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.

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.

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.

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.

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.

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.

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.

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 D3 and its metabolites in patients with and without type 2 diabetes and their relationship to diabetes complications.

BACKGROUND: Epidemiological studies have suggested that vitamin D deficiency is associated with the development of type 2 diabetes (T2DM) and is related to diabetes complications. This study was undertaken to determine the relationship between diabetes complications and cardiovascular risk factors with vitamin D3 and its metabolites: 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 25-hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3); and 25-hydroxy-3epi-vitamin D3 (3epi25(OH)D3). METHODS: 750 Qatari subjects, 460 (61.3%) with and 290 (38.7%) without T2DM, who were not taking vitamin D3 supplements, participated in this cross-sectional, observational study. Plasma concentrations of vitamin D3 and its metabolites were measured by liquid chromatography tandem mass spectrometry analysis. RESULTS: T2DM subjects had lower concentrations of all vitamin D3 metabolites (p < 0.001) except 3epi25(OH)D3 (p < 0.071). Males had higher concentrations of all vitamin D3 metabolites (p < 0.001). In the T2DM subjects, lower 25(OH)D3 was associated with retinopathy (p < 0.03) and dyslipidemia (p < 0.04), but not neuropathy or vascular complications; lower 1,25(OH)2D3 was associated with hypertension (p < 0.009), dyslipidemia (p < 0.003) and retinopathy (p < 0.006), and coronary artery disease (p < 0.012), but not neuropathy; lower 24,25(OH)2D3 concentrations were associated with dyslipidemia alone (p < 0.019); 3epi25(OH)D3 associated with diabetic neuropathy alone (p < 0.029). In nondiabetics, 25(OH)D3, 1,25(OH)2D3 and 24,25(OH)2D3 were associated with dyslipidemia (p < 0.001, p < 0.001, p < 0.015, respectively) and lower 1,25(OH)2D3 was associated with hypertension (p < 0.001). Spearman's correlation showed 1,25(OH)2D3 to be negatively correlated to age and diabetes duration. CONCLUSIONS: Different diabetes complications were associated with differing vitamin D parameters, with diabetic retinopathy related to lower 25(OH)D3 and 1,25(OH)2D3 levels, hypertension significantly associated with lower 1,25(OH)2D3, while dyslipidemia was associated with lower 25(OH)D3, 1,25(OH)2D3 and 24,25(OH)2D3. While 25(OH)D metabolites were lower in females, there was not an exaggeration in complications.

Qatari Genotype May Contribute to Complications in Type 2 Diabetes.

OBJECTIVE: There is increasing evidence of a strong genetic component in type 2 diabetes (T2DM) that may contribute to diabetes complications. Given the high prevalence of diabetes with its associated complications in the Middle East, we sought to determine if the genotype within a Middle East population may be contributory. Therefore, three genotype-based Qatari ancestral groups, Q1 Arab Bedouin, Q2 Asian/Persian, and Q3 sub-Saharan African, with a fourth admixed group were correlated with T2DM prevalence and its complications to determine if they differed between the 4 Qatari ancestries, particularly for the SLMAP allele-associated diabetic retinopathy. METHODS: In this cross-sectional study, 398 Qatari subjects, 220 with and 178 without T2DM, were genotyped by Affymetrix 500k SNP arrays. Ancestry was correlated with diabetes complications. RESULTS: 398 subjects were included, the mean age was 49.8 years, and 56.8% were male. The genotype-based ancestry and T2DM prevalence were as follows: 164 (41.2%) with ancestry Q1, 60.4% with T2DM; 149 (37.4%) with ancestry Q2, 49.7% with T2DM; 31 (7.8%) with ancestry Q3, 61.3% with T2DM; and 54 (13.6%) with "admixed" ancestry, 51.9% with T2DM. For patients with diabetes, hypertension (p < 0.035) and retinopathy (p < 0.016) were greater in the Q3 ancestry. CONCLUSION: These data suggest that the genotype may contribute to complication risk, as exemplified by the increase in hypertension and retinopathy in the Q3 ancestry, though the SLMAP allele was not implicated; however, diabetes prevalence did not differ between the four Qatari ancestries.

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.

Association of Differing Qatari Genotypes with Vitamin D Metabolites.

OBJECTIVE: Genetic studies have identified four Qatari genotypes: Q1 Arab, Bedouin; Q2 Asian/Persian; Q3 African; and a fourth admixed group not fitting into the previous 3 groups. This study was undertaken to determine if there was an increased risk of deficiency of vitamin D and its metabolites associated with differing genotypes, perhaps due to genetic differences in skin pigmentation. METHODS: 398 Qatari subjects (220 type 2 diabetes and 178 controls) had their genotype determined by Affymetrix 500 k SNP arrays. Total values of 1,25-dihydroxyvitamin D (1,25(OH)2D), 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D), and 25-hydroxy-3epi-vitamin D (3epi-25(OH)D) concentrations were measured by the LC-MS/MS analysis. RESULTS: The distribution was as follows: 164 (41.2%) genotyped Q1, 149 (37.4%) genotyped Q2, 31 (7.8%) genotyped Q3, and 54 (13.6%) genotyped "admixed." Median levels of 25(OH)D and 3epi-25(OH)D did not differ across Q1, Q2, Q3, and "admixed" genotypes, respectively. 1,25(OH)2D levels were lower (p < 0.04) between Q2 and the admixed groups, and 24,25(OH)2D levels were lower (p < 0.05) between Q1 and the admixed groups. Vitamin D metabolite levels were lower in females for 25(OH)D, 1,25(OH)2D (p < 0.001), and 24,25(OH)2D (p < 0.006), but 3epi-25(OH)D did not differ (p < 0.26). Diabetes prevalence was not different between genotypes. Total 1,25(OH)2D (p < 0.001), total 24,25(OH)2D (p < 0.001), and total 3epi-25(OH)D (p < 0.005) were all significantly lower in diabetes patients compared to controls whilst the total 25(OH)D was higher in diabetes than controls (p < 0.001). CONCLUSION: Whilst 25(OH)D levels did not differ between genotype groups, 1,25(OH)2D and 24,25(OH)2D were lower in the admixed group, suggesting that there are genetic differences in vitamin D metabolism that may be of importance in a population that may allow a more targeted approach to vitamin D replacement. This may be of specific importance in vitamin D replacement strategies with the Q2 genotype requiring less, and the other genotypes requiring more to increase 1,25(OH)2D. Whilst overall the group was vitamin D deficient, total 25(OH)D was higher in diabetes, but 1,25(OH)2D, 24,25(OH)2D, and 3epi-25(OH)D were lower in diabetes that did not affect the relationship to genotype.

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.

Whole-methylome analysis of circulating monocytes in acute diabetic Charcot foot reveals differentially methylated genes involved in the formation of osteoclasts.

AIM: To assess whether DNA methylation of monocytes play a role in the development of acute diabetic Charcot foot (CF). PATIENTS & METHODS: We studied the whole methylome (WM) of circulating monocytes in 18 patients with Type 2 diabetes (T2D) and acute CF, 18 T2D patients with equivalent neuropathy and 18 T2D patients without neuropathy, using the enhanced reduced representation bisulfite sequencing technique. RESULTS & CONCLUSION: WM analysis demonstrated that CF monocytes are differentially methylated compared with non-CF monocytes, in both CpG-site and gene-mapped analysis approaches. Among the methylated genes, several are involved in the migration process during monocyte differentiation into osteoclasts or are indirectly involved through the regulation of inflammatory pathways. Finally, we demonstrated an association between methylation and gene expression in cis- and trans-association.

Exome sequencing-based identification of novel type 2 diabetes risk allele loci in the Qatari population.

BACKGROUND: Type 2 diabetes (T2D) susceptibility is influenced by genetic and lifestyle factors. To date, the majority of genetic studies of T2D have been in populations of European and Asian descent. The focus of this study is on genetic variations underlying T2D in Qataris, a population with one of the highest incidences of T2D worldwide. RESULTS: Illumina HiSeq exome sequencing was performed on 864 Qatari subjects (574 T2D cases, 290 controls). Sequence kernel association test (SKAT) gene-based analysis identified an association for low frequency potentially deleterious variants in 6 genes. However, these findings were not replicated by SKAT analysis in an independent cohort of 12,699 exomes, primarly due to the absence of low frequency potentially deleterious variants in 5 of the 6 genes. Interestingly one of the genes identified, catenin beta 1 (CTNNB1, ß-catenin), is the key effector of the Wnt pathway and interacts with the nuclear receptor transcription factor 7-like 2 (TCF7L2), variants which are the most strongly associated with risk of developing T2D worldwide. Single variant analysis did not identify any associated variants, suggesting the SKAT association signal was not driven by individual variants. None of the 6 associated genes were among 634 previously described T2D genes. CONCLUSIONS: The observation that genes not previously linked to T2D in prior studies of European and Asian populations are associated with T2D in Qatar provides new insights into the complexity of T2D pathogenesis and emphasizes the importance of understudied populations when assessing genetic variation in the pathogenesis of common disorders.

Differentially expressed circulating microRNAs in the development of acute diabetic Charcot foot.

AIM: Charcot foot (CF) is a rare complication of Type 2 diabetes (T2D). MATERIALS & METHODS: We assessed circulating miRNAs in 17 patients with T2D and acute CF (G1), 17 patients with T2D (G2) and equivalent neuropathy and 17 patients with T2D without neuropathy (G3) using the high-throughput miRNA expression profiling. RESULTS: 51 significantly deregulated miRNAs were identified in G1 versus G2, 37 in G1 versus G3 and 64 in G2 versus G3. Furthermore, we demonstrated that 16 miRNAs differentially expressed between G1 versus G2 could be involved in osteoclastic differentiation. Among them, eight are key factors involved in CF pathophysiology. CONCLUSION: Our data reveal that CF patients exhibit an altered expression profile of circulating miRNAs.

A systematic review on the genetics of male infertility in the era of next-generation sequencing.

OBJECTIVES: To identify the role of next-generation sequencing (NGS) in male infertility, as advances in NGS technologies have contributed to the identification of novel genes responsible for a wide variety of human conditions and recently has been applied to male infertility, allowing new genetic factors to be discovered. MATERIALS AND METHODS: PubMed was searched for combinations of the following terms: 'exome', 'genome', 'panel', 'sequencing', 'whole-exome sequencing', 'whole-genome sequencing', 'next-generation sequencing', 'azoospermia', 'oligospermia', 'asthenospermia', 'teratospermia', 'spermatogenesis', and 'male infertility', to identify studies in which NGS technologies were used to discover variants causing male infertility. RESULTS: Altogether, 23 studies were found in which the primary mode of variant discovery was an NGS-based technology. These studies were mostly focused on patients with quantitative sperm abnormalities (non-obstructive azoospermia and oligospermia), followed by morphological and motility defects. Combined, these studies uncover variants in 28 genes causing male infertility discovered by NGS methods. CONCLUSIONS: Male infertility is a condition that is genetically heterogeneous, and therefore remarkably amenable to study by NGS. Although some headway has been made, given the high incidence of this condition despite its detrimental effect on reproductive fitness, there is significant potential for further discoveries.

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.

Whole-exome sequencing identifies common and rare variant metabolic QTLs in a Middle Eastern population.

Metabolomics-genome-wide association studies (mGWAS) have uncovered many metabolic quantitative trait loci (mQTLs) influencing human metabolic individuality, though predominantly in European cohorts. By combining whole-exome sequencing with a high-resolution metabolomics profiling for a highly consanguineous Middle Eastern population, we discover 21 common variant and 12 functional rare variant mQTLs, of which 45% are novel altogether. We fine-map 10 common variant mQTLs to new metabolite ratio associations, and 11 common variant mQTLs to putative protein-altering variants. This is the first work to report common and rare variant mQTLs linked to diseases and/or pharmacological targets in a consanguineous Arab cohort, with wide implications for precision medicine in the Middle East.

Circulating microparticles in acute diabetic Charcot foot exhibit a high content of inflammatory cytokines, and support monocyte-to-osteoclast cell induction.

Circulating microparticles (MPs) are major mediators in cardiovascular complications of type 2 diabetes (T2D); however, their contribution to Charcot foot (CF) disease is not known. Here, we purified and assessed the origin, concentration and content of circulating MPs from 33 individuals: 11 with T2D and acute CF, 11 T2D patients with equivalent neuropathy and 11 non-diabetic controls. First, we demonstrated that there were no differences in the distribution of MPs of endothelial, platelet origin among the 3 groups. However, MPs from leukocytes and monocytes origin were increased in CF patients. Moreover, we demonstrated that monocytes-derived MPs originated more frequently from intermediate and non-classical monocytes in CF patients. Five cytokines (G-CSF, GM-CSF, IL-1-ra, IL-2 and IL-16) were significantly increased in MPs from acute CF patients. Applying ingenuity pathways analysis, we found that those cytokines interacted well and induced the activation of pathways that are involved in osteoclast formation. Further, we treated THP-1 monocytes and monocytes sorted from healthy patients with CF-derived MPs during their differentiation into osteoclasts, which increased their differentiation into multinucleated osteoclast-like cells. Altogether, our study suggests that circulating MPs in CF disease have a high content of inflammatory cytokines and could increase osteoclast differentiation in vitro.