Selected publications

@article{nokey,

title = {Genome sequencing of Pakistani families with male infertility identifies deleterious genotypes in SPAG6, CCDC9, TKTL1, TUBA3C, and M1AP},

author = {MR Khan and A Akbari and TJ Nicholas and H Castillo-Madeen and M Ajmal and TUI Haq and M Laan and and AR Quinlan and JS Ahuja and AA Shah and DF Conrad},

url = {https://onlinelibrary.wiley.com/doi/10.1111/andr.13570},

doi = {10.1111/andr.13570},

year  = {2023},

date = {2023-12-10},

journal = {Andrology},

abstract = {Background: There are likely to be hundreds of monogenic forms of human male infertility. Whole genome sequencing (WGS) is the most efficient way to make progress in mapping the causative genetic variants, and ultimately improve clinical management of the disease in each patient. Recruitment of consanguineous families is an effective approach to ascertain the genetic forms of many diseases.

Objectives: To apply WGS to large consanguineous families with likely hereditary male infertility and identify potential genetic cases.

Materials and methods: We recruited seven large families with clinically diagnosed male infertility from rural Pakistan, including five with a history of consanguinity. We generated WGS data on 26 individuals (3-5 per family) and analyzed the resulting data with a computational pipeline to identify potentially causal single nucleotide variants, indels, and copy number variants.

Results: We identified plausible genetic causes in five of the seven families, including a homozygous 10 kb deletion of exon 2 in a well-established male infertility gene (M1AP), and biallelic missense substitutions (SPAG6, CCDC9, TUBA3C) and an in-frame hemizygous deletion (TKTL1) in genes with emerging relevance.

Discussion and conclusion: The rate of genetic findings using the current approach (71%) was much higher than what we recently achieved using whole-exome sequencing (WES) of unrelated singleton cases (20%). Furthermore, we identified a pathogenic single-exon deletion in M1AP that would be undetectable by WES. Screening more families with WGS, especially in underrepresented populations, will further reveal the types of variants underlying male infertility and accelerate the use of genetics in the patient management.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {A naturally occurring variant of MBD4 causes maternal germline hypermutation in primates},

author = {AM Stendahl and R Sanghvi and S Peterson and K Ray and AC Lima and and R Rahbari and DF Conrad},

url = {https://genome.cshlp.org/content/early/2023/12/08/gr.277977.123.long},

doi = {10.1101/gr.277977.123},

year  = {2023},

date = {2023-12-08},

journal = {Genome Research},

abstract = {As part of an ongoing genome sequencing project at the Oregon National Primate Research Center, we identified a rhesus macaque with a rare homozygous frameshift mutation in the gene methyl-CpG binding domain 4, DNA glycosylase (MBD4). MBD4 is responsible for the repair of C > T deamination mutations at CpG dinucleotides and has been linked to somatic hypermutation and cancer predisposition in humans. We show here that MBD4-associated hypermutation also affects the germline: The six offspring of the MBD4-null dam have a fourfold to sixfold increase in de novo mutation burden. This excess burden was predominantly C > T mutations at CpG dinucleotides consistent with MBD4 loss of function in the dam. There was also a significant excess of C > T at CpA sites, indicating an important, unappreciated role for MBD4 to repair deamination in CpA contexts. The MBD4-null dam developed sustained eosinophilia later in life, but we saw no other signs of neoplastic processes associated with MBD4 loss of function in humans nor any obvious disease in the hypermutated offspring. This work provides the first evidence for a genetic factor causing hypermutation in the maternal germline of a mammal and adds to the very small list of naturally occurring variants known to modulate germline mutation rates in mammals.},

keywords = {primate genetics},

pubstate = {published},

tppubtype = {article}

}

@article{Lewinsohn2023,

title = {Consensus label propagation with graph convolutional networks for single-cell RNA sequencing cell type annotation},

author = {DP Lewinsohn and KA Vigh-Conrad and DF Conrad and CB Scott},

url = {https://doi.org/10.1093/bioinformatics/btad360},

doi = {https://doi.org/10.1093/bioinformatics/btad360},

year  = {2023},

date = {2023-06-02},

journal = {Bioinformatics},

volume = {39},

issue = {6},

abstract = {Motivation

Single-cell RNA sequencing (scRNA-seq) data, annotated by cell type, is useful in a variety of downstream biological applications, such as profiling gene expression at the single-cell level. However, manually assigning these annotations with known marker genes is both time-consuming and subjective.



Results

We present a Graph Convolutional Network (GCN)-based approach to automate the annotation process. Our process builds upon existing labeling approaches, using state-of-the-art tools to find cells with highly confident label assignments through consensus and spreading these confident labels with a semi-supervised GCN. Using simulated data and two scRNA-seq datasets from different tissues, we show that our method improves accuracy over a simple consensus algorithm and the average of the underlying tools. We also compare our method to a nonparametric neighbor majority approach, showing comparable results. We then demonstrate that our GCN method allows for feature interpretation, identifying important genes for cell type classification. We present our completed pipeline, written in PyTorch, as an end-to-end tool for automating and interpreting the classification of scRNA-seq data.



Availability and implementation

Our code for conducting the experiments in this paper and using our model is available at https://github.com/lewinsohndp/scSHARP.},

keywords = {single cell genomics},

pubstate = {published},

tppubtype = {article}

}

@article{Rockweiler2023,

title = {The origins and functional effects of postzygotic mutations throughout the human life span},

author = {NB Rockweiler and A Ramu and L Nagirnaja and WH Wong and MJ Noordam and CW Drubin and N Huang and B Miller and EZ Todres and KA Vigh-Conrad and A Zito and KS Small and KG Ardlie and BA Cohen and DF Conrad},

url = {https://www.science.org/doi/10.1126/science.abn7113},

doi = {10.1126/science.abn7113},

year  = {2023},

date = {2023-04-14},

journal = {Science},

volume = {380},

issue = {6641},

abstract = {Postzygotic mutations (PZMs) begin to accrue in the human genome immediately after fertilization, but how and when PZMs affect development and lifetime health remain unclear. To study the origins and functional consequences of PZMs, we generated a multitissue atlas of PZMs spanning 54 tissue and cell types from 948 donors. Nearly half the variation in mutation burden among tissue samples can be explained by measured technical and biological effects, and 9% can be attributed to donor-specific effects. Through phylogenetic reconstruction of PZMs, we found that their type and predicted functional impact vary during prenatal development, across tissues, and through the germ cell life cycle. Thus, methods for interpreting effects across the body and the life span are needed to fully understand the consequences of genetic variants.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}

@article{Nagirnaja2022,

title = {Diverse monogenic subforms of human spermatogenic failure},

author = {L Nagirnaja and AM Lopes and WL Charng and B Miller and R Stakaitis and T Luan and C Friedrich and E Mahyari and E Fadial and L Kasak and K Vigh-Conrad and MS Oud and MJ Xavier and SR Cheers and ER James and J Guo and and TG Jenkins and A Riera-Escamilla and A Barros and F Carvalho and S Fernandes and J Concalves and CA Gurnett and and N Jørgensen and D Jezek and ES Jungheim and S Kliesch and RI McLachlan and KR Omurtag and A Pilatz and and JI Sandlow and J Smith and ML Eisenberg and JM Hotaling and KA Jarvi and M Punab and E Rajpert-De Meyts and DT Carrell and C Krausz and M Laan and MK O'Bryan and PN Schlegel and F Tüttelmann and JA Veltman and K Almstrup and KI Aston and DF Conrad },

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9792524/pdf/41467_2022_Article_35661.pdf},

doi = {10.1038/s41467-022-35661-z},

year  = {2022},

date = {2022-12-26},

journal = {Nat. Commun.},

volume = {13},

number = {7953},

issue = {1},

abstract = {Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Yang2022,

title = {SATINN: An automated neural network-based classification of testicular sections allows for high-throughput histopathology of mouse mutants},

author = {R Yang and A Stendahl and KA Vigh-Conrad and M Held and AC Lima and DF Conrad},

doi = {https://doi.org/10.1093/bioinformatics/btac673},

year  = {2022},

date = {2022-10-10},

journal = {Bioinformatics},

abstract = {Motivation

The mammalian testis is a complex organ with a cellular composition that changes smoothly and cyclically in normal adults. While testis histology is already an invaluable tool for identifying and describing developmental differences in evolution and disease, methods for standardized, digital image analysis of testis are needed to expand the utility of this approach.



Results

We developed SATINN (Software for Analysis of Testis Images with Neural Networks), a multi-level framework for automated analysis of multiplexed immunofluorescence images from mouse testis. This approach uses residual learning to train convolutional neural networks (CNNs) to classify nuclei from seminiferous tubules into 7 distinct cell types with an accuracy of 81.7%. These cell classifications are then used in a second-level tubule CNN, which places seminiferous tubules into one of 12 distinct tubule stages with 57.3% direct accuracy and 94.9% within ±1 stage. We further describe numerous cell- and tubule-level statistics that can be derived from wildtype testis. Finally, we demonstrate how the classifiers and derived statistics can be used to rapidly and precisely describe pathology by applying our methods to image data from two mutant mouse lines. Our results demonstrate the feasibility and potential of using computer-assisted analysis for testis histology, an area poised to evolve rapidly on the back of emerging, spatially-resolved genomic and proteomic technologies.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36150389,

title = {The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans},

author = {MJ Wyrwoll and CM Gaasbeek and I Golubickaite and R Stakaitis and MS Oud and L Nagirnaja and C Dion and EB Sindi and HG Leitch and CN Jayasena and A Sironen and A Dicke and N Rotte and B Stallmeyer and S Kliesch and CHP Grangeiro and TF Araujo and P Lasko and GEMINI Consortium, K D'Hauwers and RM Smits and L Ramos and MJ Xavier and DF Conrad and K Almstrup and JA Veltman and F Tüttelmann and GW van der Heijdenand },

doi = {10.1016/j.ajhg.2022.09.002},

issn = {1537-6605},

year  = {2022},

date = {2022-10-01},

urldate = {2022-10-01},

journal = {Am J Hum Genet},

volume = {109},

number = {10},

pages = {1850-1866},

abstract = {Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35013161,

title = {A de novo paradigm for male infertility},

author = {MS Oud and RM Smits and HE Smith and FK Mastrorosa and GS Holt and BJ Houston and PF de Vries and BKS Alobaidi and LE Batty and H Ismail and J Greenwood and H Sheth and A Mikulasova and GDN Astuti and C Gilissen and K McEleny and H Turner and J Coxhead and S Cockell and DDM Braat and K Fleischer and KWM D'Hauwers and E Schaafsma and GEMINI Consortium, and L Nagirnaja and DF Conrad and C Friedrich and S Kliesch and KI Aston and A Riera-Escamilla and C Krausz and C Gonzaga-Jauregui and M Santibanez-Koref and DJ Elliott and LELM Vissers and F Tüttelmann and MK O'Bryan and L Ramos and MJ Xavier and GW van der Heijden and JA Veltman},

doi = {10.1038/s41467-021-27132-8},

issn = {2041-1723},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Nat Commun},

volume = {13},

number = {1},

pages = {154},

abstract = {De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35022746,

title = {Human INHBB Gene Variant (c.1079T>C:p.Met360Thr) Alters Testis Germ Cell Content, but Does Not Impact Fertility in Mice},

author = {BJ Houston and AE O'Connor and D Wang and G Goodchild and DJ Merriner and H Luan and DF Conrad and L Nagirnaja and KI Aston and S Kliesch and MJ Wyrwoll and C Friedrich and F Tüttelmann and C Harrison and MK O'Bryan and K Walton},

doi = {10.1210/endocr/bqab269},

issn = {1945-7170},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Endocrinology},

volume = {163},

number = {3},

abstract = {Testicular-derived inhibin B (α/β B dimers) acts in an endocrine manner to suppress pituitary production of follicle-stimulating hormone (FSH), by blocking the actions of activins (β A/B/β A/B dimers). Previously, we identified a homozygous genetic variant (c.1079T>C:p.Met360Thr) arising from uniparental disomy of chromosome 2 in the INHBB gene (β B-subunit of inhibin B and activin B) in a man suffering from infertility (azoospermia). In this study, we aimed to test the causality of the p.Met360Thr variant in INHBB and testis function. Here, we used CRISPR/Cas9 technology to generate InhbbM364T/M364T mice, where mouse INHBB p.Met364 corresponds with human p.Met360. Surprisingly, we found that the testes of male InhbbM364T/M364T mutant mice were significantly larger compared with those of aged-matched wildtype littermates at 12 and 24 weeks of age. This was attributed to a significant increase in Sertoli cell and round spermatid number and, consequently, seminiferous tubule area in InhbbM364T/M364T males compared to wildtype males. Despite this testis phenotype, male InhbbM364T/M364T mutant mice retained normal fertility. Serum hormone analyses, however, indicated that the InhbbM364T variant resulted in reduced circulating levels of activin B but did not affect FSH production. We also examined the effect of this p.Met360Thr and an additional INHBB variant (c.314C>T: p.Thr105Met) found in another infertile man on inhibin B and activin B in vitro biosynthesis. We found that both INHBB variants resulted in a significant disruption to activin B in vitro biosynthesis. Together, this analysis supports that INHBB variants that limit activin B production have consequences for testis composition in males.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35535697,

title = {Actionable secondary findings following exome sequencing of 836 non-obstructive azoospermia cases and their value in patient management},

author = {L Kasak and K Lillepea and L Nagirnaja and KI Aston and PN Schlegel and J Gonçalves and F Carvalho and D Moreno-Mendoza and K Almstrup and ML Eisenberg and KA Jarvi and MK O'Bryan and AM Lopes and DF Conrad and GEMINI Consortium and M Punab and M Laan},

doi = {10.1093/humrep/deac100},

issn = {1460-2350},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Hum Reprod},

volume = {37},

number = {7},

pages = {1652--1663},

abstract = {STUDY QUESTION: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)?



SUMMARY ANSWER: One in 28 NOA cases carried an identifiable, medically actionable SF.



WHAT IS KNOWN ALREADY: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited.



STUDY DESIGN, SIZE, DURATION: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study.



PARTICIPANTS/MATERIALS, SETTING, METHODS: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed.



MAIN RESULTS AND THE ROLE OF CHANCE: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population.



LIMITATIONS, REASONS FOR CAUTION: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants.



WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA.



STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Estonian Research Council grants IUT34-12 and PRG1021 (M.L. and M.P.); National Institutes of Health of the United States of America grant R01HD078641 (D.F.C., K.I.A. and P.N.S.); National Institutes of Health of the United States of America grant P50HD096723 (D.F.C. and P.N.S.); National Health and Medical Research Council of Australia grant APP1120356 (M.K.O'B., D.F.C. and K.I.A.); Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-007274 (A.M.L., F.C. and J.G.) and FCT: IF/01262/2014 (A.M.L.). J.G. was partially funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Centre for Toxicogenomics and Human Health-ToxOmics (grants UID/BIM/00009/2016 and UIDB/00009/2020). M.L.E. is a consultant for, and holds stock in, Roman, Sandstone, Dadi, Hannah, Underdog and has received funding from NIH/NICHD. Co-authors L.K., K.L., L.N., K.I.A., P.N.S., J.G., F.C., D.M.-M., K.A., K.A.J., M.K.O'B., A.M.L., D.F.C., M.P. and M.L. declare no conflict of interest.



TRIAL REGISTRATION NUMBER: N/A.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35679955,

title = {Zinc finger RNA binding protein 2 (ZFR2) is not required for male fertility in the mouse},

author = {LM Cauchi and BJ Houston and L Nagirnaja and AE O'Connor and DJ Merriner and KI Aston and PN Schlegel and DF Conrad and R Burke and MK O'Bryan},

doi = {10.1016/j.ydbio.2022.05.020},

issn = {1095-564X},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Dev Biol},

volume = {489},

pages = {55-61},

abstract = {BACKGROUND: Thousands of genes are expressed during spermatogenesis and male infertility has a strong genetic component. Within this study, we focus on the role of Zfr2 in male fertility, a gene previously implicated in human male fertility. To date, very little is known about the role of ZFR2 in either humans or mice. To this end, the requirement for ZFR2 in male fertility was assessed using a knockout mouse model.



RESULTS: Zfr2 was found to be expressed in the testes of both humans and mice. Deletion of Zfr2 was achieved via removal of exon 2 using CRISPR-Cas9 methods. The absence of Zfr2 did not result in a reduction in any fertility parameters assessed. Knockout males were capable of fostering litter sizes equal to wild type males, and there were no effects of Zfr2 knockout on sperm number or motility. We note Zfr2 knockout females were also fertile.



CONCLUSIONS: The absence of Zfr2 alone is not sufficient to cause a reduction in male fertility in mice.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35809576,

title = {Large-scale analyses of the X chromosome in 2,354 infertile men discover recurrently affected genes associated with spermatogenic failure},

author = {A Riera-Escamilla and M Vockel and L Nagirnaja and MJ Xavier and A Carbonell and D Moreno-Mendoza and M Pybus and G Farnetani and V Rosta and F Cioppi and C Friedrich and MS Oud and GW van der Heijden and A Soave and T Diemer and E Ars and J Sánchez-Curbelo and S Kliesch and MK O'Bryan and E Ruiz-Castañe and GEMINI Consortium and F Azorín and JA Veltman and KI Aston and DF Conrad and F Tüttelmann and C Krausz},

doi = {10.1016/j.ajhg.2022.06.007},

issn = {1537-6605},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Am J Hum Genet},

volume = {109},

number = {8},

pages = {1458-1471},

abstract = {Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. We aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, we propose 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and our functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, our study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35850260,

title = {DDB1- and CUL4-associated factor 12-like protein 1 (Dcaf12l1) is not essential for male fertility in mice},

author = {BJ Houston and AM Lopes and M Laan and L Nagirnaja and AE O'Connor and DJ Merriner and J Nguyen and M Punab and A Riera-Escamilla and C Krausz and KI Aston and DF Conrad and MK O'Bryan},

doi = {10.1016/j.ydbio.2022.07.006},

issn = {1095-564X},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Dev Biol},

volume = {490},

pages = {66-72},

abstract = {Male infertility is a common condition affecting at least 7% of men worldwide and is often genetic in origin. Using whole exome sequencing, we recently discovered three hemizygous, likely damaging variants in DDB1- and CUL4-associated factor 12-like protein 1 (DCAF12L1) in men with azoospermia. DCAF12L1 is located on the X-chromosome and as identified by single cell sequencing studies, its expression is enriched in human testes and specifically in Sertoli cells and spermatogonia. However, very little is known about the role of DCAF12L1 in spermatogenesis, thus we generated a knockout mouse model to further explore the role of DCAF12L1 in male fertility. Knockout mice were generated using CRISPR/Cas9 technology to remove the entire coding region of Dcaf12l1 and were assessed for fertility over a broad range of ages (2-8 months of age). Despite outstanding genetic evidence in men, loss of DCAF12L1 had no discernible impact on male fertility in mice, as highlighted by breeding trials, histological assessment of the testis and epididymis, daily sperm production and evaluation of sperm motility using computer assisted methods. This disparity is likely due to the parallel evolution, and subsequent divergence, of DCAF12 family members in mice and men or the presence of compounding environmental factors in men.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Mahyari2021,

title = {Comparative single-cell analysis of biopsies clarifies pathogenic mechanisms in Klinefelter syndrome},

author = {E Mahyari and J Guo and AC Lima and DP Lewinsohn and AM Stendahl and KA Vigh-Conrad and X Nie and L Nagirnaja and NB Rockweiler and DT Carrell and JM Hotaling and KI Aston and DF Conrad},

url = {https://www.cell.com/ajhg/fulltext/S0002-9297(21)00337-2},

doi = {10.1016/j.ajhg.2021.09.001},

year  = {2021},

date = {2021-10-07},

journal = {Am J Hum Genet},

volume = {108},

number = {10},

pages = {1924-1945},

abstract = {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.},

keywords = {human reproduction, single cell genomics},

pubstate = {published},

tppubtype = {article}

}

@article{Nagirnaja2021,

title = {Variant PNLDC1, Defective piRNA Processing, and Azoospermia},

author = {L Nagirnaja and N Mørup and JE Nielsen and R Stakaitis and I Golubickaite and MS Oud and and SB Winge and F Carvalho and and KI Aston and F Khani and GW van der Heijden and J Marques and NE Skakkebaek and E Rajpert-De Meyts and PN Schlegel and and N Jørgensen and JA Veltman and and AM Lopes and DF Conrad and K Almstrup},

url = {https://www.nejm.org/doi/10.1056/NEJMoa2028973?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub++0pubmed},

doi = {10.1056/NEJMoa2028973},

year  = {2021},

date = {2021-08-19},

journal = {N Engl J Med.},

volume = {385},

number = {8},

pages = {707-719},

abstract = {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 PNLDC1: 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 PNLDC1 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.).},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Hardy2021,

title = {Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure},

author = {JJ Hardy and MJ Wyrwoll and W Mcfadden and A Malcher and N Rotte and NC Pollock and S Munyoki and MV Veroli and BJ Houston and MJ Xavier and L Kasak and M Punab and M Laan and S Kliesch and P Schlegel and T Jaffe and K Hwang and J Vukina and MA Brieño-Enríquez and K Orwig and J Yanowitz and M Buszczak and JA Veltman and M Oud and L Nagirnaja and M Olszewska and MK O'Bryan and DF Conrad and M Kurpisz and F Tüttelmann and AN Yatsenko and GEMINI Consortium},

url = {https://link.springer.com/article/10.1007%2Fs00439-021-02287-y},

doi = {10.1007/s00439-021-02287-y},

year  = {2021},

date = {2021-05-07},

journal = {Hum Genet. },

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Oud2021,

title = {Lack of evidence for a role of PIWIL1 variants in human male infertility},

author = {MS Oud and L Volozonoka and C Friedrich and S Kliesch and L Nagirnaja and C Gilissen and MK O'Bryan and RI McLachlan and KI Aston and F Tüttelmann and DF Conrad and JA Veltman

},

url = {https://www.cell.com/cell/fulltext/S0092-8674(21)00286-5},

doi = {10.1016/j.cell.2021.03.001},

year  = {2021},

date = {2021-04-15},

journal = {Cell},

volume = {184},

number = {8},

pages = {1941-1942},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Houston2021,

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

author = {BJ Houston and L Nagirnaja and DR Merriner DJ and AE O'Connor and H Okuda and K Omurtag and C Smith and KI Aston and DF Conrad and MK O'Bryan},

url = {https://anatomypubs.onlinelibrary.wiley.com/doi/10.1002/dvdy.299},

doi = {10.1002/dvdy.299},

year  = {2021},

date = {2021-01-13},

journal = {Dev Dyn. },

abstract = {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.



Keywords: sertoli cell; sex development; spermatogenesis.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Laisk2020,

title = {The genetic architecture of sporadic and multiple consecutive miscarriage.},

author = {T Laisk and ALG Soares and T Ferreira and JN Painter and JC Censin and S Laber and J Bacelis and CY Chen and M Lepamets and K Lin and S Liu and IY Millwood and A Ramu and J Southcombe and MS Andersen and L Yang and CM Becker and AD Børglum and SD Gordon and J Bybjerg-Grauholm and Ø Helgeland and DM Hougaard and S Jin and S Johansson and J Juodakis and C Kartsonaki and V Kukushkina and PA Lind and A Metspalu and GW Montgomery and AP Morris and O Mors and PB Mortensen and PR Njølstad and M Nordentoft and DR Nyholt and M Lippincott and S Seminara and A Salumets and H Snieder and K Zondervan and T Werge and A Chen and DR Conrad and B Jacobsson and L Li and NG Martin and BM Neale and R Nielsen and RG Walters and I Granne and SE Medland and R Mägi and DA Lawlor and CM Lindgren },

url = {https://www.nature.com/articles/s41467-020-19742-5},

doi = {10.1038/s41467-020-19742-5},

year  = {2020},

date = {2020-11-25},

journal = {Nat Commun. },

volume = {11},

number = {1},

abstract = {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 × 10-8, 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 × 10-8, OR = 1.7; rs143445068, MAF = 0.8%, P = 5.2 × 10-9, OR = 3.4; rs183453668, MAF = 0.5%, P = 2.8 × 10-8, 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.},

keywords = {genomic variation, human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Salas-Huetos2020,

title = {Disruption of human meiotic telomere complex genes TERB1, TERB2 and MAJIN in men with non-obstructive azoospermia},

author = {A Salas-Huetos and F Tüttelmann and MJ Wyrwoll and S Kliesch and AM Lopes and J Concalves and SE Boyden and M Woste and JM Hotaling and GEMINI Consortium and L Nagirnaja and DF Conrad and DT Carrell and KI Aston},

url = {https://link.springer.com/article/10.1007/s00439-020-02236-1},

doi = {10.1007/s00439-020-02236-1},

year  = {2020},

date = {2020-11-19},

journal = {Hum Genet},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Bustamante-Marin2020,

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

author = {XM Bustamante-Marin and A Horani and M Stoyanova and W Charng and M Bottier and PR Sears and W Yin and L Daniels and H Bowen and DF Conrad and MR Knowles and LE Ostrowski and MA Zariwala and SK Dutcher},

url = {https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008691},

doi = {10.1371/journal.pgen.1008691},

year  = {2020},

date = {2020-08-07},

journal = {PLoS Genet},

volume = {16},

number = {8},

abstract = {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.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}

@article{Wyrwoll2020,

title = {Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility},

author = {MJ Wyrwoll and ŞG Temel and L Nagirnaja and MS Oud and AM Lopes and GW van der Heijden and JS Heald and N Rotte and J Wistuba and M Wöste and S Ledig and H Krenz and RM Smits and F Carvalho and J Gonçalves and D Fietz and B Türkgenç and MC Ergören and M Çetinkaya and M Başar and S Kahraman and K McEleny and MJ Xavier and H Turner and A Pilatz and Al Röpke and M Dugas and S Kliesch and N Neuhaus and GEMINI Consortium and KI Aston and DF Conrad and JA Veltman and C Friedrich and F Tüttelmann },

url = {https://linkinghub.elsevier.com/retrieve/pii/S0002-9297(20)30198-1},

doi = {10.1016/j.ajhg.2020.06.010},

year  = {2020},

date = {2020-08-06},

journal = {Am J Hum Genet},

volume = {107},

number = {2},

pages = {342-351},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Krausz2020,

title = {Genetic dissection of spermatogenic arrest through exome analysis: clinical implications for the management of azoospermic men},

author = {C Krausz and A Riera-Escamilla and D Moreno-Mendoza and K Holleman and F Cioppi and F Algaba and M Pybus and C Friedrich and MJ Wyrwoll and E Casamonti and S Pietroforte and L Nagirnaja and AM Lopes and S Kliesch and A Pilatz and DT Carrell and DF Conrad and E Ars and E Ruiz-Castañé and KI Aston and WM Baarends and F Tüttelmann},

url = {https://www.nature.com/articles/s41436-020-0907-1},

doi = {doi: 10.1038/s41436-020-0907-1},

year  = {2020},

date = {2020-08-03},

journal = {Genet Med},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Houston2020,

title = {A framework for high-resolution phenotyping of candidate male infertility mutants: from human to mouse},

author = {BJ Houston and DF Conrad and MK O'Bryan},

url = {https://link.springer.com/article/10.1007/s00439-020-02159-x},

doi = {10.1007/s00439-020-02159-x},

year  = {2020},

date = {2020-04-04},

journal = {Hum Genet},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Bustamante-Marin2019,

title = {Identification of genetic variants in CFAP221 as a cause of primary ciliary dyskinesia},

author = {XM Bustamante-Marin and A Shapiro and PR Sears and W Charng and DF Conrad and MW Leigh and MR Knowles and LE Ostrowski and MA Zariwala},

doi = {10.1038/s10038-019-0686-1},

year  = {2019},

date = {2019-10-21},

journal = {Journal of Human Genetics},

abstract = {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.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}

@article{Lee2019,

title = {Rare mutations in the complement regulatory gene CSMD1 are associated with male and female infertility},

author = {AS Lee and J Rusch and AC Lima and AR Usmani and N Huang and M Lepamets and K Vigh-Conrad and RE Worthington and R Magi and X Wu and KI Aston and and JP Atkinson and DT Carrell and and RA Hess and MK O'Bryan and DF Conrad},

url = {https://www.nature.com/articles/s41467-019-12522-w},

doi = {10.1038/s41467-019-12522-w},

year  = {2019},

date = {2019-10-11},

journal = {Nature Communications},

volume = {10},

number = {1},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Jung2019,

title = {Unified single-cell analysis of testis gene regulation and pathology in 5 mouse strains},

author = {M Jung and D Wells and J Rusch and S Ahmad and J Marchini and SR Myers and DF Conrad},

doi = {10.7554/eLife.43966},

year  = {2019},

date = {2019-06-25},

journal = {eLife},

volume = {8},

abstract = {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 analyse a novel 57,600-cell dataset from the testes of wild-type mice and mice with gonadal defects due to disruption of the genes Mlh3, Hormad1, Cul4a or Cnp. By jointly analysing mutant and wild-type cells we decomposed our data into 46 components that identify novel meiotic gene-regulatory programmes, 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 Mlh3-/- and Hormad1-/- mice, an area typically associated with immune privilege.},

keywords = {human reproduction, single cell genomics},

pubstate = {published},

tppubtype = {article}

}

@article{Kasak2018,

title = {Bi-allelic Recessive Loss-of-Function Variants in FANCM Cause Non-obstructive Azoospermia},

author = {L Kasak and M Punab and L Nagirnaja and M Grigorova and A Minajeva and AM Lopes and AM Punab and KI Aston and F Carvalho and E Laasik and LB Smith and GEMINI Consortium and DF Conrad and M Laan},

url = {https://www.sciencedirect.com/science/article/pii/S0002929718302325?via%3Dihub},

year  = {2018},

date = {2018-08-02},

journal = {American Journal of Human Genetics},

volume = {103},

number = {2},

pages = {200-212},

abstract = {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.Gln498Thrfs∗7 (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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Nagirnaja2018,

title = {Genetic intersection of male infertility and cancer},

author = {L Nagirnaja and KI Aston and DF Conrad},

doi = {10.1016/j.fertnstert.2017.10.028},

year  = {2018},

date = {2018-01-01},

journal = {Fertility and Sterility},

volume = {109},

number = {1},

pages = {20-26},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Wu2017,

title = {Estimating error models for whole genome sequencing using mixtures of Dirichlet-multinomial distributions},

author = {SH Wu and RS Schwartz and DJ Winter and DF Conrad and RA Cartwright},

doi = {10.1093/bioinformatics/btx133},

year  = {2017},

date = {2017-08-01},

journal = {Bioinformatics},

volume = {33},

number = {15},

pages = {2322-2329},

abstract = {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.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}

@article{Lima2017c,

title = {The long and short of translational control in male germ cells},

author = {AC Lima and DF Conrad},

doi = {10.1093/biolre/iox075},

year  = {2017},

date = {2017-07-01},

journal = {Biology of Reproduction},

volume = {97},

number = {1},

pages = {2-4},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Lima2017b,

title = {A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry},

author = {AC Lima and M Jung and J Rusch and AR Usmani and AM Lopes and DF Conrad},

doi = {10.3791/55913},

year  = {2017},

date = {2017-07-01},

journal = {Journal of Visualized Experiments},

volume = {12},

number = {125},

abstract = {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.},

keywords = {human reproduction, single cell genomics},

pubstate = {published},

tppubtype = {article}

}

@article{Nagirnaja2017,

title = {How to Map the Genetic Basis for Conditions that are Comorbid with Male Infertility},

author = {L Nagirnaja and K Vigh-Conrad and DF Conrad},

doi = {10.1055/s-0037-1603567},

year  = {2017},

date = {2017-05-01},

journal = {Seminars in Reproductive Medicine},

volume = {35},

number = {3},

pages = {225-230},

keywords = {genomic variation, human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Chiang2017,

title = {The impact of structural variation on human gene expression},

author = {C Chiang and AJ Scott and JR Davis and EK Tsang and X Li and Y Kim and T Hadzic and FN Damani and L Ganel and GTEx Consortium and SB Montgomery and A Battle and DF Conrad and IM Hall},

doi = {10.1038/ng.3834},

year  = {2017},

date = {2017-05-01},

journal = {Nature Genetics},

volume = {49},

number = {5},

pages = {692-699},

abstract = {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.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}

@article{Ho2017,

title = {Multiplex shRNA Screening of Germ Cell Development by in Vivo Transfection of Mouse Testis},

author = {NR Ho and AR Usmani and Y Yin and L Ma and DF Conrad},

doi = {10.1534/g3.116.036087},

year  = {2017},

date = {2017-01-07},

journal = {G3 (Bethesda)},

volume = {7},

number = {1},

pages = {247-255},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Wilfert2016,

title = {Genome-wide significance testing of variation from single case exomes},

author = {AB Wilfert and KR Chao and M Kaushal and S Jain and S Zollner and R Adams and DF Conrad},

doi = {10.1038/ng.3697},

year  = {2016},

date = {2016-12-01},

journal = {Nature Genetics},

volume = {48},

number = {12},

pages = {1455-1461},

abstract = {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.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}

@article{Lima2017,

title = {Multispecies Purification of Testicular Germ Cells},

author = {AC Lima and M Jung and J Rusch and AR Usmani and AM Lopes and DF Conrad},

doi = {10.1095/biolreprod.116.140566},

year  = {2016},

date = {2016-10-01},

journal = {Biology of Reproduction},

volume = {95},

number = {4},

pages = {85},

abstract = {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.},

keywords = {human reproduction, single cell genomics},

pubstate = {published},

tppubtype = {article}

}

@article{Ho2015,

title = {Improved detection of disease-associated variation by sex-specific characterization and prediction of genes required for fertility},

author = {NR Ho and N Huang and DF Conrad },

doi = {10.1111/andr.12109},

year  = {2015},

date = {2015-11-01},

journal = {Andrology},

volume = {3},

number = {6},

pages = {1140-9},

abstract = {Despite its great potential, high-throughput functional genomic data are rarely integrated and applied to characterizing the genomic basis of fertility. We obtained and reprocessed over 30 functional genomics datasets from human and mouse germ cells to perform genome-wide prediction of genes underlying various reproductive phenotypes in both species. Genes involved in male fertility are easier to predict than their female analogs. Of the multiple genomic data types examined, protein-protein interactions are by far the most informative for gene prediction, followed by gene expression, and then epigenetic marks. As an application of our predictions, we show that copy number variants (CNVs) disrupting predicted fertility genes are more strongly associated with gonadal dysfunction in male and female case-control cohorts when compared to all gene-disrupting CNVs (OR = 1.64, p < 1.64 × 10(-8) vs. OR = 1.25, p < 4 × 10(-6)). Using gender-specific fertility gene annotations further increased the observed associations (OR = 2.31, p < 2.2 × 10(-16)). We provide our gene predictions as a resource with this article.},

keywords = {genomic variation, human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Ni2015,

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

author = {B Ni and Y Lin and L Sun and M Zhu and Z Li and J Wang and J Yu and X Guo and X Zuo and J Dong and Y Xia and Y Wen and H Wu and H Li and Y Zhu and P Ping and X Chen and J Dai and Y Jiang and P Xu and Q Du and B Yao and N Weng and H Lu and Z Wang and X Zhu and A Yang and C Xiong and H Ma and G Jin and J Xu and X Wang and Z Zhou and J Liu and X Zhang and DF Conrad and Z Hu and J Sha},

doi = {10.1093/hmg/ddv257},

year  = {2015},

date = {2015-10-01},

journal = {Human Molecular Genetics},

volume = {24},

number = {19},

pages = {5628-36},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Huang2015,

title = {A Screen for Genomic Disorders of Infertility Identifies MAST2 Duplications Associated with Nonobstructive Azoospermia in Humans},

author = {N Huang and Y Wen and X Guo and Z Li and J Dai and B Ni and J Yu and Y Lin and W Zhou and B Yao and Y Jiang and J Sha and DF Conrad and Z Hu},

doi = {10.1095/biolreprod.115.131185},

year  = {2015},

date = {2015-09-01},

journal = {Biology of Reproduction},

volume = {93},

number = {3},

pages = {61},

abstract = {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.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Lima2015,

title = {Rare double sex and mab-3-related transcription factor 1 regulatory variants in severe spermatogenic failure},

author = {AC Lima and F Carvalho and J Gonçalves and S Fernandes and PI Marques and M Sousa and A Barros and S Seixas and A Amorim and DF Conrad and AM Lopes},

doi = {10.1111/andr.12063},

year  = {2015},

date = {2015-09-01},

journal = {Andrology},

volume = {3},

number = {5},

pages = {825-33},

abstract = {The double sex and mab-3-related transcription factor 1 (DMRT1) gene has long been linked to sex-determining pathways across vertebrates and is known to play an essential role in gonadal development and maintenance of spermatogenesis in mice. In humans, the genomic region harboring the DMRT gene cluster has been implicated in disorders of sex development and recently DMRT1 deletions were shown to be associated with non-obstructive azoospermia (NOA). In this work, we have employed different methods to screen a cohort of Portuguese NOA patients for DMRT1 exonic insertions and deletions [by multiplex ligation probe assay (MLPA); n = 68] and point mutations (by Sanger sequencing; n = 155). We have found three novel patient-specific non-coding variants in heterozygosity that were absent from 357 geographically matched controls. One of these is a complex variant with a putative regulatory role (c.-223_-219CGAAA>T), located in the promoter region within a conserved sequence involved in Dmrt1 repression. Moreover, while DMRT1 domains are highly conserved across vertebrates and show reduced levels of diversity in human populations, two rare synonymous substitutions (rs376518776 and rs34946058) and two rare non-coding variants that potentially affect DMRT1 expression and splicing (rs144122237 and rs200423545) were overrepresented in patients when compared with 376 Portuguese controls (301 fertile and 75 normozoospermic). Overall our previous and present results suggest a role of changes in DMRT1 dosage in NOA potentially also through a process of gene misregulation, even though DMRT1 deleterious variants seem to be rare.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Jain2014,

title = {Validating single-cell genomics for the study of renal development},

author = {S Jain and MJ Noordam and M Hoshi and FL Vallania and DF Conrad},

doi = {10.1038/ki.2014.104},

year  = {2014},

date = {2014-11-01},

journal = {Kidney International},

volume = {86},

number = {5},

pages = {1049-55},

abstract = {Single-cell genomics will enable studies of the earliest events in kidney development, although it is unclear if existing technologies are mature enough to generate accurate and reproducible data on kidney progenitors. Here we designed a pilot study to validate a high-throughput assay to measure the expression levels of key regulators of kidney development in single cells isolated from embryonic mice. Our experiment produced 4608 expression measurements of 22 genes, made in small cell pools, and 28 single cells purified from the RET-positive ureteric bud. There were remarkable levels of concordance with expression data generated by traditional microarray analysis on bulk ureteric bud tissue with the correlation between our average single-cell measurements and GUDMAP measurements for each gene of 0.82-0.85. Nonetheless, a major motivation for single-cell technology is to uncover dynamic biology hidden in population means. There was evidence for extensive and surprising variation in expression of Wnt11 and Etv5, both downstream targets of activated RET. The variation for all genes in the study was strongly consistent with burst-like promoter kinetics. Thus, our results can inform the design of future single-cell experiments, which are poised to provide important insights into kidney development and disease.},

keywords = {single cell genomics},

pubstate = {published},

tppubtype = {article}

}

@article{MacArthur2014,

title = {Guidelines for investigating causality of sequence variants in human disease},

author = {DG MacArthur and TA Manolio and DP Dimmock and HL Rehm and J Shendure and GR Abecasis and DR Adams and RB Altman and SE Antonarakis and EA Ashley and JC Barrett and LG Biesecker and DF Conrad and GM Cooper and NJ Cox and MJ Daly and MB Gerstein and DB Goldstein and JN Hirschhorn and SM Leal and LA Pennacchio and JA Stamatoyannopoulos and SR Sunyaev and D Valle and BF Voight and W Winckler and C Gunter},

doi = {10.1038/nature13127},

year  = {2014},

date = {2014-04-24},

journal = {Nature},

volume = {508},

number = {7497},

pages = {469-76},

abstract = {The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}

@article{Ramu2013,

title = {DeNovoGear: de novo indel and point mutation discovery and phasing},

author = {A Ramu and MJ Noordam and RS Schwartz and A Wuster and ME Hurles and RA Cartwright and DF Conrad},

doi = {10.1038/nmeth.2611},

year  = {2013},

date = {2013-10-01},

journal = {Nature Methods},

volume = {10},

number = {10},

pages = {985-7},

abstract = {We present DeNovoGear software for analyzing de novo mutations from familial and somatic tissue sequencing data. DeNovoGear uses likelihood-based error modeling to reduce the false positive rate of mutation discovery in exome analysis and fragment information to identify the parental origin of germ-line mutations. We used DeNovoGear on human whole-genome sequencing data to produce a set of predicted de novo insertion and/or deletion (indel) mutations with a 95% validation rate.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}

@article{Lopes2013,

title = {Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex chromosomes, including the gene DMRT1},

author = {AM Lopes and KI Aston and E Thompson and F Carvalho and J Gonçalves and N Huang and R Matthiesen and MJ Noordam and I Quintela and A Ramu and C Seabra and AB Wilfert and J Dai and JM Downie and S Fernandes and X Guo and J Sha and A Amorim and A Barros and A Carracedo and Z Hu and ME Hurles and S Moskovtsev and C Ober and DA Paduch and JD Schiffman and PN Schlegel and M Sousa and DT Carrell and DF Conrad},

doi = {10.1371/journal.pgen.1003349},

year  = {2013},

date = {2013-03-01},

journal = {PLoS Genetics},

volume = {9},

number = {3},

abstract = {Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man's risk of disease by 10% (OR 1.10 [1.04-1.16], p<2 × 10(-3)), rare X-linked CNVs by 29%, (OR 1.29 [1.11-1.50], p<1 × 10(-3)), and rare Y-linked duplications by 88% (OR 1.88 [1.13-3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2 × 10(-5)). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.},

keywords = {human reproduction},

pubstate = {published},

tppubtype = {article}

}

@article{Conrad2011,

title = {Variation in genome-wide mutation rates within and between human families},

author = {DF Conrad and JE Keebler and MA DePristo and SJ Lindsay and Y Zhang and F Casals and Y Idaghdour and CL Hartl and C Torroja and KV Garimella and M Zilversmit and RA Cartwright and GA Rouleau and MJ Daly and EA Stone and ME Hurles and P Awadalla and 1000 Genomes Project},

doi = {10.1038/ng.862},

year  = {2011},

date = {2011-06-12},

journal = {Nature Genetics},

volume = {43},

number = {7},

pages = {712-4},

abstract = {J.B.S. Haldane proposed in 1947 that the male germline may be more mutagenic than the female germline. Diverse studies have supported Haldane's contention of a higher average mutation rate in the male germline in a variety of mammals, including humans. Here we present, to our knowledge, the first direct comparative analysis of male and female germline mutation rates from the complete genome sequences of two parent-offspring trios. Through extensive validation, we identified 49 and 35 germline de novo mutations (DNMs) in two trio offspring, as well as 1,586 non-germline DNMs arising either somatically or in the cell lines from which the DNA was derived. Most strikingly, in one family, we observed that 92% of germline DNMs were from the paternal germline, whereas, in contrast, in the other family, 64% of DNMs were from the maternal germline. These observations suggest considerable variation in mutation rates within and between families.},

keywords = {genomic variation},

pubstate = {published},

tppubtype = {article}

}