Jérôme Toutain

Confined placental mosaicism and pregnancy outcome: a distinction needs to be made between types 2 and 3.

To study the influence of types 2 and 3 confined placental mosaicism (CPM) on pregnancy outcome.

New candidate loci identified by array‐CGH in a cohort of 100 children presenting with syndromic obesity

Syndromic obesity is defined by the association of obesity with one or more feature(s) including developmental delay, dysmorphic traits, and/or congenital malformations. Over 25 syndromic forms of obesity have been identified. However, most cases remain of unknown etiology. The aim of this study was to identify new candidate loci associated with syndromic obesity to find new candidate genes and to better understand molecular mechanisms involved in this pathology. We performed oligonucleotide microarray‐based comparative genomic hybridization in a cohort of 100 children presenting with syndromic obesity of unknown etiology, after exhaustive clinical, biological, and molecular studies. Chromosomal copy number variations were detected in 42% of the children in our cohort, with 23% of patients with potentially pathogenic copy number variants. Our results support that chromosomal rearrangements are frequently associated with syndromic obesity with a variety of contributory genes having relevance to either obesity or developmental delay. A list of inherited or apparently de novo duplications and deletions including their enclosed genes and not previously linked to syndromic obesity was established. Proteins encoded by several of these genes are involved in lipid metabolism (ACOXL, MSMO1, MVD, and PDZK1) linked with nervous system function (BDH1 and LINGO2), neutral lipid storage (PLIN2), energy homeostasis and metabolic processes (CDH13, CNTNAP2, CPPED1, NDUFA4, PTGS2, and SOCS6).

Prenatal diagnosis using array-CGH: a French experience.

Array-CGH or Chromosomal Microarray Analysis (CMA) is increasingly used in prenatal diagnosis throughout the world. However, routine practices are very different among centers and countries, regarding CMA indications, design and resolution of microarrays, notification and interpretation of Copy Number Alterations (CNA). We present our data and experience from our Fetal Medicine Center on 224 prospective prenatal diagnoses. Our approach is practical, and aims to propose a strategy to offer Chromosomal Microarray Analysis (CMA) to selected fetuses and to help to interpret CNA. We hope that this publication could encourage development of CMA in centers that have not started yet this activity in prenatal routine, and could contribute to edict guidelines in this field.

Reduced placental telomere length during pregnancies complicated by intrauterine growth restriction.

Objectives Recent studies have shown that telomere length was significantly reduced in placentas collected at delivery from pregnancies complicated by intrauterine growth restriction secondary to placental insufficiency. Placental telomere length measurement during ongoing pregnancies complicated by intrauterine growth restriction has never been reported. This was the main objective of our study. Methods In our center, late chorionic villus samplings were performed between 18 and 37 weeks of amenorrhea in 24 subjects with severe intrauterine growth restriction (cases) and in 28 subjects with other indications for prenatal diagnosis (controls). Placental insufficiency was assessed by histo-pathological examination. Relative measurement of telomere length was carried out prospectively by quantitative Fluorescent In Situ Hybridization using fluorescent Peptide Nucleic Acid probes on interphase nuclei obtained from long-term cultured villi and with an automated epifluorescent microscope. A quantitative Polymerase Chain Reaction technique was performed to confirm the quantitative Fluorescent In Situ Hybridization results. The number of copies of gene loci encoding the RNA template (hTERC) and the catalytic subunit (hTERT) of the enzyme complex telomerase were also estimated in these placentas by Fluorescent In Situ Hybridization. Results Mean fluorescence intensity of telomere probes estimated by quantitative Fluorescent In Situ Hybridization was significantly less for cases compared to controls (p<0.001). This result indicated that mean telomere length was significantly reduced in placentas during pregnancies complicated by intrauterine growth restriction. Reduced telomere length was confirmed by the quantitative Polymerase Chain Reaction technique. No copy number variation of the hTERC and hTERT loci was noticed for cases, or for controls. Conclusion This study clearly demonstrates a reduction of placental telomere length in ongoing pregnancies (from 18 to 37 weeks of amenorrhea) complicated by severe intrauterine growth restriction secondary to placental insufficiency.

A freehand ultrasonographically guided technique in transabdominal chorionic villus sampling in more than 24 000 consecutive cases

We read with a great deal of interest the article by Calda and Brestak about chorionic villus vacu-sampling using the Vacutainer system (Calda and Brestak, 2009). We take this opportunity to reply to the authors as the published technique and the results obtained were not in agreement with our own practical experience. Since 1983, in our prenatal diagnostic center we have carried out chorionic villus sampling on more than 24 000 women using first the transcervical route and then the transabdominal route. The transcervical route was used from 1983 to 1988, but we then abandoned this method because of a very high rate of miscarriages linked with the procedure (∼4%). Since 1988, we have carried out transabdominal chorionic villus sampling exclusively, using the same technique between the 12th and the 37th week of amenorrhea. The technique we use is based on that described by Smidt-Jensen and Hahnemann (1984). It consists of an extra-amniotic transabdominal chorionic villus sampling puncture, monitored with ultrasound, using a 20-gauge needle rinsed with heparin serum then linked to a 20mL syringe by a plastic extension tube (Saura et al., 1992). The rate of miscarriage when using the 20-gauge needle in our medical fetal center is the same as for amniocentesis (Brun et al., 2003). Our first point is that the 20-gauge needle we use for chorionic villus sampling causes no more pain than amniocentesis, and therefore has the advantage of not requiring a local anesthetic prior to the chorionic villus puncture, which is necessary with an 18-gauge needle (Calda and Brestak, 2009; Vandenbossche et al., 2007). Next, we stress the importance of the quantity of chorionic villi sampled. It is the quantity of chorionic villi fragments that will determine the culture time, and thus ultimately, the advantage of using the chorionic villus sampling method. The smaller this quantity, the longer it takes to obtain conventional karyotyping of the culture. It may take up to 15–20 days, or even longer, if the quantity sampled is less than 5 mg, as reported by the authors. In this case, the advantage to be obtained from chorionic villus sampling is very much reduced and the benefits of choriocentesis over amniocentesis become debatable. Thus, the threshold amount of chorionic villi that is sufficient, and which the authors set at 5 mg, seems to us to be completely inadequate. We believe that a large amount of chorionic material should be sampled (more than 15–20 mg) mainly in order to obtain the conventional karyotype after 6–8 days culture. In our opinion, it is essential to obtain about 15–20 mg of chorionic villi in order to:

A French multicenter study of over 700 patients with 22q11 deletions diagnosed using FISH or aCGH

Although 22q11.2 deletion syndrome (22q11.2DS) is the most recurrent human microdeletion syndrome associated with a highly variable phenotype, little is known about the condition’s true incidence and the phenotype at diagnosis. We performed a multicenter, retrospective analysis of postnatally diagnosed patients recruited by members of the Association des Cytogénéticiens de Langue Française (the French-Speaking Cytogeneticists Association). Clinical and cytogenetic data on 749 cases diagnosed between 1995 and 2013 were collected by 31 French cytogenetics laboratories. The most frequent reasons for referral of postnatally diagnosed cases were a congenital heart defect (CHD, 48.6%), facial dysmorphism (49.7%) and developmental delay (40.7%). Since 2007 (the year in which array comparative genomic hybridization (aCGH) was introduced for the routine screening of patients with intellectual disability), almost all cases have been diagnosed using FISH (96.1%). Only 15 cases (all with an atypical phenotype) were diagnosed with aCGH; the deletion size ranged from 745 to 2904 kb. The deletion was inherited in 15.0% of cases and was of maternal origin in 85.5% of the latter. This is the largest yet documented cohort of patients with 22q11.2DS (the most commonly diagnosed microdeletion) from the same population. French cytogenetics laboratories diagnosed at least 108 affected patients (including fetuses) per year from among a national population of ∼66 million. As observed for prenatal diagnoses, CHDs were the most frequently detected malformation in postnatal diagnoses. The most common CHD in postnatal diagnoses was an isolated septal defect.

High‐resolution array‐CGH in patients with oculocutaneous albinism identifies new deletions of the TYR, OCA2, and SLC45A2 genes and a complex rearrangement of the OCA2 gene

Oculocutaneous albinism (OCA) is caused by mutations in six different genes, and their molecular diagnosis encompasses the search for point mutations and intragenic rearrangements. Here, we used high‐resolution array‐comparative genome hybridization (CGH) to search for rearrangements across exons, introns and regulatory sequences of four OCA genes: TYR, OCA2, TYRP1, and SLC45A2. We identified a total of ten new deletions in TYR, OCA2, and SLC45A2. A complex rearrangement of OCA2 was found in two unrelated patients. Whole‐genome sequencing showed deletion of a 184‐kb fragment (identical to a deletion previously found in Polish patients), whereby a large portion of the deleted sequence was re‐inserted after severe reshuffling into intron 1 of OCA2. The high‐resolution array‐CGH presented here is a powerful tool to detect gene rearrangements. Finally, we review all known deletions of the OCA1–4 genes reported so far in the literature and show that deletions or duplications account for 5.6% of all mutations identified in the OCA1–4 genes.

1.5 Mb microdeletion in 15q24 in a patient with mild OAVS phenotype.

We report on a boy presenting with features of OAVS (Oculoauriculovertebral spectrum) and carrying a 1.5 Mb microdeletion in 15q24.1q24.2. This recurrent deletion usually leads to a broad clinical spectrum but has never been found associated with features of OAVS such as ear agenesis. This observation is in accordance with OAVS being a genetically heterogeneous disorder, and points out the importance of array-CGH screening in this disorder.

Expanding the clinical phenotype at the 3q13.31 locus with a new case of microdeletion and first characterization of the reciprocal duplication

Congenital deletions at the 3q13.31 locus have been recently described as a novel microdeletion syndrome characterized by developmental delay, postnatal overgrowth, hypoplastic male genitalia and characteristic facial features. A common critical region of overlapping of 580kb was delineated including two strong candidate genes for developmental delay: DRD3 and ZBTB20. In this report, we describe a new case of 3q13.31 microdeletion identified by array-CGH in a 16year-old girl sharing clinical features commonly observed in the 3q13.31 microdeletion syndrome. This girl had a microdeletion of 7.39Mb spanning the common critical region of overlapping. More interestingly, we report for the first time the existence of a microduplication reciprocal to the microdeletion syndrome. This familial 2.76Mb microduplication identified by array-CGH was carried by two brothers and their father. The phenotype shared by the brothers resembled the phenotype related to the 3q13.31 microdeletion syndrome including especially severe intellectual disability, developmental delay, behavioral abnormalities and obesity. This microduplication involves three strong candidate genes for the developmental delay ZBTB20, LSAMP and GAP43. Further molecular characterization showed that DRD3, another strong candidate gene for developmental delay, was not included in the duplicated region. However, a dosage alteration of this gene cannot be completely excluded as the duplication was inverted at proximity of this gene, as revealed by FISH analysis. Finally, we hypothesized that the phenotype shared by the two brothers could be related to a gene dosage imbalance even if gene expression could not be measured in relevant tissues such as brain or adipocytes.

Prenatal diagnosis of 24 cases of microduplication 22q11.2: an investigation of phenotype‐genotype correlations

Microduplication 22q11.2 is primarily characterized by a highly variable clinical phenotype, which ranges from apparently normal or slightly dysmorphic features (in the presence or absence of learning disorders) to severe malformations with profound mental retardation. Hence, genetic counseling is particularly challenging when microduplication 22q11.2 is identified in a prenatal diagnosis. Here, we report on 24 prenatal cases of microduplication 22q11.2.