Deciphering balanced translocations in infertile males by next-generation sequencing to identify candidate genes for spermatogenesis disorders.

Male infertility affects about 7% of the general male population. Balanced structural chromosomal rearrangements are observed in 0.4-1.4% of infertile males and are considered as a well-established cause of infertility. However, underlying pathophysiological mechanisms still need to be clarified. A strategy combining standard and high throughput cytogenetic and molecular technologies was applied in order to identify the candidate genes that might be implicated in the spermatogenesis defect in three male carriers of different balanced translocations. Fluorescence in situ hybridization (FISH) and whole-genome paired-end sequencing were used to characterize translocation breakpoints at the molecular level while exome sequencing was performed in order to exclude the presence of any molecular event independent from the chromosomal rearrangement in the patients. All translocation breakpoints were characterized in the three patients. We identified four variants: a position effect on LACTB2 gene in Patient 1, a heterozygous CTDP1 gene disruption in Patient 2, two single-nucleotide variations (SNVs) in DNAH5 gene and a heterozygous 17q12 deletion in Patient 3. The variants identified in this study need further validation to assess their roles in male infertility. This study shows that beside the mechanical effect of structural rearrangement on meiosis, breakpoints could result in additional alterations such as gene disruption or position effect. Moreover, additional SNVs or copy number variations may be fortuitously present and could explain the variable impact of chromosomal rearrangements on spermatogenesis. In conclusion, this study confirms the relevance of combining different cytogenetic and molecular techniques to investigate patients with spermatogenesis disorders and structural rearrangements on genomic scale.

Performance of semiconductor sequencing platform for non-invasive prenatal genetic screening for fetal aneuploidy: results from a multicenter prospective cohort study in a clinical setting.

OBJECTIVE: To validate and evaluate the performance metrics of the high-throughput semiconductor sequencing platform, Ion Proton®, in non-invasive prenatal genetic screening (NIPS) for common fetal aneuploidies in a clinical setting. METHODS: This prospective cohort study included 2505 pregnant women from eight academic genetics laboratories (695 high risk for trisomy 21 (risk ≥ 1/250) pregnancies in a validation study, and 1810 such pregnancies, without ultrasound anomalies, in a real-life NIPS clinical setting). Outcome was available for all cases in the validation cohort and for 521 in the clinical cohort. Cell-free DNA from plasma samples was sequenced using the Ion Proton sequencer, and sequencing data were analyzed using the open-access software, WISECONDOR. Performance metrics for detection of trisomies 21, 18 and 13 were calculated based on either fetal karyotype result or clinical data collected at birth. We also evaluated the failure rate and compared three methods of fetal fraction quantification (RASSF1A assay, and DEFRAG and SANEFALCON software). RESULTS: Results from both cohorts were consistent and their gestational age was not significantly different so their data were combined to increase the sample size for analysis. Sensitivities and specificities, respectively, were as follows: for trisomy 21, 98.3% (95% CI, 93.5-99.7%) and 99.9% (95% CI, 99.4-100%); for trisomy 18, 96.7% (95% CI, 80.9-99.8%) and 100% (95% CI, 99.6-100%); and for trisomy 13, 94.1% (95% CI, 69.2-99.7%) and 100% (95% CI, 99.6-100%). Our failure rate was 1.2% initially and as low as 0.6% after retesting some of the failed samples. Fetal fraction estimation by the RASSF1A assay was consistent with DEFRAG results, and both were adequate for routine diagnosis. CONCLUSIONS: We describe one of the largest studies evaluating Ion Proton-based NIPS and the first clinical study reporting pregnancy outcome in a large series of patients. This platform is highly efficient in detecting the three most common trisomies. Our protocol is robust and can be implemented easily in any medical genetics laboratory. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Prenatal microarray comparative genomic hybridization: Experience from the two first years of activity at the Lyon university-hospital.

OBJECTIVES: This study aims to describe how microarray comparative genomic hybridization (aCGH) has shifted to become a prenatal diagnosis tool at the Lyon university-hospital. MATERIALS AND METHODS: This retrospective study included all patients who were referred in the 3 pluridisciplinary centers for prenatal diagnosis of the Lyon university-hospital and who received a prenatal aCGH between June 2013 and June 2015. aCGH was systematically performed in parallel with a karyotype, using the PréCytoNEM array design. RESULTS: A total of 260 microarrays were performed for the following indications: 249 abnormal ultrasounds (95.8%), 7 characterizations of chromosomal rearrangements (2.7%), and 4 twins with no abnormal ultrasounds (1.5%). With a resolution of 1 mega base, we found 235 normal results (90.4%), 23 abnormal results (8.8%) and 2 non-returns (0.8%). For the chromosomal rearrangements visible on the karyotype, aCGH identified all of the 12 unbalanced rearrangements and did not identify the 2 balanced rearrangements. Among the fetuses with normal karyotypes, 11 showed abnormal microarray results, corresponding to unbalanced cryptic chromosomal rearrangements (4.2%). CONCLUSION: Transferring aCGH to a prenatal diagnosis at the Lyon university-hospital has increased the detection rate of chromosomal abnormalities by 4.2% compared to the single karyotype.

[New technologies for the human genome exploration]. / Nouvelles méthodes d'analyse globale du génome humain.

Human genome consists of 23 pairs of chromosomes, bearing our genetic information. Basically, there are two main approaches to analyse our genome: molecular genetics with direct sequencing, which detects genic mutations, and cytogenetics with the karyotype, which detects number and structural chromosomal anomalies. The main limitation of the karyotype is its level of resolution: it cannot detect abnormalities smaller than five megabases. The combined use of cytogenetics and molecular genetics has allowed the development of several new techniques that provide a comprehensive analysis of the genome with a very high level of resolution. Currently, the most efficient of those techniques is comparative genomic hybridization on microarray (array CGH), which already has diagnostic applications. However, those new methods are challenging to interpret and they raise ethical problems. Therefore they must be cautiously supervised.

[New chromosomal syndromes]. / Syndromes chromosomiques émergents.

Mental retardation occurs in 2-3% of the general population either in isolation or in combination with facial dysmorphism and/or malformations. Chromosomal abnormalities are a most common etiology. Karyotype displays chromosome aberrations in about 10% of patients but it has a limited resolution (5 Mb). Recently, the development of new molecular cytogenetic tools, especially array CGH, allowed to detect smaller abnormalities and increased the diagnosis capability of 15-20%. Among these newly detected rearrangements, some of them are recurrent and define new recognized syndromes. We will first briefly explain the non-allelic homologous recombination (NAHR) mechanism that underlines the origin of recurrent microdeletions and microduplications. Then we will describe eight new syndromes, four microdeletions (del 17q21.31, del 3q29, del 15q24, del 2q32.3q33) and four microduplications (dup 22q11.2, dup 7q11.23, dup 17p11.2, duplication of MECP2). A better knowledge of these new recurrent chromosomal syndromes will allow to improve care for patients, to develop targeted chromosomal diagnosis and to identify genes involved in neurocognitive functions.