Rotational motion and rheotaxis of human sperm do not require functional CatSper channels and transmembrane Ca2+ signaling.

Navigation of sperm in fluid flow, called rheotaxis, provides long-range guidance in the mammalian oviduct. The rotation of sperm around their longitudinal axis (rolling) promotes rheotaxis. Whether sperm rolling and rheotaxis require calcium (Ca2+ ) influx via the sperm-specific Ca2+ channel CatSper, or rather represent passive biomechanical and hydrodynamic processes, has remained controversial. Here, we study the swimming behavior of sperm from healthy donors and from infertile patients that lack functional CatSper channels, using dark-field microscopy, optical tweezers, and microfluidics. We demonstrate that rolling and rheotaxis persist in CatSper-deficient human sperm. Furthermore, human sperm undergo rolling and rheotaxis even when Ca2+ influx is prevented. Finally, we show that rolling and rheotaxis also persist in mouse sperm deficient in both CatSper and flagellar Ca2+ -signaling domains. Our results strongly support the concept that passive biomechanical and hydrodynamic processes enable sperm rolling and rheotaxis, rather than calcium signaling mediated by CatSper or other mechanisms controlling transmembrane Ca2+ flux.

WWC2 expression in the testis: Implications for spermatogenesis and male fertility.

The family of WWC proteins is known to regulate cell proliferation and organ growth control via the Hippo signaling pathway. As WWC proteins share a similar domain structure and a common set of interacting proteins, they are supposed to fulfill compensatory functions in cells and tissues. While all three WWC family members WWC1, WWC2, and WWC3 are found co-expressed in most human organs including lung, brain, kidney, and liver, in the testis only WWC2 displays a relatively high expression. In this study, we investigated the testicular WWC2 expression in spermatogenesis and male fertility. We show that the Wwc2 mRNA expression level in mouse testes is increased during development in parallel with germ cell proliferation and differentiation. The cellular expression of each individual WWC family member was evaluated in published single-cell mRNA datasets of murine and human testes demonstrating a high WWC2 expression predominantly in early spermatocytes. In line with this, immunohistochemistry revealed cytosolic WWC2 protein expression in primary spermatocytes from human testes displaying full spermatogenesis. In accordance with these findings, markedly lower WWC2 expression levels were detected in testicular tissues from mice and men lacking germ cells. Finally, analysis of whole-exome sequencing data of male patients affected by infertility and unexplained severe spermatogenic failure revealed several heterozygous, rare WWC2 gene variants with a proposed damaging function and putative impact on WWC2 protein structure. Taken together, our findings provide novel insights into the testicular expression of WWC2 and show its cell-specific expression in spermatocytes. As rare WWC2 variants were identified in the background of disturbed spermatogenesis, WWC2 may be a novel candidate gene for male infertility.

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

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.

Congenital Limb Defects: A Retrospective Cohort Study and Overview of the Literature.

PURPOSE: Congenital limb defects are common malformations that are often associated with other organ defects and genetic disorders. Since prenatal detection is challenging and classification is often complex, the aim of this study was to describe a large cohort of fetuses with congenital limb defects and to identify characteristics that are essential for prenatal evaluation, counselling, and management. MATERIALS AND METHODS: In this retrospective cohort study, all cases of confirmed fetal limb defects from two centers for prenatal ultrasound between 2001 and 2021 were evaluated. Cases with skeletal dysplasia were excluded from this study. Demographic data, association with genetic disorders, and correlation with maternal parameters were analyzed statistically. RESULTS: 170 fetuses were included in this study. 60% were diagnosed with a reduction anomaly and 40% with a duplication anomaly. The majority of fetuses were male, and in 73.5% of all cases, additional malformations were present. Among the genetic causes, trisomy 13 and 18 were the most common in this cohort. CONCLUSION: Congenital limb malformations are important markers for complex fetal disorders that warrant referral to specialists in prenatal ultrasound. To improve prenatal detection, care should be taken to visualize all fetal extremities already in early pregnancy.

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA.

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

Extrinsic immune cell-derived, but not intrinsic oligodendroglial factors contribute to oligodendroglial differentiation block in multiple sclerosis.

Multiple sclerosis (MS) is the most frequent demyelinating disease in young adults and despite significant advances in immunotherapy, disease progression still cannot be prevented. Promotion of remyelination, an endogenous repair mechanism resulting in the formation of new myelin sheaths around demyelinated axons, represents a promising new treatment approach. However, remyelination frequently fails in MS lesions, which can in part be attributed to impaired differentiation of oligodendroglial progenitor cells into mature, myelinating oligodendrocytes. The reasons for impaired oligodendroglial differentiation and defective remyelination in MS are currently unknown. To determine whether intrinsic oligodendroglial factors contribute to impaired remyelination in relapsing-remitting MS (RRMS), we compared induced pluripotent stem cell-derived oligodendrocytes (hiOL) from RRMS patients and controls, among them two monozygous twin pairs discordant for MS. We found that hiOL from RRMS patients and controls were virtually indistinguishable with respect to remyelination-associated functions and proteomic composition. However, while analyzing the effect of extrinsic factors we discovered that supernatants of activated peripheral blood mononuclear cells (PBMCs) significantly inhibit oligodendroglial differentiation. In particular, we identified CD4+ T cells as mediators of impaired oligodendroglial differentiation; at least partly due to interferon-gamma secretion. Additionally, we observed that blocked oligodendroglial differentiation induced by PBMC supernatants could not be restored by application of oligodendroglial differentiation promoting drugs, whereas treatment of PBMCs with the immunomodulatory drug teriflunomide prior to supernatant collection partly rescued oligodendroglial differentiation. In summary, these data indicate that the oligodendroglial differentiation block is not due to intrinsic oligodendroglial factors but rather caused by the inflammatory environment in RRMS lesions which underlines the need for drug screening approaches taking the inflammatory environment into account. Combined, these findings may contribute to the development of new remyelination promoting strategies.

Initial experience with [18F]DPA-714 TSPO-PET to image inflammation in primary angiitis of the central nervous system.

PURPOSE: Primary angiitis of the central nervous system (PACNS) is a heterogeneous, rare, and poorly understood inflammatory disease. We aimed at non-invasive imaging of activated microglia/macrophages in patients with PACNS by PET-MRI targeting the translocator protein (TSPO) with [18F]DPA-714 to potentially assist differential diagnosis, therapy monitoring, and biopsy planning. METHODS: In total, nine patients with ischemic stroke and diagnosed or suspected PACNS underwent [18F]DPA-714-PET-MRI. Dynamic PET scanning was performed for 60 min after injection of 233 ± 19 MBq [18F]DPA-714, and MRI was simultaneously acquired. RESULTS: In two PACNS patients, [18F]DPA-714 uptake patterns exceeded MRI correlates of infarction, whereas uptake was confined to the infarct in four patients where initial suspicion of PACNS could not be confirmed. About three patients with PACNS or cerebral predominant lymphocytic vasculitis showed no or only faintly increased uptake. Short-term [18F]DPA-714-PET follow-up in a patient with PACNS showed reduced lesional [18F]DPA-714 uptake after anti-inflammatory treatment. Biopsy in the same patient pinpointed the source of tracer uptake to TSPO-expressing immune cells. CONCLUSIONS: [18F]DPA-714-PET imaging may facilitate the diagnosis and treatment monitoring of PACNS. Further studies are needed to fully understand the potential of TSPO-PET in deciphering the heterogeneity of the disease.

The human RHOX gene cluster: target genes and functional analysis of gene variants in infertile men.

The X-linked reproductive homeobox (RHOX) gene cluster encodes transcription factors preferentially expressed in reproductive tissues. This gene cluster has important roles in male fertility based on phenotypic defects of Rhox-mutant mice and the finding that aberrant RHOX promoter methylation is strongly associated with abnormal human sperm parameters. However, little is known about the molecular mechanism of RHOX function in humans. Using gene expression profiling, we identified genes regulated by members of the human RHOX gene cluster. Some genes were uniquely regulated by RHOXF1 or RHOXF2/2B, while others were regulated by both of these transcription factors. Several of these regulated genes encode proteins involved in processes relevant to spermatogenesis; e.g. stress protection and cell survival. One of the target genes of RHOXF2/2B is RHOXF1, suggesting cross-regulation to enhance transcriptional responses. The potential role of RHOX in human infertility was addressed by sequencing all RHOX exons in a group of 250 patients with severe oligozoospermia. This revealed two mutations in RHOXF1 (c.515G > A and c.522C > T) and four in RHOXF2/2B (-73C > G, c.202G > A, c.411C > T and c.679G > A), of which only one (c.202G > A) was found in a control group of men with normal sperm concentration. Functional analysis demonstrated that c.202G > A and c.679G > A significantly impaired the ability of RHOXF2/2B to regulate downstream genes. Molecular modelling suggested that these mutations alter RHOXF2/F2B protein conformation. By combining clinical data with in vitro functional analysis, we demonstrate how the X-linked RHOX gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility.

X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.

BACKGROUND: The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men. METHODS: We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls. RESULTS: We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression. CONCLUSIONS: In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).

Disorders of spermatogenesis: Perspectives for novel genetic diagnostics after 20 years of unchanged routine.

Infertility is a common condition estimated to affect 10-15% of couples. The clinical causes are attributed in equal parts to the male and female partners. Diagnosing male infertility mostly relies on semen (and hormone) analysis, which results in classification into the two major phenotypes of oligo- and azoospermia. The clinical routine analyses have not changed over the last 20 years and comprise screening for chromosomal aberrations and Y­chromosomal azoospermia factor deletions. These tests establish a causal genetic diagnosis in about 4% of unselected men in infertile couples and 20% of azoospermic men. Gene sequencing is currently only performed in very rare cases of hypogonadotropic hypogonadism and the CFTR gene is routinely analysed in men with obstructive azoospermia. Still, a large number of genes have been proposed to be associated with male infertility by, for example, knock-out mouse models. In particular, those that are exclusively expressed in the testes are potential candidates for further analyses. However, the genome-wide analyses (a few array-CGH, six GWAS, and some small exome sequencing studies) performed so far have not lead to improved clinical diagnostic testing. In 2017, we started to routinely analyse the three validated male infertility genes: NR5A1, DMRT1, and TEX11. Preliminary analyses demonstrated highly likely pathogenic mutations in these genes as a cause of azoospermia in 4 men, equalling 5% of the 80 patients analysed so far, and increasing the diagnostic yield in this group to 25%. Over the past few years, we have observed a steep increase in publications on novel candidate genes for male infertility, especially in men with azoospermia. In addition, concerted efforts to achieve progress in elucidating genetic causes of male infertility and to introduce novel testing strategies into clinical routine have been made recently. Thus, we are confident that major breakthroughs concerning the genetics of male infertility will be achieved in the near future and will translate into clinical routine to improve patient/couple care.

A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family.

PURPOSE: The purpose of this study was to identify mutations that cause non-syndromic male infertility using whole exome sequencing of family cases. METHODS: We recruited a consanguineous Turkish family comprising nine siblings with male triplets; two of the triplets were infertile as well as one younger infertile brother. Whole exome sequencing (WES) performed on two azoospermic brothers identified a mutation in the melanoma antigen family B4 (MAGEB4) gene which was confirmed via Sanger sequencing and then screened for on control groups and unrelated infertile subjects. The effect of the mutation on messenger RNA (mRNA) and protein levels was tested after in vitro cell transfection. Structural features of MAGEB4 were predicted throughout the conserved MAGE domain. RESULTS: The novel single-base substitution (c.1041A>T) in the X-linked MAGEB4 gene was identified as a no-stop mutation. The mutation is predicted to add 24 amino acids to the C-terminus of MAGEB4. Our functional studies were unable to detect any effect either on mRNA stability, intracellular localization of the protein, or the ability to homodimerize/heterodimerize with other MAGE proteins. We thus hypothesize that these additional amino acids may affect the proper protein interactions with MAGEB4 partners. CONCLUSION: The whole exome analysis of a consanguineous Turkish family revealed MAGEB4 as a possible new X-linked cause of inherited male infertility. This study provides the first clue to the physiological function of a MAGE protein.

Universal cardiac induction of human pluripotent stem cells in two and three-dimensional formats: implications for in vitro maturation.

Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large-scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high-efficiency procedure for generating CMs both in two-dimensional (2D) and three-dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost-efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin-free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time-course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture-induced maturation of the resulting CMs. This suggested that hPSC-CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC-CMs to functional readouts and thus define the cornerstones of culture-induced maturation.

Genetic Variants of DICE1/INTS6 in German Prostate Cancer Families with Linkage to 13q14.

INTRODUCTION: Prostate cancer is the most frequent malignancy found to occur in Caucasian men, but its genetic basis remains elusive. A prostate cancer-susceptibility locus has been identified on chromosome 13q14. The tumour suppressor gene deleted in cancer cells 1 (DICE1/INTS6) is located within this interval on 13q14.3. MATERIALS AND METHODS: We performed mutation analysis of the DICE1/INTS6 gene in thirteen German prostate cancer families. RESULTS AND CONCLUSION: None of the patients harboured DICE1 mutations, and similar frequencies of the previously identified 13 bp deletion polymorphism in the DICE1 promoter were observed in the familial prostate cancer patients as compared with sporadic prostate cancer patients and controls. However, in one family with three affected brothers, the variations c.1215A>C (p.T405T) in exon 10 and c.2568A>G (p.S856S) in exon 17 were detected in a heterozygous pattern. In sporadic prostate cancer patients, variant c.2568A>G (p.S856S) was detected in 10/325 (3.08%) compared with 5/207 (2.42%) control samples (p > 0.05). We conclude that DICE1 appears to be involved in prostate cancer progression rather than in the initiation of prostate cancer.

Severe, very early onset preeclampsia in a Covid 19-positive woman with a twin pregnancy presenting with a hydatidiform mole and coexisting normal fetus: a case report.

Cases of hydatidiform moles with a coexisting fetus are sparse and patients are at high risk for severe complications. Patients and physicians often face the dilemma of the wish to continue pregnancy until viability of the fetus while the risk for maternal complications increases. We present an educational case of a twin pregnancy presenting with a hydatidiform mole and coexisting normal fetus with a placenta praevia. The patient developed severe, early onset preeclampsia with beginning HELLP-syndrome and was tested Covid-19 positive in the further course. Termination of pregnancy was conducted via caesarean section at 18 + 6 weeks of pregnancy. Histopathology and genetic analysis confirmed a complete hydatidiform mole next to a normal placenta. Close follow-up examinations were conducted and showed normal findings including ß HCG levels normalizing within 5 months. This case combines several rare, difficult and severe medical conditions and demonstrates how an individualized therapy by an interdisciplinary team covering a highly sensitive topic was developed in a situation where no guidelines exist.

Human fertilization in vivo and in vitro requires the CatSper channel to initiate sperm hyperactivation.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study.

BACKGROUND: The genetic cause of intellectual disability in most patients is unclear because of the absence of morphological clues, information about the position of such genes, and suitable screening methods. Our aim was to identify de-novo variants in individuals with sporadic non-syndromic intellectual disability. METHODS: In this study, we enrolled children with intellectual disability and their parents from ten centres in Germany and Switzerland. We compared exome sequences between patients and their parents to identify de-novo variants. 20 children and their parents from the KORA Augsburg Diabetes Family Study were investigated as controls. FINDINGS: We enrolled 51 participants from the German Mental Retardation Network. 45 (88%) participants in the case group and 14 (70%) in the control group had de-novo variants. We identified 87 de-novo variants in the case group, with an exomic mutation rate of 1·71 per individual per generation. In the control group we identified 24 de-novo variants, which is 1·2 events per individual per generation. More participants in the case group had loss-of-function variants than in the control group (20/51 vs 2/20; p=0·022), suggesting their contribution to disease development. 16 patients carried de-novo variants in known intellectual disability genes with three recurrently mutated genes (STXBP1, SYNGAP1, and SCN2A). We deemed at least six loss-of-function mutations in six novel genes to be disease causing. We also identified several missense alterations with potential pathogenicity. INTERPRETATION: After exclusion of copy-number variants, de-novo point mutations and small indels are associated with severe, sporadic non-syndromic intellectual disability, accounting for 45-55% of patients with high locus heterogeneity. Autosomal recessive inheritance seems to contribute little in the outbred population investigated. The large number of de-novo variants in known intellectual disability genes is only partially attributable to known non-specific phenotypes. Several patients did not meet the expected syndromic manifestation, suggesting a strong bias in present clinical syndrome descriptions. FUNDING: German Ministry of Education and Research, European Commission 7th Framework Program, and Swiss National Science Foundation.

Genetic Architecture of Azoospermia-Time to Advance the Standard of Care.

BACKGROUND: Crypto- and azoospermia (very few/no sperm in the semen) are main contributors to male factor infertility. Genetic causes for spermatogenic failure (SPGF) include Klinefelter syndrome and Y-chromosomal azoospermia factor microdeletions, and CFTR mutations for obstructive azoospermia (OA). However, the majority of cases remain unexplained because monogenic causes are not analysed. OBJECTIVE: To elucidate the monogenic contribution to azoospermia by prospective exome sequencing and strict application of recent clinical guidelines. DESIGN, SETTING, AND PARTICIPANTS: Since January 2017, we studied crypto- and azoospermic men without chromosomal aberrations and Y-chromosomal microdeletions attending the Centre of Reproductive Medicine and Andrology, Münster. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We performed exome sequencing in 647 men, analysed 60 genes having at least previous limited clinical validity, and strictly assessed variants according to clinical guidelines. RESULTS AND LIMITATIONS: Overall, 55 patients (8.5%) with diagnostic genetic variants were identified. Of these patients, 20 (3.1%) carried mutations in CFTR or ADGRG2, and were diagnosed with OA. In 35 patients (5.4%) with SPGF, mutations in 20 different genes were identified. According to ClinGen criteria, 19 of the SPGF genes now reach at least moderate clinical validity. As limitations, only one transcript per gene was considered, and the list of genes is increasing rapidly so cannot be exhaustive. CONCLUSIONS: The number of diagnostic genes in crypto-/azoospermia was almost doubled to 21 using exome-based analyses and clinical guidelines. Application of this procedure in routine diagnostics will significantly improve the diagnostic yield and clinical workup as the results indicate the success rate of testicular sperm extraction. PATIENT SUMMARY: When no sperm are found in the semen, a man cannot conceive naturally. The causes are often unknown, but genetics play a major role. We searched for genetic variants in a large group of patients and found causal mutations for one in 12 men; these predict the chances for fatherhood.

WNT10A mutations are a frequent cause of a broad spectrum of ectodermal dysplasias with sex-biased manifestation pattern in heterozygotes.

Odonto-onycho-dermal dysplasia (OODD), a rare autosomal-recessive inherited form of ectodermal dysplasia including severe oligodontia, nail dystrophy, palmoplantar hyperkeratosis, and hyperhidrosis, was recently shown to be caused by a homozygous nonsense WNT10A mutation in three consanguineous Lebanese families. Here, we report on 12 patients, from 11 unrelated families, with ectodermal dysplasia caused by five previously undescribed WNT10A mutations. In this study, we show that (1) WNT10A mutations cause not only OODD but also other forms of ectodermal dysplasia, reaching from apparently monosymptomatic severe oligodontia to Schöpf-Schulz-Passarge syndrome, which is so far considered a unique entity by the findings of numerous cysts along eyelid margins and the increased risk of benign and malignant skin tumors; (2) WNT10A mutations are a frequent cause of ectodermal dysplasia and were found in about 9% of an unselected patient cohort; (3) about half of the heterozygotes (53.8%) show a phenotype manifestation, including mainly tooth and nail anomalies, which was not reported before in OODD; and (4) heterozygotes show a sex-biased manifestation pattern, with a significantly higher proportion of tooth anomalies in males than in females, which may implicate gender-specific differences of WNT10A expression.

Generation of a human iPSC line (MPIi008-A) from a patient with Denys-Drash syndrome.

An induced pluripotent stem cell (hiPSC) line (MPIi008-A) was generated from fibroblasts of a 1-year-old male patient with Denys-Drash syndrome using lentiviral delivery of reprogramming factors OCT4, SOX2, KLF4 and c-MYC. The MPIi008-A iPSC line exhibited typical iPSC morphology and normal karyotype, expressed pluripotent stem cell markers, and showed developmental potential to differentiate into derivatives of all three germ layers in vivo. The hiPSC line harbours a heterozygous missense mutation (R394L) in exon 9 of the WT1 gene.