Robust Detection of Somatic Mosaicism and Repeat Interruptions by Long-Read Targeted Sequencing in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is the most complex and variable trinucleotide repeat disorder caused by an unstable CTG repeat expansion, reaching up to 4000 CTG in the most severe cases. The genetic and clinical variability of DM1 depend on the sex and age of the transmitting parent, but also on the CTG repeat number, presence of repeat interruptions and/or on the degree of somatic instability. Currently, it is difficult to simultaneously and accurately determine these contributing factors in DM1 patients due to the limitations of gold standard methods used in molecular diagnostics and research laboratories. Our study showed the efficiency of the latest PacBio long-read sequencing technology to sequence large CTG trinucleotides, detect multiple and single repeat interruptions and estimate the levels of somatic mosaicism in DM1 patients carrying complex CTG repeat expansions inaccessible to most methods. Using this innovative approach, we revealed the existence of de novo CCG interruptions associated with CTG stabilization/contraction across generations in a new DM1 family. We also demonstrated that our method is suitable to sequence the DM1 locus and measure somatic mosaicism in DM1 families carrying more than 1000 pure CTG repeats. Better characterization of expanded alleles in DM1 patients can significantly improve prognosis and genetic counseling, not only in DM1 but also for other tandem DNA repeat disorders.

Single Circulating Fetal Trophoblastic Cells Eligible for Non Invasive Prenatal Diagnosis: the Exception Rather than the Rule.

Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations that frequently concern prenatal diagnosis requests. The objective of this study was to develop an approach to isolate and analyze Circulating Trophoblastic Fetal Cells (CFTCs) for NIPD of monogenic diseases caused by triplet repeat expansion or point mutations. We developed a method for CFTC isolation based on DEPArray sorting and used Huntington's disease as the clinical model for CFTC-based NIPD. Then, we investigated whether CFTC isolation and Whole Genome Amplification (WGA) could be used for NIPD in couples at risk of transmitting different monogenic diseases. Our data show that the allele drop-out rate was 3-fold higher in CFTCs than in maternal cells processed in the same way. Moreover, we give new insights into CFTCs by compiling data obtained by extensive molecular testing by microsatellite multiplex PCR genotyping and by WGA followed by mini-exome sequencing. CFTCs appear to be often characterized by a random state of genomic degradation.

Male partners of infertile couples with congenital unilateral absence of the vas deferens are mainly non-azoospermic.

BACKGROUND: Men with congenital unilateral absence of vas deferens were reported to be mainly azoospermic, with both unilateral renal absence and mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) but some have neither. OBJECTIVES: To assess whether in infertile couples the male partners with congenital unilateral absence of vas deferens are mainly azoospermic men. MATERIAL AND METHODS: Retrospective study in a unique university hospital; reproductive, clinical, CFTR analysis and seminal data of male partners of infertile couples (from 1998 to 2018) were analysed. Diagnosis of congenital unilateral absence of vas deferens was based on transrectal ultrasounds (TRUS): complete or partial absence of one vas deferens with complete contralateral vas deferens confirmed in 63 men. Distribution of sperm count in three classes: azoospermia, oligozoospermia or normozoospermia. Ultrasound determination of renal status; seminal biomarkers assays; and search for CFTR mutations. RESULTS: Among the 63 men, 39.7% displayed azoospermia, 27% oligozoospermia and 33.3% normozoospermia; 42% of the non-azoospermic men (16/38) had previously obtained a natural pregnancy. We found unilateral renal absence in 17/59 patients (29%). Among 50 men with CFTR testing, five carried an allele associated with cystic fibrosis belonging to the 29 men without renal anomalies, indicating a high allelic frequency (8.6%). The 63 patients displayed high rates of surgical histories for undescended testicles or inguinal hernia, low values of semen volume and of total seminal glycerophosphocholine. CONCLUSIONS: Our results indicate that men with congenital unilateral absence of vas deferens mainly display oligozoospermia or normozoospermia and that they were previously fertile. They clearly confirm, first, that CFTR testing is recommended in congenital unilateral absence of vas deferens men and it should be mandatory for those with normal kidneys; and, second, that TRUS is needed for the diagnosis of congenital unilateral absence of vas deferens. As congenital unilateral absence of vas deferens may be present whatever the sperm count, biological warnings are represented by semen volume and seminal epididymal markers and clinical warnings by surgical histories of undescended testes or inguinal hernia.

Evaluation of the template letter regarding the disclosure of genetic information within the family in France.

The 2011 French Bioethics Law regarding disclosure of genetic information within families enables health professionals to notify any at-risk relatives directly, with the patient's consent, using a template letter. To assess the impact of this template letter in terms of understanding, personal feelings and intent to contact a health professional, we conducted a study interviewing patients, members of the public and genetic professionals. Although the main response to the letter was anxiety, this was associated with good understanding of the content and most individuals mentioned intention to contact a health professional.

Sperm aneuploidy and DNA fragmentation in unexplained recurrent pregnancy loss: a multicenter case-control study.

BACKGROUND: Recurrent pregnancy loss (RPL) is defined as the loss of at least three pregnancies in the first trimester. Although the most common cause is embryo aneuploidy, and despite female checkup and couple karyotyping, in about 50% of cases RPL remain unexplained. Male implication has little been investigated and results are discordant. In this context, we conducted a multi-center prospective case-control study to investigate male gamete implication in unexplained RPL. METHODS: A total of 33 cases and 27 controls were included from three university hospitals. We investigated environmental and family factors with a detailed questionnaire and andrological examination, sperm characteristics, sperm DNA/chromatin status using the sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and sperm aneuploidy using fluorescence in situ hybridization (FISH). The Mann-Whitney test and the Wilcoxon or Fisher exact tests were used. A non-parametric Spearman correlation was performed in order to analyze the relationship between various sperm parameters and FISH and sperm DNA fragmentation results. RESULTS: We found significant differences between cases and controls in time to conceive, body mass index (BMI), family history of infertility and living environment. In cases, total sperm motility and the percentage of morphologically normal spermatozoa were significantly decreased. No difference was found between cases and controls in sperm DNA fragmentation or chromatin integrity. In cases, spermatozoa with aneuploidy, hyperhaploidy and chromosome 18 disomy were significantly increased. CONCLUSIONS: This prospective case-control study is one of the largest to examine environmental factors, sperm characteristics, sperm DNA fragmentation and chromatin, and chromosome anomalies in spermatozoa in relation to unexplained recurrent pregnancy loss. The originality of our study lies in the comprehensive andrological examination and search for risk factors and fertility history. Further studies are needed to confirm the links between unexplained RPL and a male family history of infertility or miscarriages. The increased sperm aneuploidy observed in unexplained RPL supports a male etiology. These data pave the way for further studies to demonstrate the value of preimplantation genetic screening in men with increased sperm aneuploidy whose partners experience unexplained RPL.

CONTEXTE: Les fausses couches à répétition (FCR) sont définies lorsqu'au moins trois fausses couches ont eu lieu au cours du premier trimestre. Bien que la cause la plus fréquente soit l'aneuploïdie embryonnaire, et malgré un bilan chez la femme et un caryotype du couple, dans environ 50% des cas, les FCR restent inexpliquées. L'implication masculine a été peu étudiée et les résultats restent discordants. Ainsi, nous avons réalisé une étude cas-témoins prospective et multicentrique afin d'investiguer l'implication du gamète mâle dans les FCR inexpliquées. MÉTHODES: Un total de 33 cas et de 27 témoins ont été inclus recrutés au sein de trois hôpitaux universitaires. Nous avons étudié les facteurs environnementaux et familiaux à partir d'un questionnaire détaillé ainsi que les données de l'examen andrologique, les caractéristiques du sperme, la fragmentation de l'ADN et la chromatine du spermatozoïde en utilisant le sperm chromatine structure assay (SCSA) et le test du TUNEL, ainsi que l'aneuploïdie des spermatozoïdes grâce à la méthode d'hybridation in situ de sonde chromosomique (FISH). Le test de Mann-Whitney et les tests exacts de Wilcoxon ou de Fisher ont été utilisés. Une corrélation de Spearman non-paramétrique a été réalisée afin d'analyser la relation entre les divers paramètres de sperme et les résultats de fragmentation d'ADN du sperme et les résultats de la FISH. RÉSULTATS: Nous avons trouvé des différences significatives entre les cas et les témoins pour le délai de conception, l'indice de masse corporelle (IMC), les antécédents familiaux d'infertilité et le milieu de vie. Chez les cas, la mobilité totale des spermatozoïdes et le pourcentage de spermatozoïdes normaux étaient significativement diminués. Aucune différence n'a été trouvée entre les cas et les témoins concernant la fragmentation de l'ADN des spermatozoïdes ou l'intégrité de la chromatine. Chez les cas, la fréquence des spermatozoïdes présentant une aneuploïdie, une hyperhaploïdie et une disomie du chromosome 18 étaient significativement augmentée. CONCLUSIONS: Cette étude cas-témoins prospective est. l'une des plus importantes ayant investigué à la fois les facteurs environnementaux, les caractéristiques des spermatozoïdes, la fragmentation et la chromatine de l'ADN des spermatozoïdes et les anomalies chromosomiques des spermatozoïdes en rapport avec les fausses couches à répétition inexpliquée. L'originalité de notre étude réside dans l'examen andrologique complet et la recherche des facteurs de risque et des antécédents reproductifs. D'autres études sont nécessaires pour confirmer les liens entre les FCR inexpliquées et les antécédents familiaux masculins d'infertilité ou de fausses couches. L'augmentation de l'aneuploïdie des spermatozoïdes observée chez les cas présentant des FCR inexpliquées plaide en faveur d'une étiologie masculine. Ces données ouvrent la voie à d'autres études pour démontrer l'utilité d'un dépistage génétique préimplantatoire chez les hommes présentant une augmentation de l'aneuploïdie des spermatozoïdes dont les partenaires présentent des FCR inexpliquées.

The spectrum of renal involvement in male patients with infertility related to excretory-system abnormalities: phenotypes, genotypes, and genetic counseling.

BACKGROUND: While reproductive technologies are increasingly used worldwide, epidemiologic, clinical and genetic data regarding infertile men with combined genital tract and renal abnormalities remain scarce, preventing adequate genetic counseling. METHODS: In a cohort-based study, we assessed the prevalence (1995-2014) and the clinical characteristics of renal disorders in infertile males with genital tract malformation. In a subset of 34 patients, we performed a detailed phenotype analysis of renal and genital tract disorders. RESULTS: Among the 180 patients with congenital uni- or bilateral absence of vas deferens (CU/BAVD), 45 (25 %) had a renal malformation. We also identified 14 infertile men with combined seminal vesicle (SV) and renal malformation but no CU/BAVD. Among the 34 patients with detailed clinical description, renal disease was unknown before the assessment of the infertility in 27 (79.4 %), and 7 (20.6 %) had chronic renal failure. Four main renal phenotypes were observed: solitary kidney (47 %); autosomal-dominant polycystic kidney disease (ADPKD, 0.6 %); uni- or bilateral hypoplastic kidneys (20.6 %); and a complex renal phenotype associated with a mutation of the HNF1B gene (5.8 %). Absence of SV and azoospermia were significantly associated with the presence of a solitary kidney, while dilatation of SV and necroasthenozoospermia were suggestive of ADPKD. CONCLUSION: A dominantly inherited renal disease (ADPKD or HNF1B-related nephropathy) is frequent in males with infertility and combined renal and genital tract abnormalities (26 %). A systematic renal screening should be proposed in infertile males with CU/BAVD or SV disorders.

Truncating Mutations in the Adhesion G Protein-Coupled Receptor G2 Gene ADGRG2 Cause an X-Linked Congenital Bilateral Absence of Vas Deferens.

In 80% of infertile men with obstructive azoospermia caused by a congenital bilateral absence of the vas deferens (CBAVD), mutations are identified in the cystic fibrosis transmembrane conductance regulator gene (CFTR). For the remaining 20%, the origin of the CBAVD is unknown. A large cohort of azoospermic men with CBAVD was retrospectively reassessed with more stringent selection criteria based on consistent clinical data, complete description of semen and reproductive excurrent ducts, extensive CFTR testing, and kidney ultrasound examination. To maximize the phenotypic prioritization, men with CBAVD and with unilateral renal agenesis were considered ineligible for the present study. We performed whole-exome sequencing on 12 CFTR-negative men with CBAVD and targeted sequencing on 14 additional individuals. We identified three protein-truncating hemizygous mutations, c.1545dupT (p.Glu516Ter), c.2845delT (p.Cys949AlafsTer81), and c.2002_2006delinsAGA (p.Leu668ArgfsTer21), in ADGRG2, encoding the epididymal- and efferent-ducts-specific adhesion G protein-coupled receptor G2, in four subjects, including two related individuals with X-linked transmission of their infertility. Previous studies have demonstrated that Adgrg2-knockout male mice develop obstructive infertility. Our study confirms the crucial role of ADGRG2 in human male fertility and brings new insight into congenital obstructive azoospermia pathogenesis. In men with CBAVD who are CFTR-negative, ADGRG2 testing could allow for appropriate genetic counseling with regard to the X-linked transmission of the molecular defect.