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.

Serial Sampling of the Small Airway Epithelium to Identify Persistent Smoking-dysregulated Genes.

Rationale: The small airway epithelium (beyond the sixth generation), the initiation site of smoking-induced airway disorders, is highly sensitive to the stress of smoking. Because of variations over time in smoking habits, the small airway epithelium transcriptome is dynamic, fluctuating not only among smokers but also within each smoker. Objectives: To perform accurate assessment of the smoking-related dysregulation of the human small airway epithelium despite the variation of smoking within the same individual and of the effects of smoking cessation on the dysregulated transcriptome. Methods: We conducted serial sampling of the same smokers and nonsmoker control subjects over time to identify persistent smoking dysregulation of the biology of the small airway epithelium over 1 year. We conducted serial sampling of smokers who quit smoking, before and after smoking cessation, to assess the effect of smoking cessation on the smoking-dysregulated genes. Measurements and Main Results: Repeated measures ANOVA of the small airway epithelium transcriptome sampled four times in the same individuals over 1 year enabled the identification of 475 persistent smoking-dysregulated genes. Most genes were normalized after 12 months of smoking cessation; however, 53 (11%) genes, including CYP1B1, PIR, ME1, and TRIM16, remained persistently abnormally expressed. Dysregulated pathways enriched with the nonreversible genes included xenobiotic metabolism signaling, bupropion degradation, and nicotine degradation. Conclusions: Analysis of repetitive sampling of the same individuals identified persistent smoking-induced dysregulation of the small airway epithelium transcriptome and the effect of smoking cessation. These results help identify targets for the development of therapies that can be applicable to smoking-related airway diseases.

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.

Smoking shifts human small airway epithelium club cells toward a lesser differentiated population.

The club cell, a small airway epithelial (SAE) cell, plays a central role in human lung host defense. We hypothesized that subpopulations of club cells with distinct functions may exist. The SAE of healthy nonsmokers and healthy cigarette smokers were evaluated by single-cell RNA sequencing, and unsupervised clustering revealed subpopulations of SCGCB1A1+KRT5loMUC5AC- club cells. Club cell heterogeneity was supported by evaluations of SAE tissue sections, brushed SAE cells, and in vitro air-liquid interface cultures. Three subpopulations included: (1) progenitor; (2) proliferating; and (3) effector club cells. The progenitor club cell population expressed high levels of mitochondrial, ribosomal proteins, and KRT5 relative to other club cell populations and included a differentiation branch point leading to mucous cell production. The small proliferating population expressed high levels of cyclins and proliferation markers. The effector club cell cluster expressed genes related to host defense, xenobiotic metabolism, and barrier functions associated with club cell function. Comparison of smokers vs. nonsmokers demonstrated that smoking limited the extent of differentiation of all three subclusters and altered SAM pointed domain-containing Ets transcription factor (SPDEF)-regulated transcription in the effector cell population leading to a change in the location of the branch point for mucous cell production, a potential explanation for the concomitant reduction in effector club cells and increase in mucous cells in smokers. These observations provide insights into both the makeup of human SAE club cell subpopulations and the smoking-induced changes in club cell biology.

Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2.

Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.

Dysregulation of club cell biology in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. "Bronchiolization", characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.

Cell-specific expression of lung disease risk-related genes in the human small airway epithelium.

BACKGROUND: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. METHODS: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. RESULTS: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. CONCLUSIONS: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

Expression of the SARS-CoV-2 ACE2 Receptor in the Human Airway Epithelium.

Rationale: Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19), a predominantly respiratory illness. The first step in SARS-CoV-2 infection is binding of the virus to ACE2 (angiotensin-converting enzyme 2) on the airway epithelium.Objectives: The objective was to gain insight into the expression of ACE2 in the human airway epithelium.Methods: Airway epithelia sampled by fiberoptic bronchoscopy of trachea, large airway epithelia (LAE), and small airway epithelia (SAE) of nonsmokers and smokers were analyzed for expression of ACE2 and other coronavirus infection-related genes using microarray, RNA sequencing, and 10x single-cell transcriptome analysis, with associated examination of ACE2-related microRNA.Measurements and Main Results:1) ACE2 is expressed similarly in the trachea and LAE, with lower expression in the SAE; 2) in the SAE, ACE2 is expressed in basal, intermediate, club, mucus, and ciliated cells; 3) ACE2 is upregulated in the SAE by smoking, significantly in men; 4) levels of miR-1246 expression could play a role in ACE2 upregulation in the SAE of smokers; and 5) ACE2 is expressed in airway epithelium differentiated in vitro on air-liquid interface cultures from primary airway basal stem/progenitor cells; this can be replicated using LAE and SAE immortalized basal cell lines derived from healthy nonsmokers.Conclusions:ACE2, the gene encoding the receptor for SARS-CoV-2, is expressed in the human airway epithelium, with variations in expression relevant to the biology of initial steps in SARS-CoV-2 infection.

Identifying novel associations in GWAS by hierarchical Bayesian latent variable detection of differentially misclassified phenotypes.

BACKGROUND: Heterogeneity in the definition and measurement of complex diseases in Genome-Wide Association Studies (GWAS) may lead to misdiagnoses and misclassification errors that can significantly impact discovery of disease loci. While well appreciated, almost all analyses of GWAS data consider reported disease phenotype values as is without accounting for potential misclassification. RESULTS: Here, we introduce Phenotype Latent variable Extraction of disease misdiagnosis (PheLEx), a GWAS analysis framework that learns and corrects misclassified phenotypes using structured genotype associations within a dataset. PheLEx consists of a hierarchical Bayesian latent variable model, where inference of differential misclassification is accomplished using filtered genotypes while implementing a full mixed model to account for population structure and genetic relatedness in study populations. Through simulations, we show that the PheLEx framework dramatically improves recovery of the correct disease state when considering realistic allele effect sizes compared to existing methodologies designed for Bayesian recovery of disease phenotypes. We also demonstrate the potential of PheLEx for extracting new potential loci from existing GWAS data by analyzing bipolar disorder and epilepsy phenotypes available from the UK Biobank. From the PheLEx analysis of these data, we identified new candidate disease loci not previously reported for these datasets that have value for supplemental hypothesis generation. CONCLUSION: PheLEx shows promise in reanalyzing GWAS datasets to provide supplemental candidate loci that are ignored by traditional GWAS analysis methodologies.

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.

Role of KRAS in regulating normal human airway basal cell differentiation.

BACKGROUND: KRAS is a GTPase that activates pathways involved in cell growth, differentiation and survival. In normal cells, KRAS-activity is tightly controlled, but with specific mutations, the KRAS protein is persistently activated, giving cells a growth advantage resulting in cancer. While a great deal of attention has been focused on the role of mutated KRAS as a common driver mutation for lung adenocarcinoma, little is known about the role of KRAS in regulating normal human airway differentiation. METHODS: To assess the role of KRAS signaling in regulating differentiation of the human airway epithelium, primary human airway basal stem/progenitor cells (BC) from nonsmokers were cultured on air-liquid interface (ALI) cultures to mimic the airway epithelium in vitro. Modulation of KRAS signaling was achieved using siRNA-mediated knockdown of KRAS or lentivirus-mediated over-expression of wild-type KRAS or the constitutively active G12 V mutant. The impact on differentiation was quantified using TaqMan quantitative PCR, immunofluorescent and immunohistochemical staining analysis for cell type specific markers. Finally, the impact of cigarette smoke exposure on KRAS and RAS protein family activity in the airway epithelium was assessed in vitro and in vivo. RESULTS: siRNA-mediated knockdown of KRAS decreased differentiation of BC into secretory and ciliated cells with a corresponding shift toward squamous cell differentiation. Conversely, activation of KRAS signaling via lentivirus mediated over-expression of the constitutively active G12 V KRAS mutant had the opposite effect, resulting in increased secretory and ciliated cell differentiation and decreased squamous cell differentiation. Exposure of BC to cigarette smoke extract increased KRAS and RAS protein family activation in vitro. Consistent with these observations, airway epithelium brushed from healthy smokers had elevated RAS activation compared to nonsmokers. CONCLUSIONS: Together, these data suggest that KRAS-dependent signaling plays an important role in regulating the balance of secretory, ciliated and squamous cell differentiation of the human airway epithelium and that cigarette smoking-induced airway epithelial remodeling is mediated in part by abnormal activation of KRAS-dependent signaling mechanisms.

Exaggerated BMP4 signalling alters human airway basal progenitor cell differentiation to cigarette smoking-related phenotypes.

Airway remodelling in chronic obstructive pulmonary disease (COPD) originates, in part, from smoking-induced changes in airway basal stem/progenitor cells (BCs). Based on the knowledge that bone morphogenetic protein 4 (BMP4) influences epithelial progenitor function in the developing and adult mouse lung, we hypothesised that BMP4 signalling may regulate the biology of adult human airway BCs relevant to COPD.BMP4 signalling components in human airway epithelium were analysed at the mRNA and protein levels, and the differentiation of BCs was assessed using the BC expansion and air-liquid interface models in the absence/presence of BMP4, BMP receptor inhibitor and/or small interfering RNAs against BMP receptors and downstream signalling.The data demonstrate that in cigarette smokers, BMP4 is upregulated in ciliated and intermediate undifferentiated cells, and expression of the BMP4 receptor BMPR1A is enriched in BCs. BMP4 induced BCs to acquire a smoking-related abnormal phenotype in vitro mediated by BMPR1A/Smad signalling, characterised by decreased capacity to differentiate into normal mucociliary epithelium, while generating squamous metaplasia.Exaggerated BMP4 signalling promotes cigarette smoking-relevant airway epithelial remodelling by inducing abnormal phenotypes in human airway BCs. Targeting of BMP4 signalling in airway BCs may represent a novel target to prevent/treat COPD-associated airway disease.

Corneal confocal microscopy: Neurologic disease biomarker in Friedreich ataxia.

OBJECTIVE: Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease caused by mutations in the gene encoding for the mitochondrial protein frataxin, is characterized by ataxia and gait instability, immobility, and eventual death. We evaluated corneal confocal microscopy (CCM) quantification of corneal nerve morphology as a novel, noninvasive, in vivo quantitative imaging biomarker for the severity of neurological manifestations in FRDA. METHODS: Corneal nerve fiber density, branch density, and fiber length were quantified in individuals with FRDA (n = 23) and healthy age-matched controls (n = 14). All individuals underwent genetic testing and a detailed neurological assessment with the Scale for the Assessment and Rating of Ataxia (SARA) and Friedreich's Ataxia Rating Scale (FARS). A subset of individuals with FRDA who were ambulatory underwent quantitative gait assessment. RESULTS: CCM demonstrated a significant reduction in nerve fiber density and length in FRDA compared to healthy controls. Importantly, CCM parameters correlated with genotype, SARA and FARS neurological scales, and linear regression modeling of CCM nerve parameter-generated equations that predict the neurologic severity of FRDA. INTERPRETATION: Together, the data suggest that CCM quantification of corneal nerve morphology is a rapid, sensitive imaging biomarker for quantifying the severity of neurologic disease in individuals with FRDA. Ann Neurol 2018;84:893-904.

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.

Ambient Pollution-related Reprogramming of the Human Small Airway Epithelial Transcriptome.

RATIONALE: Epidemiologic studies have demonstrated that exposure to particulate matter ambient pollution has adverse effects on lung health, exacerbated by cigarette smoking. Particulate matter less than or equal to 2.5 µm in aerodynamic diameter (PM2.5) is among the most harmful urban pollutants and is closely linked to respiratory disease. OBJECTIVES: Based on the knowledge that the small airway epithelium (SAE) plays a central role in the pathogenesis of smoking-related lung disease, we hypothesized that elevated PM2.5 levels are associated with dysregulation of SAE gene expression, which may contribute to the development of respiratory disease. METHODS: From 2009 to 2012, healthy nonsmoker (n = 29) and smoker (n = 129) residents of New York City underwent bronchoscopy with SAE brushing (2.6 ± 1.3 samples/subject; total of 405 samples). SAE gene expression was assessed by Affymetrix HG-U133 Plus 2.0 microarray. New York City PM2.5 levels (Environmental Protection Agency data) were averaged for the 30 days before bronchoscopy. A linear mixed model was used to assess PM2.5-related gene dysregulation accounting for multiple clinical and methodologic variables. MEASUREMENTS AND MAIN RESULTS: Thirty-day mean PM2.5 levels varied from 6.2 to 18 µg/m3. In nonsmokers, there was no dysregulation of SAE gene expression associated with ambient PM2.5 levels. In marked contrast, n = 219 genes were significantly dysregulated in association with PM2.5 levels in the SAE of smokers. Many of these genes relate to cell growth and transcription regulation. Interestingly, 11% of genes were mitochondria associated. CONCLUSIONS: PM2.5 exposure contributes to significant dysregulation of the SAE transcriptome of smokers, linking pollution and airway epithelial biology in the risk of development of respiratory disease in susceptible individuals.

Inferring Identical-by-Descent Sharing of Sample Ancestors Promotes High-Resolution Relative Detection.

As genetic datasets increase in size, the fraction of samples with one or more close relatives grows rapidly, resulting in sets of mutually related individuals. We present DRUID-deep relatedness utilizing identity by descent-a method that works by inferring the identical-by-descent (IBD) sharing profile of an ungenotyped ancestor of a set of close relatives. Using this IBD profile, DRUID infers relatedness between unobserved ancestors and more distant relatives, thereby combining information from multiple samples to remove one or more generations between the deep relationships to be identified. DRUID constructs sets of close relatives by detecting full siblings and also uses an approach to identify the aunts/uncles of two or more siblings, recovering 92.2% of real aunts/uncles with zero false positives. In real and simulated data, DRUID correctly infers up to 10.5% more relatives than PADRE when using data from two sets of distantly related siblings, and 10.7%-31.3% more relatives given two sets of siblings and their aunts/uncles. DRUID frequently infers relationships either correctly or within one degree of the truth, with PADRE classifying 43.3%-58.3% of tenth degree relatives in this way compared to 79.6%-96.7% using DRUID.

Ontogeny and Biology of Human Small Airway Epithelial Club Cells.

RATIONALE: Little is known about human club cells, dome-shaped cells with dense cytoplasmic granules and microvilli that represent the major secretory cells of the human small airways (at least sixth-generation bronchi). OBJECTIVES: To define the ontogeny and biology of the human small airway epithelium club cell. METHODS: The small airway epithelium was sampled from the normal human lung by bronchoscopy and brushing. Single-cell transcriptome analysis and air-liquid interface culture were used to assess club cell ontogeny and biology. MEASUREMENTS AND MAIN RESULTS: We identified the club cell population by unbiased clustering using single-cell transcriptome sequencing. Principal component gradient analysis uncovered an ontologic link between KRT5 (keratin 5)+ basal cells and SCGB1A1 (secretoglobin family 1A member 1)+ club cells, a hypothesis verified by demonstrating in vitro that a pure population of human KRT5+ SCGB1A1- small airway epithelial basal cells differentiate into SCGB1A1+KRT5- club cells on air-liquid interface culture. Using SCGB1A1 as the marker of club cells, the single-cell analysis identified novel roles for these cells in host defense, xenobiotic metabolism, antiprotease, physical barrier function, monogenic lung disorders, and receptors for human viruses. CONCLUSIONS: These observations provide novel insights into the molecular phenotype and biologic functions of the human club cell population and identify basal cells as the human progenitor cells for club cells.

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.

Mandatory role of HMGA1 in human airway epithelial normal differentiation and post-injury regeneration.

Due to high levels of expression in aggressive tumors, high mobility group AT-hook 1 (HMGA1) has recently attracted attention as a potential anti-tumor target. However, HMGA1 is also expressed in normal somatic progenitor cells, raising the question: how might systemic anti-HMGA1 therapies affect the structure and function of normal tissue differentiation? In the present study, RNA sequencing data demonstrated HMGA1 is highly expressed in human airway basal stem/progenitor cells (BC), but decreases with BC differentiation in air-liquid interface cultures (ALI). BC collected from nonsmokers, healthy smokers, and smokers with chronic obstructive pulmonary disease (COPD) displayed a range of HMGA1 expression levels. Low initial expression levels of HMGA1 in BC were associated with decreased ability to maintain a differentiated ALI epithelium. HMGA1 down-regulation in BC diminished BC proliferation, suppressed gene expression related to normal proliferation and differentiation, decreased airway epithelial resistance, suppressed junctional and cell polarity gene expression, and delayed wound closure of airway epithelium following injury. Furthermore, silencing of HMGA1 in airway BC in ALI increased the expression of genes associated with airway remodeling in COPD including squamous, epithelial-mesenchymal transition (EMT), and inflammatory genes. Together, the data suggests HMGA1 plays a central role in normal airway differentiation, and thus caution should be used to monitor airway epithelial structure and function in the context of systemic HMGA1-targeted therapies.