Teratozoospermia: spotlight on the main genetic actors in the human

BACKGROUND Male infertility affects >20 million men worldwide and represents a major health concern. Although multifactorial, male infertility has a strong genetic basis which has so far not been extensively studied. Recent studies of consanguineous families and of small cohorts of phenotypically homogeneous patients have however allowed the identification of a number of autosomal recessive causes of teratozoospermia. Homozygous mutations of aurora kinase C (AURKC) were first described to be responsible for most cases of macrozoospermia. Other genes defects have later been identified in spermatogenesis associated 16 (SPATA16) and dpy-19-like 2 (DPY19L2) in patients with globozoospermia and more recently in dynein, axonemal, heavy chain 1 (DNAH1) in a heterogeneous group of patients presenting with flagellar abnormalities previously described as dysplasia of the fibrous sheath or short/stump tail syndromes, which we propose to call multiple morphological abnormalities of the flagella (MMAF). METHODS A comprehensive review of the scientific literature available in PubMed/Medline was conducted for studies on human genetics, experimental models and physiopathology related to teratozoospermia in particular globozoospermia, large headed spermatozoa and flagellar abnormalities. The search included all articles with an English abstract available online before September 2014. RESULTS Molecular studies of numerous unrelated patients with globozoospermia and large-headed spermatozoa confirmed that mutations in DPY19L2 and AURKC are mainly responsible for their respective pathological phenotype. In globozoospermia, the deletion of the totality of the DPY19L2 gene represents ∼81% of the pathological alleles but point mutations affecting the protein function have also been described. In macrozoospermia only two recurrent mutations were identified in AURKC, accounting for almost all the pathological alleles, raising the possibility of a putative positive selection of heterozygous individuals. The recent identification of DNAH1 mutations in a proportion of patients with MMAF is promising but emphasizes that this phenotype is genetically heterogeneous. Moreover, the identification of mutations in a dynein strengthens the emerging point of view that MMAF may be a phenotypic variation of the classical forms of primary ciliary dyskinesia. Based on data from human and animal models, the MMAF phenotype seems to be favored by defects directly or indirectly affecting the central pair of axonemal microtubules of the sperm flagella. CONCLUSIONS The studies described here provide valuable information regarding the genetic and molecular defects causing infertility, to improve our understanding of the physiopathology of teratozoospermia while giving a detailed characterization of specific features of spermatogenesis. Furthermore, these findings have a significant influence on the diagnostic strategy for teratozoospermic patients allowing the clinician to provide the patient with informed genetic counseling, to adopt the best course of treatment and to develop personalized medicine directly targeting the defective gene product

Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella.

International audienceTen to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum

Genetic and molecular characterization of male infertility : applications to different forms of severe teratozoospermia

L'infertilité masculine concerne plus de 20 millions d'homme à travers le monde et représente un véritable enjeu de santé public. Bien que multifactorielle, l'infertilité masculine a une composante génétique importante qui jusqu'à présent n'a été que peu étudiée. L'objectif de mon travail a été d'initier et de poursuivre les investigations génétiques sur trois phénotypes de tératozoospermie: les spermatozoïdes macrocéphales, la globozoospermie et les anomalies morphologiques multiples des flagelles (AMMF).Pour le premier phénotype, nous avons étudié 87 patients, dont 83 cas-index, présentant un phénotype de macrozoospermie. Nous avons trouvé la mutation c.144delC dans le gène AURKC chez 82% des patients (68/83) confirmant qu'il s'agit de l'évènement génétique prépondérant pour ce phénotype. Une nouvelle mutation récurrente, p.Y248*, entrainant la dégradation totale du transcrit anormal par nonsense-mediated mRNA decay, a été retrouvée chez 10 patients non‐apparentés. L'identification de deux mutations ancestrales dans AURKC maintenues au cours de l'évolution malgré leur effet délétère sur la reproduction chez l'homme homozygote, ouvre la question d'un potentiel avantage sélectif procuré par l'haplo-insuffisance d'AURKC.Pour le second phénotype, nous avons analysé une cohorte de 34 patients globozoospermiques par séquençage et MLPA (multiplex ligation-dependent probe amplification). Au total, la délétion homozygote de DPY19L2 a été retrouvée chez 22 patients sur 30 cas non apparentés (73.3%) et 3 nouvelles mutations ponctuelles ont été identifiées. Ces résultats indiquent que l'analyse moléculaire de DPY19L2 des patients globozoospermiques ne devrait pas être limitée à la recherche de la délétion homozygote de DPY19L2. Dans un second temps, nous avons démontré que la délétion récurrente de DPY19L2 était médiée par le mécanisme de recombinaison homologue non allélique (NAHR) entre deux séquences répétées homologues (LCR) de 28kb situées de chaque côté du gène. La très grande majorité des points de cassure surviennent dans une région de 1,2 kb située dans la partie centrale des LCRs. Cette région minimale de recombinaison est elle‐même centrée sur une séquence consensus de 13 nucléotides reconnue par PRDM9, une protéine à doigts de zinc qui favorise la survenue des cassures doubles brins initiant les processus de recombinaisons. Les modèles théoriques prédisent que, lors de la méiose, le mécanisme NAHR génère de novo plus d'allèles recombinés délétés que dupliqués. Étonnamment, dans la population générale les allèles DPY19L2 dupliqués sont trois fois plus fréquents que les allèles délétés. Nous avons développé une PCR digitale sur le sperme afin de mesurer le taux de délétions et de duplications de novo à ce locus chez des témoins. Tel qu'il était prédit par le modèle de NAHR, nous avons identifié un taux de délétions supérieur à celui des duplications. Ce paradoxe peut s'expliquer par la sélection qui s'opère à l'encontre des hommes infertiles porteurs de la délétion homozygote et potentiellement des hommes porteurs d'une délétion hétérozygote.Enfin pour le troisième phénotype, nous avons réalisé l'analyse par cartographie par homozygotie de 20 patients infertiles, dont 18 cas-index, présentant des anomalies morphologiques du flagelle. Cinq mutations homozygotes ont été identifiées dans le gène DNAH1 parmi les 18 patients non-apparentés (28%). Ce gène code pour une chaine lourde des bras internes de dynéine exprimée dans le testicule. Des analyses d'immunofluorescence et de RT-PCR ont confirmé le caractère pathogène d'une de ces mutations situées sur un site donneur d'épissage. Les analyses par microscopie électronique ont révélé une désorganisation générale de l'axonème incluant une disparition des doublets centraux et des bras internes de dynéine suggérant que DNAH1 est une protéine clé dans la biogenèse du flagelle du spermatozoïde.Male infertility affects more than 20 million men worldwide and represents a major health concern. Although multifactorial, male infertility has a strong genetic basis which has so far not been extensively studied. The objectives of my thesis were to initiate and conduct some genetic investigations on three specific phenotypes of teratozoospermia: macrozoospermia, globozoospermia and multiple morphological abnormalities of the flagella (MMAF).For the first phenotype, we studied 87 patients with macrozoospermia, including 83 index cases, and identified c.144delC, a pathogenic mutation in the AURKC gene in 82% of patients (68/83) confirming that this variant is the main cause of macrozoospermia. A new recurrent mutation, p.Y248*, leading to degradation of the mutant transcripts by non-sense mediated mRNA decay was identified in 10 unrelated patients. Patients with no identified AURKC mutation have a decreased rate of spermatozoa with a large head and multiple flagella. Identification of two ancestral mutations in AURKC maintained during evolution despite their negative effect on reproduction in homozygous men, raises the question of a potential selective advantage provided by the AURKC haploinsufficiency.For the second phenotype, we first analyzed 34 patients presenting with globozoospermia using MLPA (multiplex ligation-dependent probe amplification) and Sanger sequencing. In total, 22 of the 30 unrelated patients where homozygous for the DPY19L2 deletion (73.3%) and 3 novel point mutations were identified. These results suggest that the molecular investigation of the DPY19L2 gene in globozoospermic patients should not be limited to the detection of the DPY19L2 genomic deletion and open interesting perspectives for the identification of DPY19L2 partners during acrosome biogenesis. Subsequently, we demonstrated that the genomic deletion was mediated by Non-Allelic Homologous Recombination (NAHR) between two homologous 28-Kb Low Copy Repeats (LCRs) located on each side of the gene. The vast majority of genomic breakpoints fell within a 1.2-Kb region central to the 28-Kb LCR. A 13-mer consensus sequence is located in the centre of that 1.2-Kb region recognized by PRDM9, a multi-unit zinc finger binding protein that promotes the formation of double-strand breaks (DSBs) initiating the homologous recombination process. The accepted theoretical NAHR model predicts that during meiosis, NAHR produces more deleted than duplicated alleles. Surprisingly, array-CGH data show that, in the general population, DPY19L2 duplicated alleles are approximately three times as frequent as deleted alleles. In order to shed light on this paradox, we developed a sperm-based digital PCR to measure the de novo rates of deletions and duplications at this locus. As predicted by the NAHR model, we identified an excess of de novo deletions over duplications. These discording results may be explained by the purifying selection against sterile, homozygous deleted men. Heterozygous deleted men might also suffer a small fitness penalty.Lastly, for the third phenotype, homozygosity mapping was carried out on a cohort of 20 North African individuals, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF). Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR and immunostaining studies confirmed the pathogenic effect of one of these mutations located on a donor splice site. Electronic microscopy revealed a general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms suggesting that DNAH1 plays a critical role in sperm flagellum biogenesis and assembly

Mise au point du dosage génique du gène OCRL1 par Multiplex Ligation-dependent Probe Amplification (MLPA) (application au syndrome de Lowe et à la maladie de Dent)

Le syndrome de Lowe et la maladie de Dent sont deux maladies génétiques très rares de transmission récessive liée au chromosome X chez lesquelles des délétions du gène OCRL1 ont été décrites. Bien que simple à détecter chez un homme, ces réarrangements sont délicats à mettre en évidence chez les femmes potentiellement conductrices de la maladie avec les techniques de diagnostic de première intention (ex : séquençage) en raison de la présence des deux chromosomes X et donc de 2 copies du gène OCRL1. Leur détection, de même que la détection d autres réarrangements génomiques potentiellement causals de la maladie tels que les duplications ou les délétions en mosaïque, imposent l utilisation de techniques quantitatives complémentaires souvent longues, fastidieuses et peu résolutives. La technique de Multiplex Ligation-dependent Probe Amplification (MLPA) se distingue des autres techniques quantitatives par sa rapidité et son excellente résolution. Ainsi, 24 sondes MLPA spécifiques ont été modélisées et validées pour quantifier rapidement et simplement le gène OCRL1. Cette technique a permis de préciser le statut de conductrice de 5 femmes avec un apparenté atteint porteur d une délétion génomique d OCRL1. Enfin, malgré sa sensibilité, aucune délétion ou duplication n a été retrouvée chez 15 patients présentant un tableau incomplet voire typique du syndrome de Lowe pour deux d entre eux, et chez lesquels l analyse préalable par séquençage d OCRL1 s était révélée non contributive. Notre technique MLPA spécifique du gène OCRL1 est fiable et robuste et ainsi applicable en routine comme méthode diagnostique complémentaire du séquençage pour le syndrome de Lowe et la maladie de Dent. C est un outil simple et performant pour déterminer le statut des femmes potentiellement conductrices de délétions. De plus les résultats de notre étude démontrent que les duplications du gène OCRL1 sont des événements probablement exceptionnels. Ils fragilisent un peu plus l hypothèse que les réarrangements génomiques en mosaïque ou les duplications contribueraient à expliquer la fraction de patients avec un phénotype plus ou moins complet et pour lesquels aucune mutation n avait pu être identifiée avec les techniques conventionnelles.The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked recessively inherited disease characterized by a severe pleiotropic phenotype including mental retardation, bilateral congenital cataract and renal Fanconi syndrome. Dent disease (DD) is an X-linked renal proximal tubulopathy. Mutations in the OCRL1 gene (oculocerebrorenal syndrome of Lowe, type 1) have been associated with both diseases but most of all with OCRL. While more than 150 mutations have been described to date only 5 % are exonic deletions. Moreover no duplication (homogen or mosaic) and no deletion mosaicism have yet been described in the both diseases. We have designed four synthetic mutiplex ligation-dependent probe amplification (MLPA) probe sets that measures exons copy number in OCRL1 gene with high precision allowing rapid gene quantification. After OCRL1 MLPA probe sets validation, we screened four female patients with an affected relative harbouring a known genomic deletion and 11 male and 4 female patients presenting with an incomplete phenotype or even typical for two of them and previously diagnosed as sequence-negative. Two of four female were detected as carrier for the mutation previously identified in the affected relative. Neither deletion nor duplication (mosaic or homogen) were found in the 15 patients with a milder or complete phenotype, and previously diagnosed as sequence-negative, thus ruling out the involvement of OCRL1 gene. These results of our study provide no further evidence for the hypothesis that duplications and somatic mosaic deletions account the fraction of patients who have no detectible mutation after the usual screening procedures.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

Sun proteins and Dpy19l2 forming LINC-like links are critical for spermiogenesis

Summary: In this response to Pasch et al.’s (2015) discovery that Sun4 is essential for sperm head formation, the authors highlight that like Sun4, Dpy19l2 has a likely LINK-like function and that it also plays a crucial role in spermiogenesis and male infertility

Development of a multiplex ligation-dependent probe amplification (MLPA) assay for quantification of the OCRL1 gene.

International audienceOBJECTIVES: To develop and evaluate the efficacy of Multiplex Ligation-dependent Probe Amplification (MLPA) technique in detection of genomic rearrangements of the OCRL1 gene associated with Oculocerebrorenal syndrome of Lowe (OCRL). DESIGN AND METHODS: Four synthetic MLPA probe sets have been designed to measure exons copy number in OCRL1 gene. After OCRL1 MLPA probe sets validation in 7 OCRL1 deleted patients, we screened 5 female patients to asses their carrier status and 15 patients with suspected OCRL, previously diagnosed as sequence-negative. RESULTS: MLPA was able to detect all the known deletions. Two of five females were detected as carrier for the family mutation. Neither mosaic deletion nor duplication was found in the 15 patients suspected of having Lowe syndrome. CONCLUSIONS: Our MLPA allows rapid and precise OCRL1 gene quantification. Moreover this study provides no further evidence for the hypothesis that duplications and deletion somatic mosaic deletions account for the fraction of patients who have no detectible mutation after the usual screening procedures

Génétique de l’infertilité masculine

L’infertilité concerne de 10 % à 15 % des couples ayant un désir d’enfant et une composante masculine est retrouvée dans près de la moitié des cas. Dans une proportion importante de cas, une base génétique chromosomique ou génique connue, parfois transmise par les parents, est en cause. L’utilisation des nouvelles techniques de génotypage pangénomique a récemment permis d’impliquer de nouveaux gènes dans des phénotypes spécifiques d’infertilité masculine. Pour les patients concernés, ces avancées permettent de réaliser un diagnostic, d’affiner le pronostic et donc de mieux orienter la prise en charge. À terme, ce travail permettra de mieux comprendre les mécanismes moléculaires de la spermatogenèse et donc de proposer des solutions thérapeutiques qui pourront être applicables au plus grand nombre

Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human

Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 (n = 6), CFAP43 (n = 10), and CFAP44 (n = 6). CRISPR/Cas9 created homozygous CFAP43/44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human