREs), as well as mitochondrial DNA variants (mtDNA) segregating with ASD in local families. RESULTS: Overall, we identify potentially pathogenic variants in known genes or regions in 27 out of 100 families (27%), of which 11 variants (40.7%) were classified as pathogenic or likely-pathogenic based on American College of Medical Genetics (ACMG) guidelines. Dominant variants, including de novo and inherited, contributed to 15 (55.6%) of these families, consisting of SNVs/indels (66.7%), CNVs (13.3%), TREs (13.3%), and mtDNA variants (6.7%). Moreover, homozygous variants were found in 7 families (25.9%), with a sixfold increase in homozygous burden in consanguineous versus non-consanguineous families (13.6% and 1.8%, respectively). Furthermore, 28 novel ASD candidate genes were identified in 20 families, 23 of which had recurrent hits in MSSNG and SSC cohorts. CONCLUSIONS: This study illustrates the value of ASD studies in under-represented populations and the importance of WGS as a comprehensive tool for establishing a molecular diagnosis for families with ASD. Moreover, it uncovers a significant role for recessive variation in ASD architecture in consanguineous settings and provides a unique resource of Middle Eastern genomes for future research to the global ASD community.

An integrated tumor, immune and microbiome atlas of colon cancer.

The lack of multi-omics cancer datasets with extensive follow-up information hinders the identification of accurate biomarkers of clinical outcome. In this cohort study, we performed comprehensive genomic analyses on fresh-frozen samples from 348 patients affected by primary colon cancer, encompassing RNA, whole-exome, deep T cell receptor and 16S bacterial rRNA gene sequencing on tumor and matched healthy colon tissue, complemented with tumor whole-genome sequencing for further microbiome characterization. A type 1 helper T cell, cytotoxic, gene expression signature, called Immunologic Constant of Rejection, captured the presence of clonally expanded, tumor-enriched T cell clones and outperformed conventional prognostic molecular biomarkers, such as the consensus molecular subtype and the microsatellite instability classifications. Quantification of genetic immunoediting, defined as a lower number of neoantigens than expected, further refined its prognostic value. We identified a microbiome signature, driven by Ruminococcus bromii, associated with a favorable outcome. By combining microbiome signature and Immunologic Constant of Rejection, we developed and validated a composite score (mICRoScore), which identifies a group of patients with excellent survival probability. The publicly available multi-omics dataset provides a resource for better understanding colon cancer biology that could facilitate the discovery of personalized therapeutic approaches.

A Complex Intrachromosomal Rearrangement Disrupting IRF6 in a Family with Popliteal Pterygium and Van der Woude Syndromes.

Clefts of the lip and/or palate (CL/P) are considered the most common form of congenital anomalies occurring either in isolation or in association with other clinical features. Van der woude syndrome (VWS) is associated with about 2% of all CL/P cases and is further characterized by having lower lip pits. Popliteal pterygium syndrome (PPS) is a more severe form of VWS, normally characterized by orofacial clefts, lower lip pits, skin webbing, skeletal anomalies and syndactyly of toes and fingers. Both syndromes are inherited in an autosomal dominant manner, usually caused by heterozygous mutations in the Interferon Regulatory Factor 6 (IRF6) gene. Here we report the case of a two-generation family where the index presented with popliteal pterygium syndrome while both the father and sister had clinical features of van der woude syndrome, but without any point mutations detected by re-sequencing of known gene panels or microarray testing. Using whole genome sequencing (WGS) followed by local de novo assembly, we discover and validate a copy-neutral, 429 kb complex intra-chromosomal rearrangement in the long arm of chromosome 1, disrupting the IRF6 gene. This variant is copy-neutral, novel against publicly available databases, and segregates in the family in an autosomal dominant pattern. This finding suggests that missing heritability in rare diseases may be due to complex genomic rearrangements that can be resolved by WGS and de novo assembly, helping deliver answers to patients where no genetic etiology was identified by other means.

A GHRHR founder mutation causes isolated growth hormone deficiency type IV in a consanguineous Pakistani family.

Background: Isolated growth hormone deficiency (IGHD) is caused by a severe shortage or absence of growth hormone (GH), which results in aberrant growth and development. Patients with IGHD type IV (IGHD4) have a short stature, reduced serum GH levels, and delayed bone age. Objectives: To identify the causative mutation of IGHD in a consanguineous family comprising four affected patients with IGHD4 (MIM#618157) and explore its functional impact in silico. Methods: Clinical and radiological studies were performed to determine the phenotypic spectrum and hormonal profile of the disease, while whole-exome sequencing (WES) and Sanger sequencing were performed to identify the disease-causing mutation. In-silico studies involved protein structural modeling and docking, and molecular dynamic simulation analyses using computational tools. Finally, data from the Qatar Genome Program (QGP) were screened for the presence of the founder variant in the Qatari population. Results: All affected individuals presented with a short stature without gross skeletal anomalies and significantly reduced serum GH levels. Genetic mapping revealed a homozygous nonsense mutation [NM_000823:c.G214T:p.(Glu72*)] in the third exon of the growth-hormone-releasing hormone receptor gene GHRHR (MIM#139191) that was segregated in all patients. The substituted amber codon (UAG) seems to truncate the protein by deleting the C-terminus GPCR domain, thus markedly disturbing the GHRHR receptor and its interaction with the growth hormone-releasing hormone. Conclusion: These data support that a p.Glu72* founder mutation in GHRHR perturbs growth hormone signaling and causes IGHD type IV. In-silico and biochemical analyses support the pathogenic effect of this nonsense mutation, while our comprehensive phenotype and hormonal profiling has established the genotype-phenotype correlation. Based on the current study, early detection of GHRHR may help in better therapeutic intervention.

The link between glycemic control measures and eye microvascular complications in a clinical cohort of type 2 diabetes with microRNA-223-3p signature.

BACKGROUND: Type 2 diabetes (T2D) is a critical healthcare challenge and priority in Qatar which is listed amongst the top 10 countries in the world, with its prevalence presently at 17% double the global average. MicroRNAs (miRNAs) are implicated in the pathogenesis of (T2D) and long-term microvascular complications including diabetic retinopathy (DR). METHODS: In this study, a T2D cohort that accurately matches the characteristics of the general population was employed to find microRNA (miRNA) signatures that are correlated with glycemic and ß cell function measurements. Targeted miRNA profiling was performed in (471) T2D individuals with or without DR and (491) (non-diabetic) healthy controls from the Qatar Biobank. Discovery analysis identified 20 differentially expressed miRNAs in T2D compared to controls, of which miR-223-3p was significantly upregulated (fold change:5.16, p = 3.6e-02) and positively correlated with glucose and hemoglobin A1c (HbA1c) levels (p-value = 9.88e-04 and 1.64e-05, respectively), but did not show any significant associations with insulin or C-peptide. Accordingly, we performed functional validation using a miR-223-3p mimic (overexpression) under control and hyperglycemia-induced conditions in a zebrafish model. RESULTS: Over-expression of miR-223-3p alone was associated with significantly higher glucose (42.7 mg/dL, n = 75 vs 38.7 mg/dL, n = 75, p = 0.02) and degenerated retinal vasculature, and altered retinal morphology involving changes in the ganglion cell layer and inner and outer nuclear layers. Assessment of retinal angiogenesis revealed significant upregulation in the expression of vascular endothelial growth factor and its receptors, including kinase insert domain receptor. Further, the pancreatic markers, pancreatic and duodenal homeobox 1, and the insulin gene expressions were upregulated in the miR-223-3p group. CONCLUSION: Our zebrafish model validates a novel correlation between miR-223-3p and DR development. Targeting miR-223-3p in T2D patients may serve as a promising therapeutic strategy to control DR in at-risk individuals.

Assessing the genetic burden of familial hypercholesterolemia in a large middle eastern biobank.

BACKGROUND: The genetic architecture underlying Familial Hypercholesterolemia (FH) in Middle Eastern Arabs is yet to be fully described, and approaches to assess this from population-wide biobanks are important for public health planning and personalized medicine. METHODS: We evaluate the pilot phase cohort (n = 6,140 adults) of the Qatar Biobank (QBB) for FH using the Dutch Lipid Clinic Network (DLCN) criteria, followed by an in-depth characterization of all genetic alleles in known dominant (LDLR, APOB, and PCSK9) and recessive (LDLRAP1, ABCG5, ABCG8, and LIPA) FH-causing genes derived from whole-genome sequencing (WGS). We also investigate the utility of a globally established 12-SNP polygenic risk score to predict FH individuals in this cohort with Arab ancestry. RESULTS: Using DLCN criteria, we identify eight (0.1%) 'definite', 41 (0.7%) 'probable' and 334 (5.4%) 'possible' FH individuals, estimating a prevalence of 'definite or probable' FH in the Qatari cohort of ~ 1:125. We identify ten previously known pathogenic single-nucleotide variants (SNVs) and 14 putatively novel SNVs, as well as one novel copy number variant in PCSK9. Further, despite the modest sample size, we identify one homozygote for a known pathogenic variant (ABCG8, p. Gly574Arg, global MAF = 4.49E-05) associated with Sitosterolemia 2. Finally, calculation of polygenic risk scores found that individuals with 'definite or probable' FH have a significantly higher LDL-C SNP score than 'unlikely' individuals (p = 0.0003), demonstrating its utility in Arab populations. CONCLUSION: We design and implement a standardized approach to phenotyping a population biobank for FH risk followed by systematically identifying known variants and assessing putative novel variants contributing to FH burden in Qatar. Our results motivate similar studies in population-level biobanks - especially those with globally under-represented ancestries - and highlight the importance of genetic screening programs for early detection and management of individuals with high FH risk in health systems.

A novel homozygous variant in homologous recombination repair gene ZSWIM7 causes azoospermia in males and primary ovarian insufficiency in females.

Infertility is a common, clinically heterogeneous reproductive disorder worldwide with a prevalence of about 15%. To date about eighty genes have been discovered to cause non-syndromic infertility, affecting males and females equally, though traditionally the genetic analysis of each group has been conducted separately. Here, we report the clinical and genetic characterization of a consanguineous family of Pakistani origin with multiple individuals, including male and female, affected with infertility. Males exhibited non-obstructive azoospermia whereas females had primary ovarian insufficiency. Whole exome sequencing revealed a missense variant [c.176C > T, p. (Ser59Leu)] in the ZSWIM7 gene which functions in homologous recombination repair. The variant was found in a homozygous form in all affected males and females. To our knowledge, this is the first family that has individuals affected with infertility in both sexes. This point to the utility of large consanguineous families with multiple affected siblings to reveal joint mechanisms affecting human reproduction.

Transcriptome Profile Identifies Actin as an Essential Regulator of Cardiac Myosin Binding Protein C3 Hypertrophic Cardiomyopathy in a Zebrafish Model.

Variants in cardiac myosin-binding protein C (cMyBP-C) are the leading cause of inherited hypertrophic cardiomyopathy (HCM), demonstrating the key role that cMyBP-C plays in the heart's contractile machinery. To investigate the c-MYBPC3 HCM-related cardiac impairment, we generated a zebrafish mypbc3-knockout model. These knockout zebrafish displayed significant morphological heart alterations related to a significant decrease in ventricular and atrial diameters at systolic and diastolic states at the larval stages. Immunofluorescence staining revealed significant hyperplasia in the mutant's total cardiac and ventricular cardiomyocytes. Although cardiac contractility was similar to the wild-type control, the ejection fraction was significantly increased in the mypbc3 mutants. At later stages of larval development, the mutants demonstrated an early cardiac phenotype of myocardium remodeling, concurrent cardiomyocyte hyperplasia, and increased ejection fraction as critical processes in HCM initiation to counteract the increased ventricular myocardial wall stress. The examination of zebrafish adults showed a thickened ventricular cardiac wall with reduced heart rate, swimming speed, and endurance ability in both the mypbc3 heterozygous and homozygous groups. Furthermore, heart transcriptome profiling showed a significant downregulation of the actin-filament-based process, indicating an impaired actin cytoskeleton organization as the main dysregulating factor associated with the early ventricular cardiac hypertrophy in the zebrafish mypbc3 HCM model.

The immune landscape of solid pediatric tumors.

BACKGROUND: Large immunogenomic analyses have demonstrated the prognostic role of the functional orientation of the tumor microenvironment in adult solid tumors, this variable has been poorly explored in the pediatric counterpart. METHODS: We performed a systematic analysis of public RNAseq data (TARGET) for five pediatric tumor types (408 patients): Wilms tumor (WLM), neuroblastoma (NBL), osteosarcoma (OS), clear cell sarcoma of the kidney (CCSK) and rhabdoid tumor of the kidney (RT). We assessed the performance of the Immunologic Constant of Rejection (ICR), which captures an active Th1/cytotoxic response. We also performed gene set enrichment analysis (ssGSEA) and clustered more than 100 well characterized immune traits to define immune subtypes and compared their outcome. RESULTS: A higher ICR score was associated with better survival in OS and high risk NBL without MYCN amplification but with poorer survival in WLM. Clustering of immune traits revealed the same five principal modules previously described in adult tumors (TCGA). These modules divided pediatric patients into six immune subtypes (S1-S6) with distinct survival outcomes. The S2 cluster showed the best overall survival, characterized by low enrichment of the wound healing signature, high Th1, and low Th2 infiltration, while the reverse was observed in S4. Upregulation of the WNT/Beta-catenin pathway was associated with unfavorable outcomes and decreased T-cell infiltration in OS. CONCLUSIONS: We demonstrated that extracranial pediatric tumors could be classified according to their immune disposition, unveiling similarities with adults' tumors. Immunological parameters might be explored to refine diagnostic and prognostic biomarkers and to identify potential immune-responsive tumors.

Patterns and distribution of de novo mutations in multiplex Middle Eastern families.

While de novo mutations (DNMs) are key to genetic diversity, they are also responsible for a high number of rare disorders. To date, no study has systematically examined the rate and distribution of DNMs in multiplex families in highly consanguineous populations. Leveraging WGS profiles of 645 individuals in 146 families, we implemented a combinatorial approach using 3 complementary tools for DNM discovery in 353 unique trio combinations. We found a total of 27,168 DNMs (median: 70 single-nucleotide and 6 insertion-deletions per individual). Phasing revealed around 80% of DNMs were paternal in origin. Notably, using whole-genome methylation data of spermatogonial stem cells, these DNMs were significantly more likely to occur at highly methylated CpGs (OR: 2.03; p value = 6.62 × 10-11). We then examined the effects of consanguinity and ethnicity on DNMs, and found that consanguinity does not seem to correlate with DNM rate, and special attention has to be considered while measuring such a correlation. Additionally, we found that Middle-Eastern families with Arab ancestry had fewer DNMs than African families, although not significant (p value = 0.16). Finally, for families with diseased probands, we examined the difference in DNM counts and putative impact across affected and unaffected siblings, but did not find significant differences between disease groups, likely owing to the enrichment for recessive disorders in this part of the world, or the small sample size per clinical condition. This study serves as a reference for DNM discovery in multiplex families from the globally under-represented populations of the Middle-East.

Analysis of incidental findings in Qatar genome participants reveals novel functional variants in LMNA and DSP.

In order to report clinically actionable incidental findings in genetic testing, the American College of Medical Genetics and Genomics (ACMG) recommended the evaluation of variants in 59 genes associated with highly penetrant mutations. However, there is a lack of epidemiological data on medically actionable rare variants in these genes in Arab populations. We used whole genome sequencing data from 6045 participants from the Qatar Genome Programme and integrated it with phenotypic data collected by the Qatar Biobank. We identified novel putative pathogenic variants in the 59 ACMG genes by filtering previously unrecorded variants based on computational prediction of pathogenicity, variant rarity and segregation evidence. We assessed the phenotypic associations of candidate variants in genes linked to cardiovascular diseases. Finally, we used a zebrafish knockdown and synthetic human mRNA co-injection assay to functionally characterize two of these novel variants. We assessed the zebrafish cardiac function in terms of heart rate, rhythm and hemodynamics, as well as the heart structure. We identified 52 492 novel variants, which have not been reported in global and disease-specific databases. A total of 74 novel variants were selected with potentially pathogenic effect. We prioritized two novel cardiovascular variants, DSP c.1841A > G (p.Asp614Gly) and LMNA c.326 T > G (p.Val109Gly) for functional characterization. Our results showed that both variants resulted in abnormal zebrafish heart rate, rhythm and structure. This study highlights medically actionable variants that are specific to the Middle Eastern Qatari population.

A recessive variant in SIM2 in a child with complex craniofacial anomalies and global developmental delay.

Rare deletions and duplications on the long arm of Chromosome 21 have previously been reported in many patients with craniofacial and developmental phenotypes. However, this Down Syndrome Critical Region (DSCR) contains multiple genes, making identifying a single causative gene difficult. Here, we report a case of a boy with bicoronal craniosynostosis, facial dysmorphism, developmental delay, and intellectual impairment who was found by whole genome sequencing to have a homozygous missense mutation in the Single-Minded Homolog 2 (SIM2) gene (c.461 A > G, p.Tyr154Cys) within the DSCR. SIM2 encodes an essential bHLH and PAS domain transcription factor expressed during fetal brain development and acts as a master regulator of neurogenesis. This variant is globally very rare, segregates in the family, and is predicted to be highly deleterious by in silico analysis, 3D molecular modeling of protein structure, and functional analysis of zebrafish models. Zebrafish expressing the human SIM2p.Y154C variant displayed a progressed microcephaly-like phenotype and head shape abnormalities. When combined with careful phenotyping of the patient vis-à-vis previously reported cases harboring structural variants in this critical 21q22 region, the data support a pathogenic role of SIM2 in this complex syndrome and demonstrates the utility of next-generation sequencing in prioritizing genes in contiguous deletions/duplications syndromes and diagnosing microarray-negative patients in the craniofacial clinic.

Clinical, Genetic and Functional Characterization of a Novel AVPR2 Missense Mutation in a Woman with X-Linked Recessive Nephrogenic Diabetes Insipidus.

Nephrogenic diabetes insipidus (NDI) is a rare disorder characterized by renal unresponsiveness to the hormone vasopressin, leading to excretion of large volumes of diluted urine. Mutations in the arginine vasopressin receptor-2 (AVPR2) gene cause congenital NDI and have an X-linked recessive inheritance. The disorder affects almost exclusively male family members, but female carriers occasionally present partial phenotypes due to skewed inactivation of the X-chromosome. Here, we report a rare case of a woman affected with X-linked recessive NDI, presenting an average urinary output of 12 L/day. Clinical and biochemical studies showed incomplete responses to water deprivation and vasopressin stimulation tests. Genetic analyses revealed a novel heterozygous missense mutation (c.493G > C, p.Ala165Pro) in the AVPR2 gene. Using a combination of in-silico protein modeling with human cellular models and molecular phenotyping, we provide functional evidence for phenotypic effects. The mutation destabilizes the helical structure of the AVPR2 transmembrane domains and disrupts its plasma membrane localization and downstream intracellular signaling pathways upon activation with its agonist vasopressin. These defects lead to deficient aquaporin 2 (AQP2) membrane translocation, explaining the inability to concentrate urine in this patient.