Publications by authors named "Donald F Conrad"

59 Publications

Comparative single-cell analysis of biopsies clarifies pathogenic mechanisms in Klinefelter syndrome.

Am J Hum Genet 2021 10;108(10):1924-1945

Division of Genetics, Oregon National Primate Research Center, Oregon Health and Science University, Portland, OR 97006, USA; Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Klinefelter syndrome (KS), also known as 47, XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely because of the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple individuals with KS that lack transcription from the XIST locus, and the consequence of this is increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known cell signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution.
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http://dx.doi.org/10.1016/j.ajhg.2021.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8546046PMC
October 2021

Variant , Defective piRNA Processing, and Azoospermia.

N Engl J Med 2021 08 4;385(8):707-719. Epub 2021 Aug 4.

From the Division of Genetics, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton (L.N., D.F.C.); the Center for Embryonic Cell and Gene Therapy, Oregon Health and Science University, Portland (D.F.C.); the Department of Growth and Reproduction (N.M., J.E.N., R.S., I.G., S.B.W., N.E.S., E.R.-D.M., N.J., K.A.) and the International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (N.M., J.E.N., R.S., I.G., S.B.W., N.E.S., E.R.-D.M., N.J., K.A.), Rigshospitalet, and the Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences (K.A.), University of Copenhagen, Copenhagen; the Laboratory of Molecular Neurooncology, Neuroscience Institute (R.S.), and the Institute of Biology Systems and Genetic Research (I.G.), Lithuanian University of Health Sciences, Kaunas, Lithuania; the Department of Human Genetics, Donders Institute for Brain, Cognition, and Behavior (M.S.O., G.W.H.), and the Department of Obstetrics and Gynecology (G.W.H.), Radboud University Medical Center, Nijmegen, the Netherlands; Serviço de Genética, Departamento de Patologia, Faculdade de Medicina da Universidade do Porto (F.C., C.J.M.), Instituto de Investigação e Inovação em Saúde, Universidade do Porto (F.C., C.J.M., A.M.L.), and the Institute of Molecular Pathology and Immunology of the University of Porto (A.M.L.) - all in Porto, Portugal; the Andrology and In Vitro Fertilization Laboratory, Department of Surgery (Urology), University of Utah School of Medicine, Salt Lake City (K.I.A.); the Departments of Pathology and Laboratory Medicine (F.K.) and Urology (P.N.S.), Weill Cornell Medicine, New York; and the Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom (J.A.V.).

Background: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility.

Methods: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing.

Results: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in : the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying mutations.

Conclusions: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).
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http://dx.doi.org/10.1056/NEJMoa2028973DOI Listing
August 2021

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Hum Genet 2021 Aug 7;140(8):1169-1182. Epub 2021 May 7.

Institute of Reproductive Genetics, University of Münster, Münster, Germany.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.
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http://dx.doi.org/10.1007/s00439-021-02287-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266742PMC
August 2021

The Sertoli cell expressed gene secernin-1 (Scrn1) is dispensable for male fertility in the mouse.

Dev Dyn 2021 Jul 25;250(7):922-931. Epub 2021 Jan 25.

School of Biological Sciences, Monash University, Clayton, Victoria, Australia.

Background: Male infertility is a prevalent clinical presentation for which there is likely a strong genetic component due to the thousands of genes required for spermatogenesis. Within this study we investigated the role of the gene Scrn1 in male fertility. Scrn1 is preferentially expressed in XY gonads during the period of sex determination and in adult Sertoli cells based on single cell RNA sequencing. We investigated the expression of Scrn1 in juvenile and adult tissues and generated a knockout mouse model to test its role in male fertility.

Results: Scrn1 was expressed at all ages examined in the post-natal testis; however, its expression peaked at postnatal days 7-14 and SCRN1 protein was clearly localized to Sertoli cells. Scrn1 deletion was achieved via removal of exon 3, and its loss had no effect on male fertility or sex determination. Knockout mice were capable of siring litters of equal size to wild type counterparts and generated equal numbers of sperm with comparable motility and morphology characteristics.

Conclusions: Scrn1 was found to be dispensable for male fertility, but this study identifies SCRN1 as a novel marker of the Sertoli cell cytoplasm.
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http://dx.doi.org/10.1002/dvdy.299DOI Listing
July 2021

The genetic architecture of sporadic and multiple consecutive miscarriage.

Nat Commun 2020 11 25;11(1):5980. Epub 2020 Nov 25.

University of Queensland, St Lucia, QLD, Australia.

Miscarriage is a common, complex trait affecting ~15% of clinically confirmed pregnancies. Here we present the results of large-scale genetic association analyses with 69,054 cases from five different ancestries for sporadic miscarriage, 750 cases of European ancestry for multiple (≥3) consecutive miscarriage, and up to 359,469 female controls. We identify one genome-wide significant association (rs146350366, minor allele frequency (MAF) 1.2%, P = 3.2 × 10, odds ratio (OR) = 1.4) for sporadic miscarriage in our European ancestry meta-analysis and three genome-wide significant associations for multiple consecutive miscarriage (rs7859844, MAF = 6.4%, P = 1.3 × 10, OR = 1.7; rs143445068, MAF = 0.8%, P = 5.2 × 10, OR = 3.4; rs183453668, MAF = 0.5%, P = 2.8 × 10, OR = 3.8). We further investigate the genetic architecture of miscarriage with biobank-scale Mendelian randomization, heritability, and genetic correlation analyses. Our results show that miscarriage etiopathogenesis is partly driven by genetic variation potentially related to placental biology, and illustrate the utility of large-scale biobank data for understanding this pregnancy complication.
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http://dx.doi.org/10.1038/s41467-020-19742-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689465PMC
November 2020

Disruption of human meiotic telomere complex genes TERB1, TERB2 and MAJIN in men with non-obstructive azoospermia.

Hum Genet 2021 Jan 19;140(1):217-227. Epub 2020 Nov 19.

Andrology and IVF Laboratory, Division of Urology, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, 84108, USA.

Non-obstructive azoospermia (NOA), the lack of spermatozoa in semen due to impaired spermatogenesis affects nearly 1% of men. In about half of cases, an underlying cause for NOA cannot be identified. This study aimed to identify novel variants associated with idiopathic NOA. We identified a nonconsanguineous family in which multiple sons displayed the NOA phenotype. We performed whole-exome sequencing in three affected brothers with NOA, their two unaffected brothers and their father, and identified compound heterozygous frameshift variants (one novel and one extremely rare) in Telomere Repeat Binding Bouquet Formation Protein 2 (TERB2) that segregated perfectly with NOA. TERB2 interacts with TERB1 and Membrane Anchored Junction Protein (MAJIN) to form the tripartite meiotic telomere complex (MTC), which has been shown in mouse models to be necessary for the completion of meiosis and both male and female fertility. Given our novel findings of TERB2 variants in NOA men, along with the integral role of the three MTC proteins in spermatogenesis, we subsequently explored exome sequence data from 1495 NOA men to investigate the role of MTC gene variants in spermatogenic impairment. Remarkably, we identified two NOA patients with likely damaging rare homozygous stop and missense variants in TERB1 and one NOA patient with a rare homozygous missense variant in MAJIN. Available testis histology data from three of the NOA patients indicate germ cell maturation arrest, consistent with mouse phenotypes. These findings suggest that variants in MTC genes may be an important cause of NOA in both consanguineous and outbred populations.
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http://dx.doi.org/10.1007/s00439-020-02236-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893731PMC
January 2021

Mutation of CFAP57, a protein required for the asymmetric targeting of a subset of inner dynein arms in Chlamydomonas, causes primary ciliary dyskinesia.

PLoS Genet 2020 08 7;16(8):e1008691. Epub 2020 Aug 7.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C>T), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the "g" inner dyneins (DHC7 and DHC3) and the "d" inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.
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http://dx.doi.org/10.1371/journal.pgen.1008691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444499PMC
August 2020

Genetic dissection of spermatogenic arrest through exome analysis: clinical implications for the management of azoospermic men.

Genet Med 2020 12 3;22(12):1956-1966. Epub 2020 Aug 3.

Institute of Human Genetics, University of Münster, Münster, Germany.

Purpose: Azoospermia affects 1% of men and it can be the consequence of spermatogenic maturation arrest (MA). Although the etiology of MA is likely to be of genetic origin, only 13 genes have been reported as recurrent potential causes of MA.

Methods: Exome sequencing in 147 selected MA patients (discovery cohort and two validation cohorts).

Results: We found strong evidence for five novel genes likely responsible for MA (ADAD2, TERB1, SHOC1, MSH4, and RAD21L1), for which mouse knockout (KO) models are concordant with the human phenotype. Four of them were validated in the two independent MA cohorts. In addition, nine patients carried pathogenic variants in seven previously reported genes-TEX14, DMRT1, TEX11, SYCE1, MEIOB, MEI1, and STAG3-allowing to upgrade the clinical significance of these genes for diagnostic purposes. Our meiotic studies provide novel insight into the functional consequences of the variants, supporting their pathogenic role.

Conclusion: Our findings contribute substantially to the development of a pre-testicular sperm extraction (TESE) prognostic gene panel. If properly validated, the genetic diagnosis of complete MA prior to surgical interventions is clinically relevant. Wider implications include the understanding of potential genetic links between nonobstructive azoospermia (NOA) and cancer predisposition, and between NOA and premature ovarian failure.
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http://dx.doi.org/10.1038/s41436-020-0907-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710580PMC
December 2020

Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility.

Am J Hum Genet 2020 08 15;107(2):342-351. Epub 2020 Jul 15.

Centre of Reproductive Medicine and Andrology, Institute of Reproductive Medicine, University of Münster, 48149 Münster, Germany.

Male infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.
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http://dx.doi.org/10.1016/j.ajhg.2020.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413853PMC
August 2020

A framework for high-resolution phenotyping of candidate male infertility mutants: from human to mouse.

Hum Genet 2021 Jan 4;140(1):155-182. Epub 2020 Apr 4.

School of Biological Sciences, Monash University, 25 Rainforest Walk, Clayton, VIC, 3800, Australia.

Male infertility is a heterogeneous condition of largely unknown etiology that affects at least 7% of men worldwide. Classical genetic approaches and emerging next-generation sequencing studies support genetic variants as a frequent cause of male infertility. Meanwhile, the barriers to transmission of this disease mean that most individual genetic cases will be rare, but because of the large percentage of the genome required for spermatogenesis, the number of distinct causal mutations is potentially large. Identifying bona fide causes of male infertility thus requires advanced filtering techniques to select for high-probability candidates, including the ability to test causality in animal models. The mouse remains the gold standard for defining the genotype-phenotype connection in male fertility. Here, we present a best practice guide consisting of (a) major points to consider when interpreting next-generation sequencing data performed on infertile men, and, (b) a systematic strategy to categorize infertility types and how they relate to human male infertility. Phenotyping infertility in mice can involve investigating the function of multiple cell types across the testis and epididymis, as well as sperm function. These findings will feed into the diagnosis and treatment of male infertility as well as male health broadly.
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http://dx.doi.org/10.1007/s00439-020-02159-xDOI Listing
January 2021

Identification of genetic variants in CFAP221 as a cause of primary ciliary dyskinesia.

J Hum Genet 2020 Jan 21;65(2):175-180. Epub 2019 Oct 21.

Department of Pathology and Laboratory Medicine, Marsico Lung Institute University of North Carolina, Chapel Hill, NC, 27599, USA.

Primary ciliary dyskinesia (PCD) is a rare disorder that affects the biogenesis or function of motile cilia resulting in chronic airway disease. PCD is genetically and phenotypically heterogeneous, with causative mutations identified in over 40 genes; however, the genetic basis of many cases is unknown. Using whole-exome sequencing, we identified three affected siblings with clinical symptoms of PCD but normal ciliary structure, carrying compound heterozygous loss-of-function variants in CFAP221. Computational analysis suggests that these variants are the most damaging alleles shared by all three siblings. Nasal epithelial cells from one of the subjects demonstrated slightly reduced beat frequency (16.5 Hz vs 17.7 Hz, p = 0.16); however, waveform analysis revealed that the CFAP221 defective cilia beat in an aberrant circular pattern. These results show that genetic variants in CFAP221 cause PCD and that CFAP221 should be considered a candidate gene in cases where PCD is suspected but cilia structure and beat frequency appear normal.
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http://dx.doi.org/10.1038/s10038-019-0686-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920546PMC
January 2020

Rare mutations in the complement regulatory gene CSMD1 are associated with male and female infertility.

Nat Commun 2019 10 11;10(1):4626. Epub 2019 Oct 11.

Department of Genetics, Washington University School of Medicine, St. Louis, MO, 63110, USA.

Infertility in men and women is a complex genetic trait with shared biological bases between the sexes. Here, we perform a series of rare variant analyses across 73,185 women and men to identify genes that contribute to primary gonadal dysfunction. We report CSMD1, a complement regulatory protein on chromosome 8p23, as a strong candidate locus in both sexes. We show that CSMD1 is enriched at the germ-cell/somatic-cell interface in both male and female gonads. Csmd1-knockout males show increased rates of infertility with significantly increased complement C3 protein deposition in the testes, accompanied by severe histological degeneration. Knockout females show significant reduction in ovarian quality and breeding success, as well as mammary branching impairment. Double knockout of Csmd1 and C3 causes non-additive reduction in breeding success, suggesting that CSMD1 and the complement pathway play an important role in the normal postnatal development of the gonads in both sexes.
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http://dx.doi.org/10.1038/s41467-019-12522-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789153PMC
October 2019

Unified single-cell analysis of testis gene regulation and pathology in five mouse strains.

Elife 2019 06 25;8. Epub 2019 Jun 25.

Department of Genetics, Washington University School of Medicine, St. Louis, United States.

To fully exploit the potential of single-cell functional genomics in the study of development and disease, robust methods are needed to simplify the analysis of data across samples, time-points and individuals. Here we introduce a model-based factor analysis method, SDA, to analyze a novel 57,600 cell dataset from the testes of wild-type mice and mice with gonadal defects due to disruption of the genes , , or . By jointly analyzing mutant and wild-type cells we decomposed our data into 46 components that identify novel meiotic gene-regulatory programs, mutant-specific pathological processes, and technical effects, and provide a framework for imputation. We identify, de novo, DNA sequence motifs associated with individual components that define temporally varying modes of gene expression control. Analysis of SDA components also led us to identify a rare population of macrophages within the seminiferous tubules of and mice, an area typically associated with immune privilege.
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http://dx.doi.org/10.7554/eLife.43966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615865PMC
June 2019

Bi-allelic Recessive Loss-of-Function Variants in FANCM Cause Non-obstructive Azoospermia.

Am J Hum Genet 2018 08;103(2):200-212

Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu 50411, Estonia. Electronic address:

Infertility affects around 7% of men worldwide. Idiopathic non-obstructive azoospermia (NOA) is defined as the absence of spermatozoa in the ejaculate due to failed spermatogenesis. There is a high probability that NOA is caused by rare genetic defects. In this study, whole-exome sequencing (WES) was applied to two Estonian brothers diagnosed with NOA and Sertoli cell-only syndrome (SCOS). Compound heterozygous loss-of-function (LoF) variants in FANCM (Fanconi anemia complementation group M) were detected as the most likely cause for their condition. A rare maternally inherited frameshift variant p.Gln498Thrfs7 (rs761250416) and a previously undescribed splicing variant (c.4387-10A>G) derived from the father introduce a premature STOP codon leading to a truncated protein. FANCM exhibits enhanced testicular expression. In control subjects, immunohistochemical staining localized FANCM to the Sertoli and spermatogenic cells of seminiferous tubules with increasing intensity through germ cell development. This is consistent with its role in maintaining genomic stability in meiosis and mitosis. In the individual with SCOS carrying bi-allelic FANCM LoF variants, none or only faint expression was detected in the Sertoli cells. As further evidence, we detected two additional NOA-affected case subjects with independent FANCM homozygous nonsense variants, one from Estonia (p.Gln1701; rs147021911) and another from Portugal (p.Arg1931; rs144567652). The study convincingly demonstrates that bi-allelic recessive LoF variants in FANCM cause azoospermia. FANCM pathogenic variants have also been linked with doubled risk of familial breast and ovarian cancer, providing an example mechanism for the association between infertility and cancer risk, supported by published data on Fancm mutant mouse models.
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http://dx.doi.org/10.1016/j.ajhg.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080835PMC
August 2018

Genetic intersection of male infertility and cancer.

Fertil Steril 2018 01;109(1):20-26

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri. Electronic address:

Recent epidemiological studies have identified an association between male factor infertility and increased cancer risk, however, the underlying etiology for the shared risk has not been investigated. It is likely that much of the association between the two disease states can be attributed to underlying genetic lesions. In this article we review the reported associations between cancer and spermatogenic defects, and through database searches we identify candidate genes and gene classes that could explain some of the observed shared genetic risk. We discuss the importance of fully characterizing the genetic basis for the relationship between cancer and male factor infertility and propose future studies to that end.
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http://dx.doi.org/10.1016/j.fertnstert.2017.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761685PMC
January 2018

Dynamic landscape and regulation of RNA editing in mammals.

Nature 2017 10;550(7675):249-254

Cardiovascular Research Institute, University of California-San Francisco, San Francisco, California 94158, USA.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.
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http://dx.doi.org/10.1038/nature24041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723435PMC
October 2017

The long and short of translational control in male germ cells.

Biol Reprod 2017 07;97(1):2-4

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, United States of America.

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http://dx.doi.org/10.1093/biolre/iox075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6248522PMC
July 2017

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry.

J Vis Exp 2017 07 12(125). Epub 2017 Jul 12.

Department of Genetics, Washington University School of Medicine;

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.
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http://dx.doi.org/10.3791/55913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612271PMC
July 2017

How to Map the Genetic Basis for Conditions that are Comorbid with Male Infertility.

Semin Reprod Med 2017 05 28;35(3):225-230. Epub 2017 Jun 28.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri.

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http://dx.doi.org/10.1055/s-0037-1603567DOI Listing
May 2017

The impact of structural variation on human gene expression.

Nat Genet 2017 May 3;49(5):692-699. Epub 2017 Apr 3.

McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA.

Structural variants (SVs) are an important source of human genetic diversity, but their contribution to traits, disease and gene regulation remains unclear. We mapped cis expression quantitative trait loci (eQTLs) in 13 tissues via joint analysis of SVs, single-nucleotide variants (SNVs) and short insertion/deletion (indel) variants from deep whole-genome sequencing (WGS). We estimated that SVs are causal at 3.5-6.8% of eQTLs-a substantially higher fraction than prior estimates-and that expression-altering SVs have larger effect sizes than do SNVs and indels. We identified 789 putative causal SVs predicted to directly alter gene expression: most (88.3%) were noncoding variants enriched at enhancers and other regulatory elements, and 52 were linked to genome-wide association study loci. We observed a notable abundance of rare high-impact SVs associated with aberrant expression of nearby genes. These results suggest that comprehensive WGS-based SV analyses will increase the power of common- and rare-variant association studies.
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http://dx.doi.org/10.1038/ng.3834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406250PMC
May 2017

Estimating error models for whole genome sequencing using mixtures of Dirichlet-multinomial distributions.

Bioinformatics 2017 Aug;33(15):2322-2329

The Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA.

Motivation: Accurate identification of genotypes is an essential part of the analysis of genomic data, including in identification of sequence polymorphisms, linking mutations with disease and determining mutation rates. Biological and technical processes that adversely affect genotyping include copy-number-variation, paralogous sequences, library preparation, sequencing error and reference-mapping biases, among others.

Results: We modeled the read depth for all data as a mixture of Dirichlet-multinomial distributions, resulting in significant improvements over previously used models. In most cases the best model was comprised of two distributions. The major-component distribution is similar to a binomial distribution with low error and low reference bias. The minor-component distribution is overdispersed with higher error and reference bias. We also found that sites fitting the minor component are enriched for copy number variants and low complexity regions, which can produce erroneous genotype calls. By removing sites that do not fit the major component, we can improve the accuracy of genotype calls.

Availability And Implementation: Methods and data files are available at https://github.com/CartwrightLab/WuEtAl2017/ (doi:10.5281/zenodo.256858).

Contact: [email protected]

Supplementary Information: Supplementary data is available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btx133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860108PMC
August 2017

Multiplex shRNA Screening of Germ Cell Development by in Vivo Transfection of Mouse Testis.

G3 (Bethesda) 2017 01 5;7(1):247-255. Epub 2017 Jan 5.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

Spermatozoa are one of the few mammalian cell types that cannot be fully derived in vitro, severely limiting the application of modern genomic techniques to study germ cell biology. The current gold standard approach of characterizing single-gene knockout mice is slow as generation of each mutant line can take 6-9 months. Here, we describe an in vivo approach to rapid functional screening of germline genes based on a new nonsurgical, nonviral in vivo transfection method to deliver nucleic acids into testicular germ cells. By coupling multiplex transfection of short hairpin RNA (shRNA) constructs with pooled amplicon sequencing as a readout, we were able to screen many genes for spermatogenesis function in a quick and inexpensive experiment. We transfected nine mouse testes with a pilot pool of RNA interference (RNAi) against well-characterized genes to show that this system is highly reproducible and accurate. With a false negative rate of 18% and a false positive rate of 12%, this method has similar performance as other RNAi screens in the well-described Drosophila model system. In a separate experiment, we screened 26 uncharacterized genes computationally predicted to be essential for spermatogenesis and found numerous candidates for follow-up studies. Finally, as a control experiment, we performed a long-term selection screen in neuronal N2a cells, sampling shRNA frequencies at five sequential time points. By characterizing the effect of both libraries on N2a cells, we show that our screening results from testis are tissue-specific. Our calculations indicate that the current implementation of this approach could be used to screen thousands of protein-coding genes simultaneously in a single mouse testis. The experimental protocols and analysis scripts provided will enable other groups to use this procedure to study diverse aspects of germ cell biology ranging from epigenetics to cell physiology. This approach also has great promise as an applied tool for validating diagnoses made from medical genome sequencing, or designing synthetic biological sequences that can act as potent and highly specific male contraceptives.
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http://dx.doi.org/10.1534/g3.116.036087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217113PMC
January 2017

Genome-wide significance testing of variation from single case exomes.

Nat Genet 2016 12 24;48(12):1455-1461. Epub 2016 Oct 24.

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA.

Standard techniques from genetic epidemiology are ill-suited to formally assess the significance of variants identified from a single case. We developed a statistical inference framework for identifying unusual functional variation from a single exome or genome, what we refer to as the 'n-of-one' problem. Using this approach we assessed our ability to identify the causal genotypes in over 5 million simulated cases of Mendelian disease, identifying 39% of disease genotypes as the most damaging unit in a typical exome background. We applied our approach to 129 n-of-one families from the Undiagnosed Diseases Program, nominating 60% of 30 disease genes determined to be diagnostic by a standard clinical workup. Our method can currently produce well-calibrated P values when applied to single genomes, can facilitate integration of multiple data types for n-of-one analyses, and, with further work, could become a widely used epidemiological method like linkage analysis or genome-wide association analysis.
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http://dx.doi.org/10.1038/ng.3697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127779PMC
December 2016

Multispecies Purification of Testicular Germ Cells.

Biol Reprod 2016 10 24;95(4):85. Epub 2016 Aug 24.

Advanced methods of cellular purification are required to apply genome technology to the study of spermatogenesis. One approach, based on flow cytometry of murine testicular cells stained with Hoechst-33342 (Ho-FACS), has been extensively optimized and currently allows the isolation of 9 germ cell types. This staining technique is straightforward to implement, highly effective at purifying specific germ cell types and yields sufficient cell numbers for high throughput studies. Ho-FACS is a technique that does not require species-specific markers, but whose applicability to other species is largely unexplored. We hypothesized that, due to the similar cell physiology of spermatogenesis across mammals, Ho-FACS could be used to produce highly purified subpopulations of germ cells in mammals other than mouse. To test this hypothesis, we applied Ho-FACS to 4 mammalian species that are widely used in testis research - Rattus norvegicus, Cavia porcellus, Canis familiaris and Sus scrofa domesticus We successfully isolated 4 germ cell populations from these species with average purity of 79% for spermatocytes, and 90% for spermatids and 66% for spermatogonia. Additionally, we compare the performance of mechanical and chemical dissociation for each species, and propose an optimized gating strategy to better discriminate round and elongating spermatids in the mouse, which can potentially be applied to other species. Our work indicates that spermatogenesis may be uniquely accessible among mammalian developmental systems, as a single set of reagents may be sufficient to isolate germ cell populations from many different mammalian species, opening new avenues in the fields of development and male reproductive biology.
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http://dx.doi.org/10.1095/biolreprod.116.140566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5176363PMC
October 2016

Defining the Phenotype and Assessing Severity in Phosphoglucomutase-1 Deficiency.

J Pediatr 2016 08 17;175:130-136.e8. Epub 2016 May 17.

Hayward Genetics Center, Tulane University School of Medicine, New Orleans, LA; Department of Pediatrics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.

Objective: To define phenotypic groups and identify predictors of disease severity in patients with phosphoglucomutase-1 deficiency (PGM1-CDG).

Study Design: We evaluated 27 patients with PGM1-CDG who were divided into 3 phenotypic groups, and group assignment was validated by a scoring system, the Tulane PGM1-CDG Rating Scale (TPCRS). This scale evaluates measurable clinical features of PGM1-CDG. We examined the relationship between genotype, enzyme activity, and TPCRS score by using regression analysis. Associations between the most common clinical features and disease severity were evaluated by principal component analysis.

Results: We found a statistically significant stratification of the TPCRS scores among the phenotypic groups (P < .001). Regression analysis showed that there is no significant correlation between genotype, enzyme activity, and TPCRS score. Principal component analysis identified 5 variables that contributed to 54% variance in the cohort and are predictive of disease severity: congenital malformation, cardiac involvement, endocrine deficiency, myopathy, and growth.

Conclusions: We established a scoring algorithm to reliably evaluate disease severity in patients with PGM1-CDG on the basis of their clinical history and presentation. We also identified 5 clinical features that are predictors of disease severity; 2 of these features can be evaluated by physical examination, without the need for specific diagnostic testing and thus allow for rapid assessment and initiation of therapy.
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http://dx.doi.org/10.1016/j.jpeds.2016.04.021DOI Listing
August 2016

A Screen for Genomic Disorders of Infertility Identifies MAST2 Duplications Associated with Nonobstructive Azoospermia in Humans.

Biol Reprod 2015 Sep 22;93(3):61. Epub 2015 Jul 22.

State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China

Since the cytogenetic identification of azoospermia factor regions 40 years ago, the Y chromosome has dominated research on the genetics of male infertility. We hypothesized that hotspots of structural rearrangement, which are dispersed across the genome, may mediate rare, recurrent copy number variations (CNVs), leading to severe infertility. We tested this hypothesis by contrasting patterns of rare CNVs in 970 Han Chinese men with idiopathic nonobstructive azoospermia and 1661 ethnicity-matched controls. Our results strongly support our previous claim that sperm production is modulated by genetic variation across the entire genome. The X chromosome in particular was enriched for loci modulating spermatogenesis--rare X-linked deletions larger than 100 kb were twice as common in patients compared with controls (odds ratio [OR] = 2.05, P = 0.01). At rearrangement hotspots across the genome, we observed a 2.4-fold enrichment of singleton CNVs in patients (P < 0.02), and we identified 117 testis genes, such as SYCE1, contained within 47 hotspots that may plausibly mediate genomic disorders of fertility. In our discovery sample we observed 3 case-specific duplications of the autosomal gene MAST2, and in a replication phase we found another 11 duplications in 1457 patients and 1 duplication in 1590 controls (P < 5 × 10(-5), combined data). With a large, polygenic genetic basis, new ways of establishing the pathogenicity of rare, large-effect mutations will be needed to fully reap the benefit of genome data in the management of azoospermia.
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http://dx.doi.org/10.1095/biolreprod.115.131185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710186PMC
September 2015

Low-frequency germline variants across 6p22.2-6p21.33 are associated with non-obstructive azoospermia in Han Chinese men.

Hum Mol Genet 2015 Oct 21;24(19):5628-36. Epub 2015 Jul 21.

Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center For Cancer Personalized Medicine, School of Public Health and.

Genome-wide association studies (GWAS) have identified several common loci contributing to non-obstructive azoospermia (NOA). However, a substantial fraction of NOA heritability remains undefined, especially those low-frequency [defined here as having a minor allele frequency (MAF) between 0.5 and 5%] and rare (MAF below 0.5%) variants. Here, we performed a 3-stage exome-wide association study in Han Chinese men to evaluate the role of low-frequency or rare germline variants in NOA development. The discovery stage included 962 NOA cases and 1348 healthy male controls genotyped by exome chips and was followed by a 2-stage replication with an additional 2168 cases and 5248 controls. We identified three low-frequency variants located at 6p22.2 (rs2298090 in HIST1H1E encoding p.Lys152Arg: OR = 0.30, P = 2.40 × 10(-16)) and 6p21.33 (rs200847762 in FKBPL encoding p.Pro137Leu: OR = 0.11, P = 3.77 × 10(-16); rs11754464 in MSH5: OR = 1.78, P = 3.71 × 10(-7)) associated with NOA risk after Bonferroni correction. In summary, we report an instance of newly identified signals for NOA risk in genes previously undetected through GWAS on 6p22.2-6p21.33 in a Chinese population and highlight the role of low-frequency variants with a large effect in the process of spermatogenesis.
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http://dx.doi.org/10.1093/hmg/ddv257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902876PMC
October 2015

Using whole-genome sequences of the LG/J and SM/J inbred mouse strains to prioritize quantitative trait genes and nucleotides.

BMC Genomics 2015 May 28;16:415. Epub 2015 May 28.

Department of Genetics, Washington University School of Medicine, Campus Box 8108, 660 S Euclid Ave, St Louis, MO, 63110, USA.

Background: The laboratory mouse is the most commonly used model for studying variation in complex traits relevant to human disease. Here we present the whole-genome sequences of two inbred strains, LG/J and SM/J, which are frequently used to study variation in complex traits as diverse as aging, bone-growth, adiposity, maternal behavior, and methamphetamine sensitivity.

Results: We identified small nucleotide variants (SNVs) and structural variants (SVs) in the LG/J and SM/J strains relative to the reference genome and discovered novel variants in these two strains by comparing their sequences to other mouse genomes. We find that 39% of the LG/J and SM/J genomes are identical-by-descent (IBD). We characterized amino-acid changing mutations using three algorithms: LRT, PolyPhen-2 and SIFT. We also identified polymorphisms between LG/J and SM/J that fall in regulatory regions and highly informative transcription factor binding sites (TFBS). We intersected these functional predictions with quantitative trait loci (QTL) mapped in advanced intercrosses of these two strains. We find that QTL are both over-represented in non-IBD regions and highly enriched for variants predicted to have a functional impact. Variants in QTL associated with metabolic (231 QTL identified in an F16 generation) and developmental (41 QTL identified in an F34 generation) traits were interrogated and we highlight candidate quantitative trait genes (QTG) and nucleotides (QTN) in a QTL on chr13 associated with variation in basal glucose levels and in a QTL on chr6 associated with variation in tibia length.

Conclusions: We show how integrating genomic sequence with QTL reduces the QTL search space and helps researchers prioritize candidate genes and nucleotides for experimental follow-up. Additionally, given the LG/J and SM/J phylogenetic context among inbred strains, these data contribute important information to the genomic landscape of the laboratory mouse.
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http://dx.doi.org/10.1186/s12864-015-1592-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445795PMC
May 2015

Human genomics. Effect of predicted protein-truncating genetic variants on the human transcriptome.

Science 2015 May;348(6235):666-9

Broad Institute of MIT and Harvard, Cambridge, MA, USA. Analytical and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA.

Accurate prediction of the functional effect of genetic variation is critical for clinical genome interpretation. We systematically characterized the transcriptome effects of protein-truncating variants, a class of variants expected to have profound effects on gene function, using data from the Genotype-Tissue Expression (GTEx) and Geuvadis projects. We quantitated tissue-specific and positional effects on nonsense-mediated transcript decay and present an improved predictive model for this decay. We directly measured the effect of variants both proximal and distal to splice junctions. Furthermore, we found that robustness to heterozygous gene inactivation is not due to dosage compensation. Our results illustrate the value of transcriptome data in the functional interpretation of genetic variants.
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http://dx.doi.org/10.1126/science.1261877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537935PMC
May 2015
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