Dysregulation of long non-coding RNA gene expression pathways in monocytes of type 2 diabetes patients with cardiovascular disease.

BACKGROUND: Monocytes play a central role in the pathophysiology of cardiovascular complications in type 2 diabetes (T2D) patients through different mechanisms. We investigated diabetes-induced changes in lncRNA genes from T2D patients with cardiovascular disease (CVD), long-duration diabetes, and poor glycemic control. METHODS: We performed paired-end RNA sequencing of monocytes from 37 non-diabetes controls and 120 patients with T2D, of whom 86 had either macro or microvascular disease or both. Monocytes were sorted from peripheral blood using flow cytometry; their RNA was purified and sequenced. Alignments and gene counts were obtained with STAR to reference GRCh38 using Gencode (v41) annotations followed by batch correction with CombatSeq. Differential expression analysis was performed with EdgeR and pathway analysis with IPA software focusing on differentially expressed genes (DEGs) with a p-value < 0.05. Additionally, differential co-expression analysis was done with csdR to identify lncRNAs highly associated with diabetes-related expression networks with network centrality scores computed with Igraph and network visualization with Cytoscape. RESULTS: Comparing T2D vs. non-T2D, we found two significantly upregulated lncRNAs (ENSG00000287255, FDR = 0.017 and ENSG00000289424, FDR = 0.048) and one significantly downregulated lncRNA (ENSG00000276603, FDR = 0.017). Pathway analysis on DEGs revealed networks affecting cellular movement, growth, and development. Co-expression analysis revealed ENSG00000225822 (UBXN7-AS1) as the highest-scoring diabetes network-associated lncRNA. Analysis within T2D patients and CVD revealed one lncRNA upregulated in monocytes from patients with microvascular disease without clinically documented macrovascular disease. (ENSG00000261654, FDR = 0.046). Pathway analysis revealed DEGs involved in networks affecting metabolic and cardiovascular pathologies. Co-expression analysis identified lncRNAs strongly associated with diabetes networks, including ENSG0000028654, ENSG00000261326 (LINC01355), ENSG00000260135 (MMP2-AS1), ENSG00000262097, and ENSG00000241560 (ZBTB20-AS1) when we combined the results from all patients with CVD. Similarly, we identified from co-expression analysis of diabetes patients with a duration ≥ 10 years vs. <10 years two lncRNAs: ENSG00000269019 (HOMER3-AS10) and ENSG00000212719 (LINC02693). The comparison of patients with good vs. poor glycemic control also identified two lncRNAs: ENSG00000245164 (LINC00861) and ENSG00000286313. CONCLUSION: We identified dysregulated diabetes-related genes and pathways in monocytes of diabetes patients with cardiovascular complications, including lncRNA genes of unknown function strongly associated with networks of known diabetes genes.

DNA methylation predicts the outcome of COVID-19 patients with acute respiratory distress syndrome.

BACKGROUND: COVID-19 infections could be complicated by acute respiratory distress syndrome (ARDS), increasing mortality risk. We sought to assess the methylome of peripheral blood mononuclear cells in COVID-19 with ARDS. METHODS: We recruited 100 COVID-19 patients with ARDS under mechanical ventilation and 33 non-COVID-19 controls between April and July 2020. COVID-19 patients were followed at four time points for 60 days. DNA methylation and immune cell populations were measured at each time point. A multivariate cox proportional risk regression analysis was conducted to identify predictive signatures according to survival. RESULTS: The comparison of COVID-19 to controls at inclusion revealed the presence of a 14.4% difference in promoter-associated CpGs in genes that control immune-related pathways such as interferon-gamma and interferon-alpha responses. On day 60, 24% of patients died. The inter-comparison of baseline DNA methylation to the last recorded time point in both COVID-19 groups or the intra-comparison between inclusion and the end of follow-up in every group showed that most changes occurred as the disease progressed, mainly in the AIM gene, which is associated with an intensified immune response in those who recovered. The multivariate Cox proportional risk regression analysis showed that higher methylation of the "Apoptotic execution Pathway" genes (ROC1, ZNF789, and H1F0) at inclusion increases mortality risk by over twofold. CONCLUSION: We observed an epigenetic signature of immune-related genes in COVID-19 patients with ARDS. Further, Hypermethylation of the apoptotic execution pathway genes predicts the outcome. TRIAL REGISTRATION: IMRPOVIE study, NCT04473131.

Angiogenic content of microparticles in patients with diabetes and coronary artery disease predicts networks of endothelial dysfunction.

BACKGROUND: Elevated endothelial microparticles (EMPs) levels are surrogate markers of vascular dysfunction. We analyzed EMPs with apoptotic characteristics and assessed the angiogenic contents of microparticles in the blood of patients with type 2 diabetes (T2D) according to the presence of coronary artery disease (CAD). METHODS: A total of 80 participants were recruited and equally classified as (1) healthy without T2D, (2) T2D without cardiovascular complications, (3) T2D and chronic coronary artery disease (CAD), and (4) T2D and acute coronary syndrome (ACS). MPs were isolated from the peripheral circulation, and EMPs were characterized using flow cytometry of CD42 and CD31. CD62E was used to determine EMPs' apoptotic/activation state. MPs content was extracted and profiled using an angiogenesis array. RESULTS: Levels of CD42- CD31 + EMPs were significantly increased in T2D with ACS (257.5 ± 35.58) when compared to healthy subjects (105.7 ± 12.96, p < 0.01). There was no significant difference when comparing T2D with and without chronic CAD. The ratio of CD42-CD62 +/CD42-CD31 + EMPs was reduced in all T2D patients, with further reduction in ACS when compared to chronic CAD, reflecting a release by apoptotic endothelial cells. The angiogenic content of the full population of MPs was analyzed. It revealed a significant differential expression of 5 factors in patients with ACS and diabetes, including TGF-ß1, PD-ECGF, platelet factor 4, serpin E1, and thrombospondin 1. Ingenuity Pathway Analysis revealed that those five differentially expressed molecules, mainly TGF-ß1, inhibit key pathways involved in normal endothelial function. Further comparison of the three diabetes groups to healthy controls and diabetes without cardiovascular disease to diabetes with CAD identified networks that inhibit normal endothelial cell function. Interestingly, DDP-IV was the only differentially expressed protein between chronic CAD and ACS in patients with diabetes. CONCLUSION: Our data showed that the release of apoptosis-induced EMPs is increased in diabetes, irrespective of CAD, ACS patients having the highest levels. The protein contents of MPs interact in networks that indicate vascular dysfunction.

Point of Care Exome Sequencing Reveals Allelic and Phenotypic Heterogeneity Underlying Mendelian disease in Qatar.

The effectiveness of next generation sequencing at solving genetic disease has motivated the rapid adoption of this technology into clinical practice around the world. In this study, we use whole exome sequencing (WES) to assess 48 patients with Mendelian disease from 30 serial families as part of the "Qatar Mendelian Disease pilot program" - a coordinated multi-center effort to build capacity and clinical expertise in genetic medicine in Qatar. By enrolling whole families (parents plus available siblings), we demonstrate significantly improved discriminatory power for candidate variant identification over trios for both de novo and recessive inheritance patterns. For the same index cases, we further demonstrate that even in the absence of families, variant prioritization is improved up to 8-fold when a modest set of population-matched controls is used vs large public databases, stressing the poor representation of Middle Eastern alleles in presently available databases. Our in-house pipeline identified candidate disease variants in 27 of 30 families (90%), 23 of which (85%) harbor novel pathogenic variants in known disease genes, pointing to significant allelic heterogeneity and founder mutations underlying Mendelian disease in the Middle East. For 6 of these families, the clinical presentation was only partially explained by the candidate gene, suggesting phenotypic expansion of known syndromes. Our pilot study demonstrates the utility of WES for Middle Eastern populations, the dramatic improvement in variant prioritization conferred by enrolling population-matched controls and/or enrolling additional unaffected siblings at the point-of-care, and 25 novel disease-causing alleles, relevant to newborn and premarital screening panels in regional populations.

A Novel STK4 Mutation Impairs T Cell Immunity Through Dysregulation of Cytokine-Induced Adhesion and Chemotaxis Genes.

PURPOSE: Human serine/threonine kinase 4 (STK4) deficiency is a rare, autosomal recessive genetic disorder leading to combined immunodeficiency; however, the extent to which immune signaling and host defense are impaired is unclear. We assessed the functional consequences of a novel, homozygous nonsense STK4 mutation (NM_006282.2:c.871C > T, p.Arg291*) identified in a pediatric patient by comparing his innate and adaptive cell-mediated and humoral immune responses with those of three heterozygous relatives and unrelated controls. METHODS: The genetic etiology was verified by whole genome and Sanger sequencing. STK4 gene and protein expression was measured by quantitative RT-PCR and immunoblotting, respectively. Cellular abnormalities were assessed by high-throughput RT-RCR, RNA-Seq, ELISA, and flow cytometry. Antibody responses were assessed by ELISA and phage immunoprecipitation-sequencing. RESULTS: The patient exhibited partial loss of STK4 expression and complete loss of STK4 function combined with recurrent viral and bacterial infections, notably persistent Epstein-Barr virus viremia and pulmonary tuberculosis. Cellular and molecular analyses revealed abnormal fractions of T cell subsets, plasmacytoid dendritic cells, and NK cells. The transcriptional responses of the patient's whole blood and PBMC samples indicated dysregulated interferon signaling, impaired T cell immunity, and increased T cell apoptosis as well as impaired regulation of cytokine-induced adhesion and leukocyte chemotaxis genes. Nonetheless, the patient had detectable vaccine-specific antibodies and IgG responses to various pathogens, consistent with a normal CD19 + B cell fraction, albeit with a distinctive antibody repertoire, largely driven by herpes virus antigens. CONCLUSION: Patients with STK4 deficiency can exhibit broad impairment of immune function extending beyond lymphoid cells.

Association of vitamin D2 and D3 with type 2 diabetes complications.

AIMS: Vitamin D measurement is a composite of vitamin D2 (25(OH)D2) and D3 (25(OH)D3) levels, and its deficiency is associated with the development of type 2 diabetes (T2DM) and diabetic complications; vitamin D deficiency may be treated with vitamin D2 supplements. This study was undertaken to determine if vitamin D2 and D3 levels differed between those with and without T2DM in this Middle Eastern population, and the relationship between diabetic microvascular complications and vitamin D2 and vitamin D3 levels in subjects with T2DM. METHODS: Four hundred ninety-six Qatari subjects, 274 with and 222 without T2DM participated in the study. Plasma levels of total vitamin D2 and D3 were measured by LC-MS/MS analysis. RESULTS: All subjects were taking vitamin D2 and none were taking D3 supplements. Vitamin D2 levels were higher in diabetics, particularly in females, and higher levels were associated with hypertension and dyslipidemia in the diabetic subjects (p < 0.001), but were not related to diabetic retinopathy or nephropathy. Vitamin D3 levels measured in the same subjects were lower in diabetics, particularly in females (p < 0.001), were unrelated to dyslipidemia or hypertension, but were associated with retinopathy (p < 0.014). Neither vitamin D2 nor vitamin D3 were associated with neuropathy. For those subjects with hypertension, dyslipidemia, retinopathy or neuropathy, comparison of highest with lowest tertiles for vitamin D2 and vitamin D3 showed no difference. CONCLUSIONS: In this Qatari cohort, vitamin D2 was associated with hypertension and dyslipidemia, whilst vitamin D3 levels were associated with diabetic retinopathy. Vitamin D2 levels were higher, whilst vitamin D3 were lower in diabetics and females, likely due to ingestion of vitamin D2 supplements.

Indigenous Arabs are descendants of the earliest split from ancient Eurasian populations.

An open question in the history of human migration is the identity of the earliest Eurasian populations that have left contemporary descendants. The Arabian Peninsula was the initial site of the out-of-Africa migrations that occurred between 125,000 and 60,000 yr ago, leading to the hypothesis that the first Eurasian populations were established on the Peninsula and that contemporary indigenous Arabs are direct descendants of these ancient peoples. To assess this hypothesis, we sequenced the entire genomes of 104 unrelated natives of the Arabian Peninsula at high coverage, including 56 of indigenous Arab ancestry. The indigenous Arab genomes defined a cluster distinct from other ancestral groups, and these genomes showed clear hallmarks of an ancient out-of-Africa bottleneck. Similar to other Middle Eastern populations, the indigenous Arabs had higher levels of Neanderthal admixture compared to Africans but had lower levels than Europeans and Asians. These levels of Neanderthal admixture are consistent with an early divergence of Arab ancestors after the out-of-Africa bottleneck but before the major Neanderthal admixture events in Europe and other regions of Eurasia. When compared to worldwide populations sampled in the 1000 Genomes Project, although the indigenous Arabs had a signal of admixture with Europeans, they clustered in a basal, outgroup position to all 1000 Genomes non-Africans when considering pairwise similarity across the entire genome. These results place indigenous Arabs as the most distant relatives of all other contemporary non-Africans and identify these people as direct descendants of the first Eurasian populations established by the out-of-Africa migrations.

Point-of-care whole-exome sequencing of idiopathic male infertility.

PURPOSE: Nonobstructive azoospermia (NOA) affects 1% of the male population; however, despite state-of-the-art clinical assessment, for most patients the cause is unknown. We capitalized on an analysis of multiplex families in the Middle East to identify highly penetrant genetic causes. METHODS: We used whole-exome sequencing (WES) in 8 consanguineous families and combined newly discovered genes with previously reported ones to create a NOA gene panel, which was used to identify additional variants in 75 unrelated idiopathic NOA subjects and 74 fertile controls. RESULTS: In five of eight families, we identified rare deleterious recessive variants in CCDC155, NANOS2, SPO11, TEX14, and WNK3 segregating with disease. These genes, which are novel to human NOA, have remarkable testis-specific expression, and murine functional evidence supports roles for them in spermatogenesis. Among 75 unrelated NOA subjects, we identified 4 (~5.3%) with additional recessive variants in these newly discovered genes and 6 with deleterious variants in previously reported NOA genes, yielding an overall genetic etiology for 13.3% subjects versus 0 fertile controls (p = 0.001). CONCLUSION: NOA affects millions of men, many of whom remain idiopathic despite extensive laboratory evaluation. The genetic etiology for a substantial fraction of these patients (>50% familial and >10% sporadic) may be discovered by WES at the point of care.

Two hits in one: whole genome sequencing unveils LIG4 syndrome and urofacial syndrome in a case report of a child with complex phenotype.

BACKGROUND: Ligase IV syndrome, a hereditary disease associated with compromised DNA damage response mechanisms, and Urofacial syndrome, caused by an impairment of neural cell signaling, are both rare genetic disorders, whose reports in literature are limited. We describe the first case combining both disorders in a specific phenotype. CASE PRESENTATION: We report a case of a 7-year old girl presenting with a complex phenotype characterized by multiple congenital abnormalities and dysmorphic features, microcephaly, short stature, combined immunodeficiency and severe vesicoureteral reflux. Whole Genome Sequencing was performed and a novel ligase IV homozygous missense c.T1312C/p.Y438H mutation was detected, and is believed to be responsible for most of the clinical features of the child, except vesicoureteral reflux which has not been previously described for ligase IV deficiency. However, we observed a second rare damaging (nonsense) homozygous mutation (c.C2125T/p.R709X) in the leucine-rich repeats and immunoglobulin-like domains 2 gene that encodes a protein implicated in neural cell signaling and oncogenesis. Interestingly, this mutation has recently been reported as pathogenic and causing urofacial syndrome, typically displaying vesicoureteral reflux. Thus, this second mutation completes the missing genetic explanation for this intriguing clinical puzzle. We verified that both mutations fit an autosomal recessive inheritance model due to extensive consanguinity. CONCLUSIONS: We successfully identified a novel ligase IV mutation, causing ligase IV syndrome, and an additional rare leucine-rich repeats and immunoglobulin-like domains 2 gene nonsense mutation, in the context of multiple autosomal recessive conditions due to extensive consanguinity. This work demonstrates the utility of Whole Genome Sequencing data in clinical diagnosis in such cases where the combination of multiple rare phenotypes results in very intricate clinical pictures. It also reports a novel causative mutation and a clinical phenotype, which will help in better defining the essential features of both ligase IV and leucine-rich repeats and immunoglobulin-like domains 2 deficiency syndromes.

Copy number variations in the genome of the Qatari population.

BACKGROUND: The populations of the Arabian Peninsula remain the least represented in public genetic databases, both in terms of single nucleotide variants and of larger genomic mutations. We present the first high-resolution copy number variation (CNV) map for a Gulf Arab population, using a hybrid approach that integrates array genotyping intensity data and next-generation sequencing reads to call CNVs in the Qatari population. METHODS: CNVs were detected in 97 unrelated Qatari individuals by running two calling algorithms on each of two primary datasets: high-resolution genotyping (Illumina Omni 2.5M) and high depth whole-genome sequencing (Illumina PE 100bp). The four call-sets were integrated to identify high confidence CNV regions, which were subsequently annotated for putative functional effect and compared to public databases of CNVs in other populations. The availability of genome sequence was leveraged to identify tagging SNPs in high LD with common deletions in this population, enabling their imputation from genotyping experiments in the future. RESULTS: Genotyping intensities and genome sequencing data from 97 Qataris were analyzed with four different algorithms and integrated to discover 16,660 high confidence CNV regions (CNVRs) in the total population, affecting ~28 Mb in the median Qatari genome. Up to 40% of all CNVs affected genes, including novel CNVs affecting Mendelian disease genes, segregating at different frequencies in the 3 major Qatari subpopulations, including those with Bedouin, Persian/South Asian, and African ancestry. Consistent with high consanguinity levels in the Bedouin subpopulation, we found an increased burden for homozygous deletions in this group. In comparison to known CNVs in the comprehensive Database of Genomic Variants, we found that 5% of all CNVRs in Qataris were completely novel, with an enrichment of CNVs affecting several known chromosomal disorder loci and genes known to regulate sugar metabolism and type 2 diabetes in the Qatari cohort. Finally, we leveraged the availability of genome sequence to find suitable tagging SNPs for common deletions in this population. CONCLUSION: We combine four independently generated datasets from 97 individuals to study CNVs for the first time at high-resolution in a Gulf Arab population.

Role of SLMAP genetic variants in susceptibility of diabetes and diabetic retinopathy in Qatari population.

BACKGROUND: Overexpression of SLMAP gene has been associated with diabetes and endothelial dysfunction of macro- and micro-blood vessels. In this study our primary objective is to explore the role of SLMAP gene polymorphisms in the susceptibility of type 2 diabetes (T2DM) with or without diabetic retinopathy (DR) in the Qatari population. METHODS: A total of 342 Qatari subjects (non-diabetic controls and T2DM patients with or without DR) were genotyped for SLMAP gene polymorphisms (rs17058639 C > T; rs1043045 C > T and rs1057719 A > G) using Taqman SNP genotyping assay. RESULTS: SLMAP rs17058639 C > T polymorphism was associated with the presence of DR among Qataris with T2DM. One-way ANOVA and multiple logistic regression analysis showed SLMAP SNP rs17058639 C > T as an independent risk factor for DR development. SLMAP rs17058639 C > T polymorphism also had a predictive role for the severity of DR. Haplotype Crs17058639Trs1043045Ars1057719 was associated with the increased risk for DR among Qataris with T2DM. CONCLUSIONS: The data suggests the potential role of SLMAP SNPs as a risk factor for the susceptibility of DR among T2DM patients in the Qatari population.

Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells.

The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggest that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes; here we investigate the mechanism by which Hsc70 acts on ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αßγ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 µg/ml doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating ENaC biogenesis and ENaC trafficking at the cell surface.

Hsp70 promotes epithelial sodium channel functional expression by increasing its association with coat complex II and its exit from endoplasmic reticulum.

The epithelial sodium channel (ENaC) plays an important role in the homeostasis of blood pressure and of the airway surface liquid, and inappropriate regulation of ENaC results in refractory hypertension (in Liddle syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsp70, is not completely understood. Building on the previous suggestion by our group that Hsp70 promotes ENaC functional and surface expression in Xenopus oocytes, we investigated the mechanism by which Hsp70 acts upon ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αßγ-ENaC and with tetracycline-inducible overexpression of Hsp70, treatment with 1 or 2 µg/ml doxycycline increased total Hsp70 expression ~2-fold and ENaC functional expression ~1.4-fold. This increase in ENaC functional expression corresponded to an increase in ENaC expression at the apical surface of the cells and was not present when an ATPase-deficient Hsp70 was similarly overexpressed. The increase in functional expression was not due to a change in the rate at which ENaC was retrieved from the apical membrane. Instead, Hsp70 overexpression increased the association of ENaC with the Sec24D cargo recognition component of coat complex II, which carries protein cargo from the endoplasmic reticulum to the Golgi. These data support the hypothesis that Hsp70 promotes ENaC biogenesis and trafficking to the apical surface of epithelial cells.

Predicted deleterious variants in the human genome relevant to gene therapy with adeno-associated virus vectors.

Based on the observation that humans have variable responses of gene expression with the same dose of an adeno-associated vector, we hypothesized that there are deleterious variants in genes coding for processes required for adeno-associated virus (AAV)-mediated gene transfer/expression that may hamper or enhance the effectiveness of AAV-mediated gene therapy. To assess this hypothesis, we evaluated 69,442 whole genome sequences from three populations (European, African/African American, and Qatari) for predicted deleterious variants in 62 genes known to play a role in AAV-mediated gene transfer/expression. The analysis identified 5,564 potentially deleterious mutations of which 27 were classified as common based on an allele frequency ≥1% in at least one population studied. Many of these deleterious variants are predicated to prevent while others enhance effective AAV gene transfer/expression, and several are linked to known hereditary disorders. The data support the hypothesis that, like other drugs, human genetic variability contributes to the person-to-person effectiveness of AAV gene therapy and the screening for genetic variability should be considered as part of future clinical trials.

ERp29 regulates DeltaF508 and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) trafficking to the plasma membrane in cystic fibrosis (CF) and non-CF epithelial cells.

Sodium 4-phenylbutyrate (4PBA) improves the intracellular trafficking of ΔF508-CFTR in cystic fibrosis (CF) epithelial cells. The underlying mechanism is uncertain, but 4PBA modulates the expression of some cytosolic molecular chaperones. To identify other 4PBA-regulated proteins that might regulate ΔF508-CFTR trafficking, we performed a differential display RT-PCR screen on IB3-1 CF bronchiolar epithelial cells exposed to 4PBA. One transcript up-regulated by 4PBA encoded ERp29, a luminal resident of the endoplasmic reticulum (ER) thought to be a novel molecular chaperone. We tested the hypothesis that ERp29 is a 4PBA-regulated ER chaperone that influences ΔF508-CFTR trafficking. ERp29 mRNA and protein expression was significantly increased (∼1.5-fold) in 4PBA-treated IB3-1 cells. In Xenopus oocytes, ERp29 overexpression increased the functional expression of both wild-type and ΔF508-CFTR over 3-fold and increased wild-type cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane expression. In CFBE41o- WT-CFTR cells, expression of and short circuit currents mediated by CFTR decreased upon depletion of ERp29 as did maturation of newly synthesized CFTR. In IB3-1 cells, ΔF508-CFTR co-immunoprecipitated with endogenous ERp29, and overexpression of ERp29 led to increased ΔF508-CFTR expression at the plasma membrane. These data suggest that ERp29 is a 4PBA-regulated ER chaperone that regulates WT-CFTR biogenesis and can promote ΔF508-CFTR trafficking in CF epithelial cells.

Novel MYO5B mutation in microvillous inclusion disease of Syrian ancestry.

Microvillus inclusion disease (MVID) is a rare autosomal recessive condition characterized by a lack of microvilli on the surface of enterocytes, resulting in severe, life-threatening diarrhea that could lead to mortality within the first year of life. We identify two unrelated families, each with one child presenting with severe MVID from birth. Using trio whole-exome sequencing, we observed that the two families share a novel nonsense variant (Glu1589*) in the MYO5B gene, a type Vb myosin motor protein in which rare damaging mutations were previously described to cause MVID. This founder mutation was very rare in public databases and is likely specific to patients of Syrian ancestry. We present a detailed account of both patients' clinical histories to fully characterize the effect of this variant and expand the genotype-phenotype databases for MVID patients from the Middle East.

Metabolic and Metabo-Clinical Signatures of Type 2 Diabetes, Obesity, Retinopathy, and Dyslipidemia.

Macro- and microvascular complications of type 2 diabetes (T2D), obesity, and dyslipidemia share common metabolic pathways. In this study, using a total of 1,300 metabolites from 996 Qatari adults (57% with T2D) and 1,159 metabolites from an independent cohort of 2,618 individuals from the Qatar BioBank (11% with T2D), we identified 373 metabolites associated with T2D, obesity, retinopathy, dyslipidemia, and lipoprotein levels, 161 of which were novel. Novel metabolites included phospholipids, sphingolipids, lysolipids, fatty acids, dipeptides, and metabolites of the urea cycle and xanthine, steroid, and glutathione metabolism. The identified metabolites enrich pathways of oxidative stress, lipotoxicity, glucotoxicity, and proteolysis. Second, we identified 15 patterns we defined as "metabo-clinical signatures." These are clusters of patients with T2D who group together based on metabolite levels and reveal the same clustering in two or more clinical variables (obesity, LDL, HDL, triglycerides, and retinopathy). These signatures revealed metabolic pathways associated with different clinical patterns and identified patients with extreme (very high/low) clinical variables associated with extreme metabolite levels in specific pathways. Among our novel findings are the role of N-acetylmethionine in retinopathy in conjunction with dyslipidemia and the possible roles of N-acetylvaline and pyroglutamine in association with high cholesterol levels and kidney function.

Altered Circulating microRNAs in Patients with Diabetic Neuropathy and Corneal Nerve Loss: A Pilot Study.

An alteration in circulating miRNAs may have important diagnostic and therapeutic relevance in diabetic neuropathy. Patients with type 2 diabetes mellitus (T2DM) underwent an assessment of neuropathic symptoms using Douleur Neuropathique 4 (DN4), the vibration perception threshold (VPT) using a Neurothesiometer, sudomotor function using the Sudoscan, corneal nerve morphology using corneal confocal microscopy (CCM) and circulating miRNAs using high-throughput miRNA expression profiling. Patients with T2DM, with (n = 9) and without (n = 7) significant corneal nerve loss were comparable in age, gender, diabetes duration, BMI, HbA1c, eGFR, blood pressure, and lipid profile. The VPT was significantly higher (p < 0.05), and electrochemical skin conductance (p < 0.05), corneal nerve fiber density (p = 0.001), corneal nerve branch density (p = 0.013), and corneal nerve fiber length (p < 0.001) were significantly lower in T2DM patients with corneal nerve loss compared to those without corneal nerve loss. Following a q-PCR-based analysis of total plasma microRNAs, we found that miR-92b-3p (p = 0.008) was significantly downregulated, while miR-22-3p (p = 0.0001) was significantly upregulated in T2DM patients with corneal nerve loss. A network analysis revealed that these miRNAs regulate axonal guidance and neuroinflammation genes. These data support the need for more extensive studies to better understand the role of dysregulated miRNAs' in diabetic neuropathy.

The QChip1 knowledgebase and microarray for precision medicine in Qatar.

Risk genes for Mendelian (single-gene) disorders (SGDs) are consistent across populations, but pathogenic risk variants that cause SGDs are typically population-private. The goal was to develop "QChip1," an inexpensive genotyping microarray to comprehensively screen newborns, couples, and patients for SGD risk variants in Qatar, a small nation on the Arabian Peninsula with a high degree of consanguinity. Over 108 variants in 8445 Qatari were identified for inclusion in a genotyping array containing 165,695 probes for 83,542 known and potentially pathogenic variants in 3438 SGDs. QChip1 had a concordance with whole-genome sequencing of 99.1%. Testing of QChip1 with 2707 Qatari genomes identified 32,674 risk variants, an average of 134 pathogenic alleles per Qatari genome. The most common pathogenic variants were those causing homocystinuria (1.12% risk allele frequency), and Stargardt disease (2.07%). The majority (85%) of Qatari SGD pathogenic variants were not present in Western populations such as European American, South Asian American, and African American in New York City and European and Afro-Caribbean in Puerto Rico; and only 50% were observed in a broad collection of data across the Greater Middle East including Kuwait, Iran, and United Arab Emirates. This study demonstrates the feasibility of developing accurate screening tools to identify SGD risk variants in understudied populations, and the need for ancestry-specific SGD screening tools.

ß1, ß2, and ß3 adrenoceptors and Na+/H+ exchanger regulatory factor 1 expression in human bronchi and their modifications in cystic fibrosis.

To date, three ß-adrenoceptor (ß-AR) subtypes have been identified, but only ß(1)-ARs and ß(2)-ARs have been characterized in human lungs. Moreover, ß(2)-ARs physically interact with the cystic fibrosis transmembrane conductance regulator (CFTR) through the Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) protein. ß(3)-ARs, which stimulate CFTR activity in transfected cells, have not been identified in human lungs. This study aimed (1) to characterize the presence of ß-AR subtypes, especially ß(3)-AR, in human bronchi, and (2) to compare their expression as well as that of NHERF1 in non-cystic fibrosis (CF) versus advanced CF lung samples. In human non-CF bronchi, ß(1)-AR, ß(2)-AR, ß(3)-AR, and NHERF1 transcripts and proteins were expressed mainly in bronchial epithelial cells. Those results were strengthened by the native expression of ß(1)-AR, ß(2)-AR, and ß(3)-AR in a human epithelial cell line, 16HBE14o(-). All ß-AR subtypes stimulated CFTR activity. In CF bronchi, we demonstrated ß(1)-AR and ß(3)-AR overexpression, and NHERF1 and ß(2)-AR underexpression. The origin of this protein remodeling (involving the physical or functional absence of CFTR, infection, inflammation, or high adrenergic tone) deserves further investigation. These results evidence for the first time, to the best of our knowledge, the presence of ß(3)-ARs in human bronchi, and suggest their usefulness as a putative new pharmacologic target in lung diseases where fluid homeostasis is altered. Furthermore, NHERF1 may be a new therapeutic target in patients with CF, to facilitate the trafficking of mutated CFTR to plasma membrane.