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.

[Analysis of prenatal follow-up strategies for trisomy 21 affected pregnancies in France]. / Analyse du parcours de dépistage prénatal des femmes ayant donné naissance en France à un enfant atteint de trisomie 21.

OBJECTIVE: The main objective of this study was to screen the prenatal follow-up of women with live birth trisomy 21 child in order to evaluate the proportion of prenatal screening failure versus cases where the women refused either the screening or the prenatal diagnosis of Down syndrome. This study covers the period of time from 2009 to 2012 when the national prenatal screening policy changed from second to first trimester and allows for a comparative assessment of the nationwide efficiency of the various maternal serum marker based strategies. METHOD: All authorized cytogenetic laboratories sent required data for all cases of trisomy 21 diagnosed in FRANCE in new-borns (less than 1-year-old) from January 2010 to July 2013. RESULTS: A total of 1253 cases of trisomy 21 were diagnosed before 1 year of age whose mother did not had prenatal diagnosis. For 861 of them, information on the prenatal follow-up was available, with 72% of cases where a prenatal screening was organized either by maternal serum marker or by ultrasound. Results of the screening strategy was positive with maternal serum marker in 28% of cases (calculated risk≥1/250), positive because of abnormal ultrasound in 5% and negative with maternal marker screening (whatever the strategy used) in 67% of cases. Detection rate over the period of the study was 82%, with similar efficiency of first and second trimester strategies (83%) but significantly lower with sequential association of first trimester Nuchal translucency measurement and second trimester serum screening (70%). CONCLUSION: Switching from second trimester to first trimester screening strategy, with as many trisomy 21 foetuses diagnosed with half invasive procedures fulfilled national health policy objectives. Analysis of these data gives useful insights to elaborate a future screening policy involving cell-free foetal DNA sequencing.

Diagnostic genetic screening for assisted reproductive technologies patients with macrozoospermia.

Macrozoospermia is characterized by a high proportion of abnormal spermatozoa with enlarged heads. So far, it has been associated with mutations only in the Aurora Kinase C gene (AURKC) in some cases. Although many publications have reported failure to conceive in couples with macrozoospermia, a few others have described successful pregnancies, thus raising questions as to whether ICSI and AURKC genetic screening should be recommended in all patients with macrozoospermia. First, we report on two monozygotic twins presenting macrozoospermia for whom the genetic status was explored (Aurora Kinase C sequencing) and whole semen and gradient-selected spermatozoa were analyzed, using Fluorescent In Situ Hybridization (FISH), Electron Microscopy and flow cytometry. Additionally, FISH analysis was performed on individually selected uniflagellate spermatozoa with normal sized heads. Second, we also provide an updated review of patients with macrozoospermia gathering the percentage of enlarged head spermatozoa, the genetic status and pregnancy outcomes. Both twins carried a homozygous mutation of AURKC. Spermocytograms showed means of 86% and 83.5% of enlarged head forms. FISH analyses showed that normal head size, uniflagellate spermatozoa had an aneuploid or polyploid nucleus despite a high level of selection. SEM analysis also showed special intranuclear inclusions in enlarged head spermatozoa. Our data together with cases reported in the literature allowed us to recommend that the AURKC gene should be sequenced when the sperm contains 30% or more of enlarged head spermatozoa, and when a mutation is found, ART should not be performed. Our analyses provide information that could greatly help practitioners in their decision-making with regard to optimal care of patients with macrozoospermia.

Aneuploidy: the impact of chromosome imbalance on nuclear organization and overall genome expression.

The organization and dynamics of chromatin within the interphase nucleus as chromosome territories (CTs) and the relationship with transcriptional regulation are not fully understood. We studied a natural example of chromosomal disorganization: aneuploidy due to trisomies 13, 18 and 21. We hypothesized that the presence of an extra copy of one chromosome alters the CT distribution, which perturbs transcriptional activity. We used 3D-FISH to study the position of the chromosomes of interest (18 and 21) in cultured amniocytes and chorionic villus cells from pregnancies with a normal or aneuploid karyotype. We studied the volumes of nuclei and CTs in both conditions and performed a compared transcriptome analysis. We did not observe any differences between euploid and aneuploid cells in terms of the radial and relative CT positions, suggesting that the same rules govern nuclear organization in cases of trisomy. We observed lower volumes for CTs 18 and 21. Overall genome expression profiles highlighted changes in the expression of a subset of genes in trisomic chromosomes, while the majority of transcriptional changes concerned genes located on euploid chromosomes. Our results suggest that a dosage imbalance of the genes on trisomic chromosomes is associated with a disturbance of overall genomic expression.

Inverted duplication with deletion: first interstitial case suggesting a novel undescribed mechanism of formation.

Inverted duplications with terminal deletions are a well-defined family of complex rearrangements already observed for most of chromosome extremities. Several mechanisms have been suggested which could lead to their occurrence, either through non-homologous end joining, non-allelic homologous recombination, or more recently through an intrastrand fold-back mechanism. We describe here a patient with intellectual disability and pharmacoresistant epilepsy, for which array CGH analysis showed the first interstitial case of inverted duplication with deletion on chromosome 1p. Furthermore, SNP array analysis revealed an associated segmental isodisomy for the distal part of 1p, which led us to consider a replicative mechanism to explain this abnormality. This observation extends the range of this once telomeric rearrangement.

3D position of pericentromeric heterochromatin within the nucleus of a patient with ICF syndrome.

ICF (immunodeficiency, centromeric region instability, facial anomalies) syndrome is a rare autosomal recessive disorder characterised by severe immunodeficiency, craniofacial anomalies and chromosome instability. Chromosome analyses from blood samples show a high frequency of decondensation of pericentromeric heterochromatin (PH) and rearrangements involving chromosomes 1 and 16. It is the first and, as far as we know, the only disease associated with a mutation in a DNA methyltransferase gene, DNMT3B, with significant hypomethylation of the classical satellite DNA, the major component of the juxtacentromeric heterochromatin. To better understand the complex links between the hypomethylation of the satellite DNA, the cytogenetic anomalies and the clinical features of ICF syndrome, we performed three-dimensional (3D) FISH on preserved cells from a patient with a suspected ICF phenotype. Analysis of DNMT3B did not reveal any mutation in our patient, making this case an ICF type 2. The results of 3D-FISH showed a statistically significant change in the intranuclear position of PH of chromosome 1 in cells of the patient as compared to normal cells. It is difficult to understand how a defect in the methylation pathway can be responsible for the various symptoms of this condition. From our observations we suggest a mechanistic link between the reorganisation of the nuclear architecture and the altered gene expression.

A randomized phase II study comparing induction or consolidation chemotherapy with cisplatin-docetaxel, plus radical concurrent chemoradiotherapy with cisplatin-docetaxel, in patients with unresectable locally advanced non-small-cell lung cancer.

BACKGROUND: In stage III non-small-cell lung cancer (NSCLC), the role of systemic chemotherapy preceding or following concurrent chemo-radiotherapy (CT-RT) is unclear. We carried out a randomized phase II study to study the toxicity involved-field CT-RT with either induction or consolidation cisplatin-docetaxel (Taxotere). PATIENTS AND METHODS: Patients were randomly assigned to receive two cycles of docetaxel (D) 75 mg/m(2) on day 1 and cisplatin (C) 40 mg/m(2) on days 1 and 2, either preceding (IND arm) or following (CON arm) concurrent CT-RT, where 66 Gy was delivered using involved-fields concurrent with weekly D 20 mg/m(2) and C 20 mg/m(2). Patients at higher risk for lung toxicity (V(20) > 35%) crossed over to IND arm. Seventy patients were needed to exclude grade (G)3-4 esophagitis in >25%. RESULTS: Of the 70 eligible patients, 26 were treated in IND and 34 CON; five with V(20) >35% switched from CON to IND. The differences in G3-4 esophagitis observed (32/2% IND versus 21/3% CON) were not significantly different from the hypothesized 25% rate. Rates of G≥2 pneumonitis were similar, but IND arm had less G3-4 neutropenia. One-year survival was 63.2% [95% confidence interval (CI) 48.4% to 78.0%] and 65.5% (95% CI 48.2% to 82.8%) for the IND and CON arms, respectively. CONCLUSION: Both study arms merit further testing in patients with limited volume stage III NSCLC.

[Genetic testing in the context of the revision of the French law on bioethics]. / Les tests génétiques à l'heure de la deuxième révision des lois de bioéthique.

This article focuses on six questions raised by genetic testing in human: (1) the use of genetic tests, (2) information given to relatives of patients affected with genetic disorders, (3) prenatal and preimplantatory diagnosis for late onset genetic diseases and the use of pangenomic tests in prenatal diagnosis, (4) direct-to-consumer genetic testing, (5) population screening in the age of genomic medicine and (6) incidental findings when genetic testing are used.

Case report: Meiotic segregation in spermatozoa of a 46,X,t(Y;10)(q11.2;p15.2) fertile translocation carrier.

Translocations involving gonosomes are frequent in azoospermic patients and sometimes in oligozoospermic ones, conditions that lead to request for assisted reproduction treatment. This study reports an unexpectedly fertile 49-year-old man bearing a de-novo translocation 46,X,t(Y;10)(q11.2;q15.2) associated with a high chromosomal risk for offspring, and referred for familial investigations after the diagnosis of an unbalanced translocation 46,XX,der(10)t(Y;10)(q11.2;p15.2) in his naturally conceived and mentally retarded daughter. Chromosome molecular investigation confirmed Y long-arm inheritance in the daughter and absence of the Yq deletion in the father. Semen analysis showed a normal sperm count associated with moderate asthenospermia and severe teratospermia. A total of 984 spermatozoa were analysed using fluorescence in-situ hybridization (FISH). Alternate segregation pattern was found in 50.31% of the spermatozoa studied. The frequencies of adjacent I, adjacent II, 3:1 segregation, and diploidy (or 4:0 segregation) were respectively 39.62, 1.63, 7.83, and 0.61%. No interchromosomal effect was observed. This patient is the first fertile man in whom the meiotic segregation pattern of a Y-autosome translocation has been analysed. The imbalance risk was close to those observed for reciprocal translocations, and emphasizes the value of FISH studies in males with a chromosomal translocation in order to provide them a personalized risk evaluation.

Early and severe amyloidosis in a patient with concurrent familial Mediterranean fever and pseudoxanthoma elasticum.

A young woman patient had early and extensive familial Mediterranean fever (FMF)-related amyloidosis and pseudoxanthoma elasticum (PXE). She had the novel G1042S mutation in the ATP-binding cassette subfamily C member 6 (ABCC6) gene, responsible for PXE, and the mutation M694I in MEFV, the FMF gene. Both mutations were homozygous, in agreement with consanguinity in the parents. ABCC6 deficiency may have increased the severity of amyloidosis by increasing the deposition in target tissues of heparan sulphate, which colocalizes spatially and temporally with amyloid proteins, and/or by decreasing the therapeutic activity of colchicine.

[Topographical organisation of the chromatin in human interphase nuclei: architecture meets function]. / Organisation de la chromatine au sein du noyau interphasique: l'art du rangement fonctionnel.

There are an estimated number of 30,000 genes in the human genome, accounting for as few as 5% of the whole DNA content. Determining the exact role of the vast majority of untranscribed DNA is a major goal for upcoming years. Among various evolutionary constrains which could explain the presence of such a quantity of so-called "junk DNA", one hypothesis is the necessary controlled topographical arrangement of the genome during interphase, leading to a non-random, reproducible position of chromosomal regions inside the nucleus. This hypothesis relies on recent progresses in imaging technologies such as fluorescence confocal microscopy, allowing for the first time the identification of each chromosome-specific chromatin during interphase. This review focuses on the past years advances leading to the actual model of chromosome territories in the interphase nucleus.

Pregnancy outcome following prenatal diagnosis of an isodicentric X chromosome: first case report.

An isodicentric X chromosome, idic (X)(q27) was found in a female fetus during cytogenetic studies performed on amniotic cells due to advanced maternal age. No mosaicism was observed. Although segmental inversion duplications have been described for several other chromosomes, isodicentric chromosomes are reported only for gonosomes. Genetic counselling was based on ultrasound findings, cytogenetic replication studies and published cases of X chromosomes duplications ascertained pre- and postnatally. The pregnancy resulted in the birth of a healthy female infant.

French multi-centric study of 2000 amniotic fluid interphase FISH analyses from high-risk pregnancies and review of the literature.

This prospective and multi-centric study confirms the accuracy and the limitations of interphase FISH and shows that any cytogenetics laboratory can perform this technique. With regard to the technical approach, we think that slides must be examined by two investigators, because the scoring may be subjective. The main problem with the AneuVysion kit concerns the alpha satellite probes, and especially the chromosome 18 probe, which is sometimes very difficult to interpret because of the high variability of the size of the spots, and this may lead to false negative and uninformative cases. The best solution would be to replace these probes by locus-specific probes. Concerning clinical management, we offer interphase FISH only in very high-risk pregnancies or/and at late gestational age because of the cost of the test. We think that an aberrant FISH result can be used for a clinical decision when it is associated with a corresponding abnormal ultrasound scan. In other cases, most of the time, we prefer to wait for the standard karyotype.

Heteromorphism 18ph+ : with or without reproductive consequences?

Heteromorphism or chromosomal variants are usually attributed to structural variations in constitutive heterochromatin. In the case of chromosome 18, 25 cases of 18ph+ have been reported to date. Using the Primed In Situ Labelling technique (PRINS) to study 2 new cases of 18ph+, we have been able to confirm their molecular nature and assuming a mechanism of formation. Although such chromosomal variants are usually thought to have no adverse clinical consequence, a review of the literature shows that many cases were diagnosed because of recurrent abortion, malformed or mentally retarded children suggesting the possible relationship between 18ph+ and such clinical outcomes.

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis.

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.

[Genetic basis of Prader-Willi and Angelman syndromes: implications for the biologic diagnosis]. / Bases génétiques des syndromes de Prader-Willi et d'Angelman: implications pour la conduite du diagnostic biologique.

Prader-Willi and Angelman syndromes are two genetic diseases whose clinical diagnosis is often impaired by a wide variability in some clinical findings. New insights in the genetic basis of these disorders allow the proposition of a biological approach to detect almost all Prader-Willi syndrome patients and over 80% of Angelman syndrome patients. Moreover, the results of these tests are indispensable for the evaluation of the recurrence risk.

Analysis of megakaryocyte growth and development factor (thrombopoietin) effects on blast cell and megakaryocyte growth in myelodysplasia.

Thrombocytopenia is a frequent feature of myelodysplastic syndromes (MDS) that could be improved by the use of recombinant human megakaryocyte growth and development factor (rHuMGDF). Using short-term liquid cultures and progenitor assays, we have found that rHuMGDF stimulated DNA synthesis and potentiated leukemic cluster growth of bone marrow mononuclear cells in 10/38 MDS cases (26%). Cytogenetically malignant colonies were detectable in rHuMGDF-stimulated cultures (n=3) by fluorescence in situ hybridization. rHuMGDF was able to stimulate CFU-MK formation in 45% of the samples tested. Finally, rHuMGDF-induced blast cell proliferation correlated with elevated expression of c-MPL, previously identified as a bad prognosis factor in MDS.