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Study Question: Does RXFP2 disruption impair male fertility?, Summary Answer: We identified biallelic variants in RXFP2 in patients with male infertility due to spermatogenic arrest at the spermatid stage, supporting a role of RXFP2 in human spermatogenesis, specifically in germ cell maturation., What Is Known Already: Since RXFP2, the receptor for INSL3, plays a crucial role in testicular descent during prenatal development, biallelic variants lead to bilateral cryptorchidism, as described in four families to date. While animal models have also suggested a function in spermatogenesis, the postnatal functions of RXFP2 and its ligand INSL3, produced in large amounts by the testes from puberty throughout adulthood, are largely unknown., Study Design, Size, Duration: A family with two male members affected by impaired fertility due to spermatogenic maturation arrest and a history of bilateral cryptorchidism underwent clinical, endocrinological, histological, genomic, in vitro cellular, and in silico investigations., Participants/materials, Setting, Methods: The endocrinological and histological findings were correlated with publicly available single-cell RNA sequencing (scRNA-seq) data. The genomic defects have been characterized using long-read sequencing and validated with in silico modeling and an in vitro cyclic AMP reporter gene assay., Main Results and the Role of Chance: An intragenic deletion of exon 1-5 of RXFP2 (NM_130806.5) was detected in trans with a hemizygous missense variant c.229G>A, p.(Glu77Lys). The p.(Glu77Lys) variant caused no clear change in cell surface expression or ability to bind INSL3, but displayed absence of a cAMP signal in response to INSL3, indicating a loss-of-function. Testicular biopsy in the proband showed a maturation arrest at the spermatid stage, corresponding to the highest level of RXFP2 expression in scRNA-seq data, thereby providing a potential explanation for the impaired fertility., Limitations, Reasons for Caution: Although this is so far the only study of human cases that supports the role of RXFP2 in spermatogenic maturation, this is corroborated by several animal studies that have already demonstrated a postnatal function of INSL3 and RXFP2 in spermatogenesis., Wider Implications of the Findings: This study corroborates RXFP2 as gene implicated in autosomal recessive congenital bilateral cryptorchidism due to biallelic variants, rather than autosomal-dominant cryptorchidism due to monoallelic RXFP2 variants. Our findings also support that RXFP2 is essential in human spermatogenesis, specifically in germ cell maturation, and that biallelic disruption can cause male infertility through spermatogenic arrest at the spermatid stage., Study Funding/competing Interest(s): Funding was provided by the Bellux Society for Pediatric Endocrinology and Diabetology (BELSPEED) and supported by a Research Foundation Flanders (FWO) senior clinical investigator grant (E.D.B., 1802220N) and a Ghent University Hospital Special Research Fund grant (M.C., FIKO-IV institutional fund). The authors declare no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of WT1 in critical developmental processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Premature ovarian insufficiency is a common form of female infertility affecting up to 4% of women and characterised by amenorrhea with elevated gonadotropin before the age of 40. Oocytes require controlled DNA breakage and repair for homologous recombination and the maintenance of oocyte integrity. Biallelic disruption of the DNA damage repair gene, Fanconi anemia complementation group A (FANCA), is a common cause of Fanconi anaemia, a syndrome characterised by bone marrow failure, cancer predisposition, physical anomalies and POI. There is ongoing dispute about the role of heterozygous FANCA variants in POI pathogenesis, with insufficient evidence supporting causation. Here, we have identified biallelic FANCA variants in French sisters presenting with POI, including a novel missense variant of uncertain significance and a likely pathogenic deletion that initially evaded detection. Functional studies indicated no discernible effect on DNA damage sensitivity in patient lymphoblasts. These novel FANCA variants add evidence that heterozygous loss of one allele is insufficient to cause DNA damage sensitivity and POI. We propose that intragenic deletions, that are relatively common in FANCA, may be missed without careful analysis, and could explain the presumed causation of heterozygous variants. Accurate variant curation is critical to optimise patient care and outcomes., (© 2024 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We generated heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of SOX9 in critical developmental processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The Cut Homeobox 1 (CUX1) gene has been implicated in a number of developmental processes and has recently emerged as an important cause of developmental delay and impaired intellectual development. Individuals with variants in CUX1 have been described with a variety of co-morbidities including variations in sex development (VSD) although these features have not been closely documented., Case Presentation: The proband is a 14-year-old male who presented with congenital complex hypospadias, neurodevelopmental differences, and subtle dysmorphism. A family history of neurodevelopmental differences and VSD was noted. Microarray testing and whole exome sequencing found the 46,XY proband had a large heterozygous in-frame deletion of exons 4-10 of the CUX1 gene., Conclusions: Our review of the literature has revealed that variants in CUX1 are associated with a range of VSD and suggest this gene should be considered in cases where a VSD is noted at birth, especially if there is a familial history of VSD and/or neurodevelopmental differences. Further work is required to fully investigate the role and regulation of CUX1 in sex development., (© 2024. The Author(s).)

: Differences/disorders of sex development can be caused by disruptions to the molecular and cellular mechanisms that control development and sex determination of the reproductive organs with 1:100 live births affected. Multiple genes are associated with 46, XY differences/disorders of sex development that can cause varying clinical phenotypes. An accurate genetic diagnosis is essential to guide clinical care for individuals with 46, XY differences/disorders of sex development and can contribute to family planning. The use of genomics in differences/disorders of sex development has grown, with several advances employed in genetic diagnosis; however, diagnostic rates have stagnated at less than 50% for these conditions. This review will discuss 46, XY differences/disorders of sex development, its molecular causes, and the genomic technologies currently utilized for diagnosis with focus on reports from the last 5 years. We also touch on the challenges in diagnosing 46, XY differences/disorders of sex development and discuss new and future technologies that promise to improved diagnostic rates for these difficult conditions., (© 2024 The Author(s). Andrology published by John Wiley & Sons Ltd on behalf of American Society of Andrology and European Academy of Andrology.)

Research Question: Premature ovarian insufficiency (POI) is characterised by amenorrhea associated with elevated follicle stimulating hormone (FSH) under the age of 40 years and affects 1-3.7% women. Genetic factors explain 20-30% of POI cases, but most causes remain unknown despite genomic advancements., Design: We used whole exome sequencing (WES) in four Iranian families, validated variants via Sanger sequencing, and conducted the Acyl-cLIP assay to measure HHAT enzyme activity., Results: Despite ethnic homogeneity, WES revealed diverse genetic causes, including a novel homozygous nonsense variant in SYCP2L, impacting synaptonemal complex (SC) assembly, in the first family. Interestingly, the second family had two independent causes for amenorrhea - the mother had POI due to a novel homozygous loss-of-function variant in FANCM (required for chromosomal stability) and her daughter had primary amenorrhea due to a novel homozygous GNRHR (required for gonadotropic signalling) frameshift variant. WES analysis also provided cytogenetic insights. WES revealed one individual was in fact 46, XY and had a novel homozygous missense variant of uncertain significance in HHAT, potentially responsible for complete sex reversal although functional assays did not support impaired HHAT activity. In the remaining individual, WES indicated likely mosaic Turners with the majority of X chromosome variants having an allelic balance of ∼85% or ∼15%. Microarray validated the individual had 90% 45,XO., Conclusions: This study demonstrates the diverse causes of amenorrhea in a small, isolated ethnic cohort highlighting how a genetic cause in one individual may not clarify familial cases. We propose that, in time, genomic sequencing may become a single universal test required for the diagnosis of infertility conditions such as POI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Disruption of meiosis and DNA repair genes is associated with female fertility disorders like premature ovarian insufficiency (POI). In this study, we identified a homozygous missense variant in the HELQ gene (c.596 A>C; p.Gln199Pro) through whole exome sequencing in a POI patient, a condition associated with disrupted ovarian function and female infertility. HELQ, an enzyme involved in DNA repair, plays a crucial role in repairing DNA cross-links and has been linked to germ cell maintenance, fertility, and tumour suppression in mice. To explore the potential association of the HELQ variant with POI, we used CRISPR/Cas9 to create a knock-in mouse model harbouring the equivalent of the human HELQ variant identified in the POI patient. Surprisingly, Helq knock-in mice showed no discernible phenotype, with fertility levels, histological features, and follicle development similar to wild-type mice. Despite the lack of observable effects in mice, the potential role of HELQ in human fertility, especially in the context of POI, should not be dismissed. Larger studies encompassing diverse ethnic populations and alternative functional approaches will be necessary to further examine the role of HELQ in POI. Our results underscore the potential uncertainties associated with genomic variants and the limitations of in vivo animal modelling.

Background: The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored., Results: By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells., Conclusions: Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency., (© 2023. The Author(s).)

Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function., (© 2023. The Author(s).)

Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition., (© 2023. The Author(s).)

Context: Premature ovarian insufficiency (POI) is a common form of female infertility that usually presents as an isolated condition but can be part of various genetic syndromes. Early diagnosis and treatment of POI can minimize comorbidity and improve health outcomes., Objective: We aimed to determine the genetic cause of syndromic POI, intellectual disability, neutropenia, and cataracts., Methods: We performed whole-exome sequencing (WES) followed by functional validation via RT-PCR, RNAseq, and quantitative proteomics, as well as clinical update of previously reported patients with variants in the caseinolytic peptidase B (CLPB) gene., Results: We identified causative variants in CLPB, encoding a mitochondrial disaggregase. Variants in this gene are known to cause an autosomal recessive syndrome involving 3-methylglutaconic aciduria, neurological dysfunction, cataracts, and neutropenia that is often fatal in childhood; however, there is likely a reporting bias toward severe cases. Using RNAseq and quantitative proteomics we validated causation and gained insight into genotype:phenotype correlation. Clinical follow-up of patients with CLPB deficiency who survived to adulthood identified POI and infertility as a common postpubertal ailment., Conclusion: A novel splicing variant is associated with CLPB deficiency in an individual who survived to adulthood. POI is a common feature of postpubertal female individuals with CLPB deficiency. Patients with CLPB deficiency should be referred to pediatric gynecologists/endocrinologists for prompt POI diagnosis and hormone replacement therapy to minimize associated comorbidities., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Background: Military service puts service members at risk for moral injury . Moral injury is an array of symptoms (e.g., guilt, shame, anger) that develop from events that violate or transgress one's moral code., Objective: We describe adaption of in-person mindfulness training program, Mindfulness to Manage Chronic Pain (MMCP), to address symptoms of moral injury to be delivered live via the web. We discuss how we will assess benchmarks (i.e., recruitment, credibility and acceptability, completion rates, and adherence) of the Mindfulness to Manage Moral Injury (MMMI) program., Methods: Aim 1: To develop and then adapt the MMCP program based on feedback from experts and veterans who took part in Study 1. Aim 2: To develop an equally intensive facilitator-led online Educational Support (ES) program to serve as a comparison intervention and conduct a run-through of each program with 20 veterans (10 MMMI; 10 ES). Aim 3: To conduct a small-scale randomized controlled trial (N = 42 veterans; 21 MMMI; 21 ES) in which we will collect pre-post-test and weekly benchmark data for both refined intervention arms., Results: Study 1 and 2 are completed. Data collection for Study 3 will be completed in 2022., Conclusion: MMMI is designed to provide a live facilitated mindfulness program to address symptoms of moral injury. If Study 3 demonstrates good benchmarks, with additional large-scale testing, MMMI may be a promising treatment that can reach veterans who may not seek traditional VAMC care and/or who prefer a web-based program., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Premature ovarian insufficiency (POI) is a leading form of female infertility, characterised by menstrual disturbance and elevated follicle-stimulating hormone before age 40. It is highly heterogeneous with variants in over 80 genes potentially causative, but the majority of cases having no known cause. One gene implicated in POI pathology is TP63. TP63 encodes multiple p63 isoforms, one of which has been shown to have a role in the surveillance of genetic quality in oocytes. TP63 C-terminal truncation variants and N-terminal duplication have been described in association with POI, however, functional validation has been lacking. Here we identify three novel TP63 missense variants in women with nonsyndromic POI, including one in the N-terminal activation domain, one in the C-terminal inhibition domain, and one affecting a unique and poorly understood p63 isoform, TA*p63. Via blue-native page and luciferase reporter assays we demonstrate that two of these variants disrupt p63 dimerization, leading to constitutively active p63 tetramer that significantly increases the transcription of downstream targets. This is the first evidence that TP63 missense variants can cause isolated POI and provides mechanistic insight that TP63 variants cause POI due to constitutive p63 activation and accelerated oocyte loss in the absence of DNA damage., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Introduction: Historically, high rates of actionable driver mutations have been reported in never-smokers with lung adenocarcinoma (ADC). In the era of modern, comprehensive cancer mutation sequencing, this relationship necessitates a more detailed analysis., Methods: All Mount Sinai patients between January 1, 2015, and June 1, 2020, with a diagnosis of ADC of any stage with known smoking status who received genomic testing were included. Most patients were analyzed using the Sema4 hotspot panel or the Oncomine Comprehensive Assay version 3 next-generation sequencing (NGS) panel conducted at Sema4. Patients were considered fully genotyped if they were comprehensively analyzed for alterations in EGFR, KRAS, MET, ALK, RET, ROS1, BRAF, NTRK1-3, and ERBB2, otherwise they were considered partially genotyped., Results: Two hundred and thirty-six never-smokers and 671 smokers met the above criteria. Of the never-smokers, 201 (85%) had a driver mutation with 167 (71%) considered actionable (ie, those with US Food and Drug Administration-approved agents). Among smokers, 439 (65%) had an identified driver mutation with 258 (38%) actionable (P < .0001). When comprehensively sequenced, 95% (70/74) of never-smokers had a driver mutation with 78% (58/74) actionable; whereas, for smokers, 75% (135/180) had a driver with only 47% (74/180) actionable (P < .0001). Within mutations groups, EGFR G719X and KRAS G12Cs were more common to smokers. For stage IV patients harboring EGFR-mutant tumors treated with EGFR-directed therapies, never-smokers had significantly improved OS compared to smokers (hazard ratio = 2.71; P = .025). In multivariable analysis, Asian ancestry and female sex remained significant predictors of (1) OS in stage IV patients and (2) likelihood of harboring a receptor of fusion-based driver., Conclusion: Comprehensive NGS revealed driver alterations in 95% of never-smokers, with the majority having an associated therapy available. All efforts should be exhausted to identify or rule out the presence of an actionable driver mutation in all metastatic lung ADC., (© The Author(s) 2022. Published by Oxford University Press.)

Purpose: Data quality and standardization remain a challenge when analyzing real-world clinical data. We built a clinical research database, using machine learning and natural learning processing, and investigated factors influencing testosterone recovery (T-recovery) in patients with localized prostate cancer (LPC) after initial androgen deprivation therapy (ADT)., Methods: Medication and treatment-associated dates missing in structured tables were extracted from patient notes using ConceptMapper, an automated data extraction tool, standardized and curated in Sema4 clinical research database. ADT usage duration was evaluated, and T-recovery in patients with LPC was analyzed by the Kaplan-Meier method and multivariable Cox proportional hazards models. We assessed the prognostic value of post-ADT T-recovery with prostate-specific antigen progression-free survival and failure-free survival., Results: In total, 4,125 of 30,832 (13.4%) patients with prostate cancer had medication exclusively from notes with high precision and recall, F1 score ≥ 0.95. Association of dates with medication usage had a F1 score of 0.76. ADT duration estimation had higher accuracy combining information from notes to tables from electronic medical record (70% v 45%). Baseline testosterone was the strongest predictor of T-recovery in these patients. Patients with a baseline testosterone ≥ 300 ng/dL recovered in 9.79 versus 38 months for patients with baseline testosterone < 300 ng/dL ( P < .0001). Shorter prostate-specific antigen progression-free interval was observed for patients with T-recovery (≥ 300 ng/dL) at 6 months after ADT cessation compared with patients without T-recovery (< 300 ng/dL; 13.7 v 25.1 months; P = .055)., Conclusion: We augmented structured electronic medical record data with data extracted from notes and improved the accuracy of medication information for patients. ADT exposure and T-recovery in patients with LPC produced results consistent with the literature and clinical experience and illustrates the power of applying machine learning methods to enhance the quality of real-world evidence in answering clinically relevant questions., Competing Interests: Sunny GuinEmployment: Sema4Stock and Other Ownership Interests: Sema4 Tomi JunEmployment: Sema4Stock and Other Ownership Interests: Sema4Research Funding: Sema4Travel, Accommodations, Expenses: Sema4Open Payments Link: https://openpaymentsdata.cms.gov/physician/7228039 Vaibhav G. PatelStock and Other Ownership Interests: CRISPR Therapeutics, Editas MedicineConsulting or Advisory Role: Seattle Genetics, Sanofi Kristin L. AyersEmployment: Sema4Stock and Other Ownership Interests: Sema4 Matthew DeitzEmployment: Sema4Stock and Other Ownership Interests: Sema4 Xiang ZhouEmployment: Sema4 Che-Kai TsaoConsulting or Advisory Role: Eisai, Exelixis, Bristol Myers Squibb/SanofiResearch Funding: Exelixis William K. OhEmployment: Sema4Stock and Other Ownership Interests: Sema4Consulting or Advisory Role: Sanofi, Janssen, AstraZeneca, Bayer, HUYA Bioscience International, TeneoBio, Advanced Accelerator Applications, Conjupro Biotherapeutics, Foundry, GlaxoSmithKline, Merck, Pfizer Rong ChenEmployment: Sema4Stock and Other Ownership Interests: Sema4, PersonalisPatents, Royalties, Other Intellectual Property: I hold patent and royalty with Personalis, Boston University, Stanford University, and Quest Diagnostics Bobby C. LiawConsulting or Advisory Role: Daiichi Sankyo, Eisai, Exelixis, Myovant Sciences, EMD Serono, Epizyme, Janssen Biotech, Bristol Myers SquibbResearch Funding: Bayer, SanofiNo other potential conflicts of interest were reported.

Background: Multiple lines of evidence suggest possible impairment of the glymphatic system in amyotrophic lateral sclerosis (ALS). To investigate this, we used in vivo magnetic resonance imaging (MRI) to assess glymphatic function early in the course of disease in a transgenic mouse with doxycycline (Dox)-controlled expression of cytoplasmic human TDP-43 (hTDP-43ΔNLS), mimicking the key pathology implicated in ALS., Methods: Adult TDP-43 transgenic and littermate monogenic control mice underwent longitudinal multimodal MRI one and three weeks after the cessation of Dox feed, together with weekly rotarod assessments of motor performance. Glymphatic function was assessed using dynamic contrast-enhanced MRI to track the clearance of an MR contrast agent injected into the cisterna magna., Results: Compared to their littermate controls, TDP-43 mice exhibited progressive neurodegeneration including that within the primary motor cortex, primary somatosensory cortex and corticospinal tract, significant weight loss including gastrocnemius atrophy, and shortened telomere length. Furthermore, in the presence of this ALS-like phenotype, these mice have significantly disrupted glymphatic function., Conclusions: Although the relationship between glymphatic clearance and ALS disease progression remains to be elucidated, these changes occurred very early in the disease course. This provides initial evidence to suggest that the glymphatic system might be a potential therapeutic target in the treatment of ALS., (© 2022. The Author(s).)

Premature ovarian insufficiency (POI), affecting 1 in 100 women, is characterised by loss of ovarian function associated with elevated gonadotropin, before the age of 40. In addition to infertility, patients face increased risk of comorbidities such as heart disease, osteoporosis, cancer and/or early mortality. We used whole exome sequencing to identify the genetic cause of POI in seven women. Each had biallelic candidate variants in genes with a primary role in DNA damage repair and/or meiosis. This includes two genes, REC8 and HROB, not previously associated with autosomal recessive POI. REC8 encodes a component of the cohesin complex and HROB encodes a factor that recruits MCM8/9 for DNA damage repair. In silico analyses, combined with concordant mouse model phenotypes support these as new genetic causes of POI. We also identified novel variants in MCM8, NUP107, STAG3 and HFM1 and a known variant in POF1B. Our study highlights the pivotal role of meiosis in ovarian function. We identify novel variants, consolidate the pathogenicity of variants previously considered of unknown significance, and propose HROB and REC8 variants as new genetic causes while exploring their link to pathogenesis., (© 2021. The Author(s), under exclusive licence to European Society of Human Genetics.)

Amyotrophic lateral sclerosis (ALS) is characterized by transactive response DNA-binding protein 43 (TDP-43) pathology, progressive loss of motor neurons and muscle dysfunction. Symptom onset can be insidious and diagnosis challenging. Conventional neuroimaging is used to exclude ALS mimics, however more advanced neuroimaging techniques may facilitate an earlier diagnosis. Here, we investigate the potential for neurite orientation dispersion and density imaging and diffusion tensor imaging (DTI) to detect microstructural changes in an experimental model of ALS with neuronal doxycycline (Dox)-suppressible overexpression of human TDP-43 (hTDP-43). In vivo diffusion-weighted imaging (DWI) was acquired 1- and 3- weeks following the initiation of hTDP-43 expression (post-Dox) to investigate whether neurite density imaging (NDI) and orientation dispersion imaging (ODI) are affected early in this preclinical model of ALS and if so, how these metrics compare to those derived from the diffusion tensor. Tract-based spatial statistics at 1-week post-Dox, i.e. very early in the disease stage, demonstrated increased NDI in TDP-43 mice but no change in ODI or DTI metrics. At 3-weeks post-Dox, a reduced pattern of increased NDI was observed along with widespread increases in ODI, and decreased fractional anisotropy (FA), apparent diffusion coefficient (ADC) and axial diffusivity (AD). A hypothesis driven analysis of the bilateral corticospinal tracts demonstrated that at 1-week post-Dox, ODI was significantly increased caudally but decreased in the motor cortex of TDP-43 mice. Decreased cortical ODI had normalized by 3-weeks post-Dox and only significant increases were observed. A similar, but inverse pattern in FA was also observed. Together, these results suggest a non-monotonic relationship between DWI metrics and pathophysiological progression with TDP-43 mice exhibiting significantly altered diffusion metrics consistent with early inflammation followed by progressive axonal degeneration. Importantly, significant group-wise changes were observed in the earliest stages of disease when subtle pathology may be more elusive to traditional structural imaging techniques., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Racial disparities among clinical trial participants present a challenge to assess whether trial results can be generalized into patients representing diverse races and ethnicities. The objective of this study was to evaluate the impact of race and ethnicity on treatment response in patients with advanced non-small cell lung cancer (aNSCLC) treated with programmed cell death-1 (PD-1) or programmed cell death-ligand 1 (PD-L1) inhibitors through analysis of real-world data (RWD)., Materials and Methods: A retrospective cohort study of 11,138 patients with lung cancer treated at hospitals within the Mount Sinai Health System was performed. Patients with confirmed aNSCLC who received anti-PD-1/PD-L1 treatment were analyzed for clinical outcomes. Our cohort included 249 patients with aNSCLC who began nivolumab, pembrolizumab, or atezolizumab treatment between November 2014 and December 2018. Time-to-treatment discontinuation (TTD) and overall survival (OS) were the analyzed clinical endpoints., Results: After a median follow-up of 14.8 months, median TTD was 7.8 months (95% confidence interval, 5.4-not estimable [NE]) in 75 African American patients versus 4.6 (2.4-7.2) in 110 White patients (hazard ratio [HR], 0.63). Median OS was not reached (18.4-NE) in African American patients versus 11.6 months (9.7-NE) in White patients (HR, 0.58). Multivariable Cox regression conducted with potential confounders confirmed longer TTD (adjusted HR, 0.65) and OS (adjusted HR, 0.60) in African American versus White patients. Similar real-world response rate (42.6% vs. 43.5%) and disease control rate (59.6% vs. 56.5%) were observed in the African American and White patient populations. Further investigation revealed the African American patient group had lower incidence (14.7%) of putative hyperprogressive diseases (HPD) upon anti-PD-1/PD-L1 treatment than the White patient group (24.5%)., Conclusion: Analysis of RWD showed longer TTD and OS in African American patients with aNSCLC treated with anti-PD-1/PD-L1 inhibitors. Lower incidence of putative HPD is a possible reason for the favorable outcomes in this patient population., Implications for Practice: There is a significant underrepresentation of minority patients in randomized clinical trials, and this study demonstrates that real-world data can be used to investigate the impact of race and ethnicity on treatment response. In retrospective analysis of patients with advanced non-small cell lung cancer treated with programmed cell death-1 or programmed cell death-ligand 1 inhibitors, African American patients had significantly longer time-to-treatment discontinuation and longer overall survival. Analysis of real-world data can yield clinical insights and establish a more complete picture of medical interventions in routine clinical practice., (© 2021 Mount Sinai Genomics, Inc., DBA Sema4 and Icahn School of Medicine at Mount Sinai. The Oncologist published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

GPR17 is a G-protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Such evidence is primarily drawn from mouse knockout studies and suggests GPR17 as a potential novel therapeutic target for the treatment of metabolic diseases. However, links between human GPR17 genetic variants, downstream cellular signaling, and metabolic diseases have yet to be reported. Here, we analyzed GPR17 coding sequences from control and disease cohorts consisting of individuals with adverse clinical metabolic deficits including severe insulin resistance, hypercholesterolemia, and obesity. We identified 18 nonsynonymous GPR17 variants, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, membrane localization, and downstream signaling profiles of nine GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). These nine GPR17 variants had similar protein expression and subcellular localization as wild-type GPR17; however, they showed diverse downstream signaling profiles. GPR17-G136S lost the capacity for agonist-mediated cAMP, Ca 2+ , and β-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT, but showed impaired Ca 2+ and β-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca 2+ signaling, but unaffected agonist-stimulated β-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with diverse signaling profiles, including differential signaling perturbations that resulted in GPCR signaling bias. Our findings provide a framework for structure-function relationship studies of GPR17 signaling and metabolic disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Immune checkpoint inhibitors (ICIs) have been incorporated into various clinical oncology guidelines for systemic treatment of advanced non-small cell lung cancers (aNSCLC). However, less than 50% (and 20%) of the patients responded to the therapy as a first (or second) line of therapy. PD-L1 immunohistochemistry (IHC) is an extensively studied biomarker of response to ICI, but results from this test have equivocal predictive power. In order to identify other biomarkers that support clinical decision-making around whether to treat with ICIs or not, we performed a retrospective study of patients with aNSCLC who underwent ICI-based therapy in the Mount Sinai Health System between 2014 and 2019., Methods: We analyzed data from standard laboratory tests performed in patients as a part of the routine clinical workup during treatment, including complete blood counts (CBC) and a comprehensive metabolic panel (CMP), to correlate test results with clinical response and survival., Results: Of 11,138 NSCLC patients identified, 249 had been treated with ICIs. We found associations between high neutrophil-to-lymphocyte ratio (NLR ≥ 5) and poor survival in ICI-treated NSCLC. We further observed that sustained high NLR after initiation of treatment had a more profound impact on survival than baseline NLR, regardless of PD-L1 status. Hazard ratios when comparing patients with NLR ≥ 5 vs. NLR < 5 are 1.7 (p = 0.02), 3.4 (p = 4.2 × 10 - 8 ), and 3.9 (p = 1.4 × 10 - 6 ) at baseline, 2-8 weeks, and 8-14 weeks after treatment start, respectively. Mild anemia, defined as hemoglobin (HGB) less than 12 g/dL was correlated with survival independently of NLR. Finally, we developed a composite NLR and HGB biomarker. Patients with pretreatment NLR ≥ 5 and HGB < 12 g/dL had a median overall survival (OS) of 8.0 months (95% CI 4.5-11.5) compared to the rest of the cohort with a median OS not reached (95% CI 15.9-NE, p = 1.8 × 10 - 5 ), and a hazard ratio of 2.6 (95% CI 1.7-4.1, p = 3.5 × 10 - 5 )., Conclusions: We developed a novel composite biomarker for ICI-based therapy in NSCLC based on routine CBC tests, which may provide meaningful clinical utility to guide treatment decision. The results suggest that treatment of anemia to elevate HGB before initiation of ICI therapy may improve patient outcomes or the use of alternative non-chemotherapy containing regimens.

Background: Complete androgen insensitivity syndrome (CAIS) is a congenital condition caused by genetic defects in the androgen receptor ( AR ) gene located on the X chromosome, which lead to a phenotypical female individual with a 46, XY karyotype. Early diagnosis of CAIS is essential for proper clinical management, allows assessment of familial risk and contributes to healthcare decisions. However, diagnosis of CAIS can be overlooked in girls with inguinal hernia, resulting in inappropriate management. Methods: Five female patients from three unrelated families presented to our genetic clinic with primary amenorrhea. Each patient had been diagnosed with inguinal hernia in childhood and had undergone hernia repair without further investigation into what was contained in the hernial sac. We carried out physical examination, cytogenetic studies, hormonal evaluation, and molecular analysis to establish a comprehensive diagnosis. Family history and pedigree were collated to identify at-risk family members. Results: All patients presented with female external genitalia. Cytogenetic studies revealed a 46, XY karyotype and hormonal analysis suggested a diagnosis of CAIS. Sequencing of the AR gene in all patients and suspected family members revealed pathogenic variants in the AR gene and confirmed the molecular diagnosis of CAIS. Conclusions: We report the delayed diagnosis of CAIS in female Indonesian patients with a history of inguinal hernia in childhood. An early diagnosis of CAIS is essential for appropriate clinical management, as well as assessing familial risk. Increasing awareness among clinicians is paramount, and we encourage a CAIS diagnosis to be considered in any patient presenting with female appearance and inguinal hernia.

Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a much-needed human gonad organoid for studying disorders/differences of sex development., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Ovarian deficiency, including premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR), represents one of the main causes of female infertility. POI is a genetically heterogeneous condition but current understanding of its genetic basis is far from complete, with the cause remaining unknown in the majority of patients. The genes that regulate DOR have been reported but the genetic basis of DOR has not been explored in depth. Both conditions are likely to lie along a continuum of degrees of decrease in ovarian reserve. We performed genomic analysis via whole exome sequencing (WES) followed by in silico analyses and functional experiments to investigate the genetic cause of ovarian deficiency in ten affected women. We achieved diagnoses for three of them, including the identification of novel variants in STAG3, GDF9, and FANCM. We identified potentially causative FSHR variants in another patient. This is the second report of biallelic GDF9 and FANCM variants, and, combined with functional support, validates these genes as bone fide autosomal recessive "POI genes". We also identified new candidate genes, NRIP1, XPO1, and MACF1. These genes have been linked to ovarian function in mouse, pig, and zebrafish respectively, but never in humans. In the case of NRIP1, we provide functional support for the deleterious nature of the variant via SUMOylation and luciferase/β-galactosidase reporter assays. Our study provides multiple insights into the genetic basis of POI/DOR. We have further elucidated the involvement of GDF9, FANCM, STAG3 and FSHR in POI pathogenesis, and propose new candidate genes, NRIP1, XPO1, and MACF1, which should be the focus of future studies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Infertility, a global problem affecting up to 15% of couples, can have varied causes ranging from natural ageing to the pathological development or function of the reproductive organs. One form of female infertility is premature ovarian insufficiency (POI), affecting up to 1 in 100 women and characterised by amenorrhoea and elevated FSH before the age of 40. POI can have a genetic basis, with over 50 causative genes identified. Non-obstructive azoospermia (NOA), a form of male infertility characterised by the absence of sperm in semen, has an incidence of 1% and is similarly heterogeneous. The genetic basis of male and female infertility is poorly understood with the majority of cases having no known cause. Here, we study a case of familial infertility including a proband with POI and her brother with NOA. We performed whole-exome sequencing (WES) and identified a homozygous STAG3 missense variant that segregated with infertility. STAG3 encodes a component of the meiosis cohesin complex required for sister chromatid separation. We report the first pathogenic homozygous missense variant in STAG3 and the first STAG3 variant associated with both male and female infertility. We also demonstrate limitations of WES for the analysis of homologous DNA sequences, with this variant being ambiguous or missed by independent WES protocols and its homozygosity only being established via long-range nested PCR., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: GATA-binding protein 4 (GATA4) and Friend of GATA 2 protein (FOG2, also known as ZFPM2) form a heterodimer complex that has been shown to influence transcription of genes in a number of developmental systems. Recent evidence has also shown these genes play a role in gonadal sexual differentiation in humans. Previously we identified four variants in GATA4 and an unexpectedly large number of variants in ZFPM2 in a cohort of individuals with 46,XY Differences/Disorders of Sex Development (DSD) (Eggers et al, Genome Biology, 2016; 17: 243)., Method: Here, we review variant curation and test the functional activity of GATA4 and ZFPM2 variants. We assess variant transcriptional activity on gonadal specific promoters (Sox9 and AMH) and variant protein-protein interactions., Results: Our findings support that the majority of GATA4 and ZFPM2 variants we identified are benign in their contribution to 46,XY DSD. Indeed, only one variant, in the conserved N-terminal zinc finger of GATA4, was considered pathogenic, with functional analysis confirming differences in its ability to regulate Sox9 and AMH and in protein interaction with ZFPM2., Conclusions: Our study helps define the genetic factors contributing to 46,XY DSD and suggests that the majority of variants we identified in GATA4 and ZFPM2/FOG2 are not causative., (© 2019 The Murdoch Children's Research Institute. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Ovarian deficiency, including diminished ovarian reserve and premature ovarian insufficiency, represents one of the main causes of female infertility. Little is known of the genetic basis of diminished ovarian reserve, while premature ovarian insufficiency often has a genetic basis, with genes affecting various processes. NR5A1 is a key gene required for gonadal function, and variants are associated with a wide phenotypic spectrum of disorders of sexual development, and are found in 0.26-8% of patients with premature ovarian insufficiency. As there is some debate about the extent of involvement of NR5A1 in the pathogenesis of ovarian deficiency, we performed an in-depth analysis of NR5A1 variants detected in a cohort of 142 patients with premature ovarian insufficiency, diminished ovarian reserve, or unexplained infertility associated with normal ovarian function. We identified rare non-synonymous protein-altering variants in 2.8 % of women with ovarian deficiency and no such variants in our small cohort of women with infertility but normal ovarian function. We observed previously reported variants associated with premature ovarian insufficiency in patients with diminished ovarian reserve, highlighting a genetic relationship between these conditions. We confirmed functional impairment resulting from a p.Val15Met variant, detected for the first time in a patient with premature ovarian insufficiency. The remaining variants were associated with preserved transcriptional activity and localization of NR5A1, indicating that rare NR5A1 variants may be incorrectly curated if functional studies are not undertaken, and/or that NR5A1 variants may have only a subtle impact on protein function and/or confer risk of ovarian deficiency via oligogenic inheritance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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Human longevity is heritable, but genome-wide association (GWA) studies have had limited success. Here, we perform two meta-analyses of GWA studies of a rigorous longevity phenotype definition including 11,262/3484 cases surviving at or beyond the age corresponding to the 90th/99th survival percentile, respectively, and 25,483 controls whose age at death or at last contact was at or below the age corresponding to the 60th survival percentile. Consistent with previous reports, rs429358 (apolipoprotein E (ApoE) ε4) is associated with lower odds of surviving to the 90th and 99th percentile age, while rs7412 (ApoE ε2) shows the opposite. Moreover, rs7676745, located near GPR78, associates with lower odds of surviving to the 90th percentile age. Gene-level association analysis reveals a role for tissue-specific expression of multiple genes in longevity. Finally, genetic correlation of the longevity GWA results with that of several disease-related phenotypes points to a shared genetic architecture between health and longevity.

Background: Genetic loss-of-function variants (LoFs) associated with disease traits are increasingly recognized as critical evidence for the selection of therapeutic targets. We integrated the analysis of genetic and clinical data from 10,511 individuals in the Mount Sinai BioMe Biobank to identify genes with loss-of-function variants (LoFs) significantly associated with cardiovascular disease (CVD) traits, and used RNA-sequence data of seven metabolic and vascular tissues isolated from 600 CVD patients in the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) study for validation. We also carried out in vitro functional studies of several candidate genes, and in vivo studies of one gene., Results: We identified LoFs in 433 genes significantly associated with at least one of 10 major CVD traits. Next, we used RNA-sequence data from the STARNET study to validate 115 of the 433 LoF harboring-genes in that their expression levels were concordantly associated with corresponding CVD traits. Together with the documented hepatic lipid-lowering gene, APOC3, the expression levels of six additional liver LoF-genes were positively associated with levels of plasma lipids in STARNET. Candidate LoF-genes were subjected to gene silencing in HepG2 cells with marked overall effects on cellular LDLR, levels of triglycerides and on secreted APOB100 and PCSK9. In addition, we identified novel LoFs in DGAT2 associated with lower plasma cholesterol and glucose levels in BioMe that were also confirmed in STARNET, and showed a selective DGAT2-inhibitor in C57BL/6 mice not only significantly lowered fasting glucose levels but also affected body weight., Conclusion: In sum, by integrating genetic and electronic medical record data, and leveraging one of the world's largest human RNA-sequence datasets (STARNET), we identified known and novel CVD-trait related genes that may serve as targets for CVD therapeutics and as such merit further investigation.

Context: Variants in bone morphogenetic protein 7 (BMP7) have been reported in patients with hypospadias. Here we report and analyze two variants in the BMP7 prodomain in monozygotic twins with hypospadias., Materials and Methods: Patients with hypospadias were prospectively recruited. After informed consent was obtained, DNA was extracted from blood. The coding regions of 1034 genes [including 64 known diagnostic genes and candidate genes for disorder/difference of sex development (DSD)] were sequenced using a targeted capture approach (HaloPlex, Agilent, Santa Clara, CA), combined with massively parallel sequencing. The resulting variants were filtered for rarity in the general population (<1%) and in our screen. Quality, depth of the reads, and predicted pathogenicity were also considered. The consequences of the identified mutations on BMP7 expression was determined by Western blot analysis on culture media from transfected cells, and activity measured using a SMAD 1/5-responsiveness luciferase assay., Results: We analyzed DNA from 46 patients with hypospadias. Two variants in BMP7 were identified in two pairs of monozygotic concordant twins exhibiting proximal hypospadias. Both variants are heterozygous, nonsynonymous, and affect highly conserved amino acids in the prodomain of BMP7 in regions predicted to be important for BMP7 assembly/folding. Functional analyses demonstrated that both variants disrupt BMP7 synthesis or secretion., Conclusion: Through our targeted DSD panel we have identified two variants in the prodomain of BMP7 in hypospadias. By decreasing BMP7 synthesis, these variants are likely to limit BMP7 bioavailability during closure of the urethral plate.Further analysis of patients with hypospadias may uncover additional variants that cause this DSD.

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the DNA-binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti-testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females., (© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

Primary Biliary Cholangitis (PBC) is a chronic autoimmune liver disease characterised by progressive destruction of intrahepatic bile ducts. The strongest genetic association is with HLA-DQA1*04:01, but at least three additional independent HLA haplotypes contribute to susceptibility. We used dense single nucleotide polymorphism (SNP) data in 2861 PBC cases and 8514 controls to impute classical HLA alleles and amino acid polymorphisms using state-of-the-art methodologies. We then demonstrated through stepwise regression that association in the HLA region can be largely explained by variation at five separate amino acid positions. Three-dimensional modelling of protein structures and calculation of electrostatic potentials for the implicated HLA alleles/amino acid substitutions demonstrated a correlation between the electrostatic potential of pocket P6 in HLA-DP molecules and the HLA-DPB1 alleles/amino acid substitutions conferring PBC susceptibility/protection, highlighting potential new avenues for future functional investigation., Competing Interests: The authors have declared that no competing interests exist.

In the version of this article originally published, the name of author Martin H. de Borst was coded incorrectly in the XML. The error has now been corrected in the HTML version of the paper.

High blood pressure is a highly heritable and modifiable risk factor for cardiovascular disease. We report the largest genetic association study of blood pressure traits (systolic, diastolic and pulse pressure) to date in over 1 million people of European ancestry. We identify 535 novel blood pressure loci that not only offer new biological insights into blood pressure regulation but also highlight shared genetic architecture between blood pressure and lifestyle exposures. Our findings identify new biological pathways for blood pressure regulation with potential for improved cardiovascular disease prevention in the future.

Context: The hypothalamic melanocortin 4 receptor (MC4R) pathway serves a critical role in regulating body weight. Loss of function (LoF) mutations in the MC4R pathway, including mutations in the pro-opiomelanocortin (POMC), prohormone convertase 1 (PCSK1), leptin receptor (LEPR), or MC4R genes, have been shown to cause early-onset severe obesity., Methods: Through a comprehensive epidemiological analysis of known and predicted LoF variants in the POMC, PCSK1, and LEPR genes, we sought to estimate the number of US individuals with biallelic MC4R pathway LoF variants., Results: We predict ~650 α-melanocyte-stimulating hormone (MSH)/POMC, 8500 PCSK1, and 3600 LEPR homozygous and compound heterozygous individuals in the United States, cumulatively enumerating >12,800 MC4R pathway-deficient obese patients. Few of these variants have been genetically diagnosed to date. These estimates increase when we include a small subset of less rare variants: β-MSH/POMC,PCSK1 N221D, and a PCSK1 LoF variant (T640A). To further define the MC4R pathway and its potential impact on obesity, we tested associations between body mass index (BMI) and LoF mutation burden in the POMC, PCSK1, and LEPR genes in various populations. We show that the cumulative allele burden in individuals with two or more LoF alleles in one or more genes in the MC4R pathway are predisposed to a higher BMI than noncarriers or heterozygous LoF carriers with a defect in only one gene., Conclusions: Our analysis represents a genetically rationalized study of the hypothalamic MC4R pathway aimed at genetic patient stratification to determine which obese subpopulations should be studied to elucidate MC4R agonist (e.g., setmelanotide) treatment responsiveness.

The Müllerian ducts are part of the embryonic urogenital system. They give rise to mature structures that serve a critical function in the transport and development of the oocyte and/or embryo. In most vertebrates, both sexes initially develop Müllerian ducts during embryogenesis, but they regress in males under the influence of testis-derived Anti-Müllerian Hormone (AMH). A number of regulatory factors have been shown to be essential for proper duct development, including Bmp and Wnt signaling molecules, together with homeodomain transcription factors such as PAX2 and LIM1. Later in development, the fate of the ducts diverges between males and females and is regulated by AMH and Wnt signaling molecules (duct regression in males) and Hox genes (duct patterning in females). Most of the genes and molecular pathways known to be involved in Müllerian duct development have been elucidated through animal models, namely, the mouse and chicken. In addition, genetic analysis of humans with reproductive tract disorders has further defined molecular mechanisms of duct formation and differentiation. However, despite our current understanding of Müllerian duct development, some questions remain to be answered at the molecular genetic level. This article is categorized under: Early Embryonic Development > Development to the Basic Body Plan., (© 2018 Wiley Periodicals, Inc.)

Variants in the NR5A1 gene encoding SF1 have been described in a diverse spectrum of disorders of sex development (DSD). Recently, we reported the use of a targeted gene panel for DSD where we identified 15 individuals with a variant in NR5A1, nine of which are novel. Here, we examine the functional effect of these changes in relation to the patient phenotype. All novel variants tested had reduced trans-activational activity, while several had altered protein level, localization, or conformation. In addition, we found evidence of new roles for SF1 protein domains including a region within the ligand binding domain that appears to contribute to SF1 regulation of Müllerian development. There was little correlation between the severity of the phenotype and the nature of the NR5A1 variant. We report two familial cases of NR5A1 deficiency with evidence of variable expressivity; we also report on individuals with oligogenic inheritance. Finally, we found that the nature of the NR5A1 variant does not inform patient outcomes (including pubertal androgenization and malignancy risk). This study adds nine novel pathogenic NR5A1 variants to the pool of diagnostic variants. It highlights a greater need for understanding the complexity of SF1 function and the additional factors that contribute., (© 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.)