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Chromoanagenesis are complex chromosomal rearrangements that are supposed to occur during a single catastrophic event. They may result in loss or gain of genetic material and may be responsible for various phenotypes. These rearrangements are usually sporadic. However, some familial cases have been reported. Here, we studied six families in which a healthy parent carrying a chromoanagenesis transmitted its rearrangement in an unbalanced manner to its descent. The rearrangements were characterized by karyotype, fluorescent in situ hybridization, chromosomal microarray and whole genome sequencing (WGS) in the parents and their offspring. We then hypothesized meiosis-pairing figures of parental chromosomes that may have led to meiotic recombination between normal and abnormal homologous chromosomes, resulting in the formation of new unbalanced rearrangements. This work underlines that chromoanagenesis can be associated with a normal phenotype and a normal fertility, even in males, and that WGS may be the unique way to identify these rearrangements in some cases. Chromoanagenesis can be transmitted in an unbalanced and unpredictable way because of meiotic recombination. Thus, genetic counseling and technical choice for prenatal diagnosis are complex. Finally this work questions the meiotic pairing mechanisms of complex rearrangements, still poorly understood.

Chromosome 1p36 deletion syndrome (1p36DS) is one of the most common terminal deletion syndromes (incidence between 1/5000 and 1/10,000 live births in the American population), due to a heterozygous deletion of part of the short arm of chromosome 1. The 1p36DS is characterized by typical craniofacial features, developmental delay/intellectual disability, hypotonia, epilepsy, cardiomyopathy/congenital heart defect, brain abnormalities, hearing loss, eyes/vision problem, and short stature. The aim of our study was to (1) evaluate the incidence of the 1p36DS in the French population compared to 22q11.2 deletion syndrome and trisomy 21; (2) review the postnatal phenotype related to microarray data, compared to previously publish prenatal data. Thanks to a collaboration with the ACLF (Association des Cytogénéticiens de Langue Française), we have collected data of 86 patients constituting, to the best of our knowledge, the second-largest cohort of 1p36DS patients in the literature. We estimated an average of at least 10 cases per year in France. 1p36DS seems to be much less frequent than 22q11.2 deletion syndrome and trisomy 21. Patients presented mainly dysmorphism, microcephaly, developmental delay/intellectual disability, hypotonia, epilepsy, brain malformations, behavioral disorders, cardiomyopathy, or cardiovascular malformations and, pre and/or postnatal growth retardation. Cardiac abnormalities, brain malformations, and epilepsy were more frequent in distal deletions, whereas microcephaly was more common in proximal deletions. Mapping and genotype-phenotype correlation allowed us to identify four critical regions responsible for intellectual disability. This study highlights some phenotypic variability, according to the deletion position, and helps to refine the phenotype of 1p36DS, allowing improved management and follow-up of patients.

International audience; Inverted duplication deletion 8p [invdupdel(8p)] is a complex and rare chromosomal rearrangement that combines a distal deletion and an inverted interstitial duplication of the short arm of chromosome 8. Carrier patients usually have developmental delay and intellectual disability (ID), associated with various cerebral and extra-cerebral malformations. Invdupdel(8p) is the most common recurrent chromosomal rearrangement in ID patients with anomalies of the corpus callosum (AnCC). Only a minority of invdupdel(8p) cases reported in the literature to date had both brain cerebral imaging and chromosomal microarray (CMA) with precise breakpoints of the rearrangements, making genotype-phenotype correlation studies for AnCC difficult. In this study, we report the clinical, radiological, and molecular data from 36 new invdupdel(8p) cases including three fetuses and five individuals from the same family, with breakpoints characterized by CMA. Among those, 97% (n = 32/33) of patients presented with mild to severe developmental delay/ID and 34% had seizures with mean age of onset of 3.9 years (2 months-9 years). Moreover, out of the 24 patients with brain MRI and 3 fetuses with neuropathology analysis, 63% (n = 17/27) had AnCC. We review additional data from 99 previously published patients with invdupdel(8p) and compare data of 17 patients from the literature with both CMA analysis and brain imaging to refine genotype-phenotype correlations for AnCC. This led us to refine a region of 5.1 Mb common to duplications of patients with AnCC and discuss potential candidate genes within this region.

Chromosomal aberrations including structural variations (SVs) are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics. Drawbacks of these tests are a very low resolution (karyotyping) and the inability to detect balanced SVs or indicate the genomic localization and orientation of duplicated segments or insertions (copy number variant [CNV] microarrays). Here, we investigated the ability of optical genome mapping (OGM) to detect known constitutional chromosomal aberrations. Ultra-high-molecular-weight DNA was isolated from 85 blood or cultured cells and processed via OGM. A de novo genome assembly was performed followed by structural variant and CNV calling and annotation, and results were compared to known aberrations from standard-of-care tests (karyotype, FISH, and/or CNV microarray). In total, we analyzed 99 chromosomal aberrations, including seven aneuploidies, 19 deletions, 20 duplications, 34 translocations, six inversions, two insertions, six isochromosomes, one ring chromosome, and four complex rearrangements. Several of these variants encompass complex regions of the human genome involved in repeat-mediated microdeletion/microduplication syndromes. High-resolution OGM reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints. This proof-of-principle study demonstrates the ability of OGM to detect nearly all types of chromosomal aberrations. We also suggest suited filtering strategies to prioritize clinically relevant aberrations and discuss future improvements. These results highlight the potential for OGM to provide a cost-effective and easy-to-use alternative that would allow comprehensive detection of chromosomal aberrations and structural variants, which could give rise to an era of "next-generation cytogenetics."

Background Idiopathic central precocious puberty (ICPP) is supposed to be non-existent in a context of testicular destruction that is typically present in Klinefelter syndrome (KS). Herein, we describe a rare case of ICPP in a Klinefelter patient (47,XXY) with 2 maternal X chromosomes. Moreover, we highlight the differences in gonadotropin levels in comparison to males with ICPP and a normal karyotype. Case presentation An 8 years old boy with a history of cryptorchidism was evaluated for precocious puberty (Tanner staging: P2/G3). Both testes measured 25x35mm. His hormonal profile confirmed a central origin of precocious puberty with high serum testosterone (4.3 ng/ml), luteinizing hormone [LH (3.5 UI/l)] and follicle stimulating hormone [FSH (7.7 UI/l)] levels. Luteinizing hormone-releasing hormone (LHRH) test amplified LH and FSH secretion to 24 and 14 UI/l respectively. Brain magnetic resonance imaging (MRI) was normal. No MKRN3 mutation was detected. He was treated for ICPP for two years. During puberty, he suffered from hypergonadotropic hypogonadism leading to the diagnosis of KS (47,XXY karyotype). Chromosomal analysis by fluorescent multiplex polymerase chain reaction (PCR) using X chromosome microsatellite markers identified 2 maternal X chromosomes. Analysing 8 cases of KS developing ICPP (our reported case and 7 other published cases) revealed that these KS patients with ICPP have higher LH and FSH levels during ICPP episode than in ICPP patients with a normal karyotype (ICPP with KS vs ICPP with a normal karyotype: LH levels 9.4 ± 12 vs 1.1 ± 0.6 UI/l; FSH levels 23.1 ± 38.5 vs 2.7 ± 1.5 UI/l). Furthermore, their response to gonadotropin-releasing hormone (GnRH) stimulation is characterized by excessive LH and FSH secretion (LH levels post-GnRH: 58 ± 48 vs 15.5 ± 0.8 UI/l; FSH levels post-GnRH: 49.1 ± 62.1 vs 5.7 ± 3.9 UI/l). Conclusions ICPP in boys is extremely rare. The pathophysiology of ICPP in KS is unknown. However, maternal X supplementary chromosome and early testicular destruction may play a significant role in the initiation of ICPP, in part explaining the relative “overrepresentation of ICPP in KS. Thus, karyotype analysis could be considered for boys suffering from ICPP, especially if testicular size is smaller or gonadotropins are significantly elevated.

Objective Principal objective of this work was to analyse the cost effectiveness of different sequences of cytogenetic techniques from the hospital's point of view, after prenatal ultrasound has identified fetal malformations. Methods Cytogenetic tests were performed for each case in 3 strategies, and their results are reported and compared to one reference strategy. Two new simulated strategies were considered: chromosomal microarrays alone and a direct test + CMA. Main outcomes measures cost-effectiveness ratio. Results A single test result was positive in 234 of the 835 pregnancies studied (28%). CMA alone would have identified 239 abnormalities. In the simulated direct test + CMA sequence, the direct test alone would have been positive for 66.1% of the abnormalities identified. When testing was indicated for NT, reference strategy (Direct + karyotyping) costs 1 084.8 euros by positive test results. Strategies Direct + CMA and CMA alone cost respectively 992.7 and 550.0 euros by positive test results. For OUM indications, reference strategy costs 2 937.8 euros by positive test results. Strategies Direct + CMA and CMA alone cost respectively, 2 118.4 and 1 304.7 euros by positive test results. Conclusions CMA appears to be the most effective test for prenatal cytogenetic diagnosis of fetal abnormalities identified by ultrasound.

Chromosomal aberrations and structural variations are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics, karyotyping and CNV-microarrays, in spite of the low resolution of the first one and the inability to detect neither balanced SVs nor to provide the genomic localization or the orientation of duplicated segments, of the latter. We here investigated the clinical utility of high resolution optical mapping by genome imaging for patients carrying known chromosomal aberrations in a context of constitutional conditions.For 85 samples, ultra-high molecular weight gDNA was isolated either from blood or cultured cells. After labeling, DNA was processed and imaged on the Saphyr instrument (Bionano Genomics). A de novo genome assembly was performed followed by SV and CNV calling and annotation. Results were compared to known aberrations from standard-of-care tests (karyotype, FISH and/or CNV-microarray).In total, we analyzed 100 chromosomal aberrations including 7 aneuploidies, 35 translocations, 6 inversions, 2 insertions, 39 copy number variations (20 deletions and 19 duplications), 6 isochromosomes, 1 ring chromosome and 4 complex rearrangements. High resolution optical mapping reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints.Our study demonstrates the ability of high resolution optical mapping to detect almost all types of chromosomal aberrations within the spectrum of karyotype, FISH and CNV-microarray. These results highlight its potential to replace these techniques, and provide a cost-effective and easy-to-use technique that would allow for comprehensive detection of chromosomal aberrations.

Background The 10q26 subtelomeric microdeletion syndrome is a rare and clinically heterogeneous disorder. The precise relationships between the causative genes and the phenotype are unclear. Case presentation We report two new cases of 860 kb deletion of 10q26.2 identified by array CGH in a fetus with intrauterine growth retardation and his mother. The deleted region encompassed only four coding genes, DOCK1, INSYN2, NPS and FOX12. The proband had dysmorphic facies characterized by a high forehead, malformed ears, a prominent nose, and retrognathia. He had bilateral club feet, clinodactily and mild psychomotor retardation. His mother had a short stature, microcephaly, a long face with a high forehead and bitemporal narrowing, arched and sparse eyebrows, strabismus, prominent nose and chin, a thin upper lip and large protruding ears, and mild intellectual disability. Conclusions This study presents the smallest 10q26.2 deletion so far identified, which further refines the minimal critical region associated with the 10q26 microdeletion syndrome. It focuses on three genes potentially responsible for the phenotype: DOCK1, which is the major candidate gene, and INSYN2 and NPS, which could be involved in cognitive functions.

Le syndrome microdeletionnel 10q26 correspond a un syndrome rare et cliniquement heterogene. La correlation genotype-phenotype reste aujourd’hui mal etablie. Nous rapportons deux nouvelles personnes (un garcon de 22 mois et sa mere) presentant une deletion 10q26.2 de 860 kb. L’ACPA avait ete realisee en prenatal en raison d’un retard de croissance intra-uterin. La region deletee contenait seulement 4 genes codants : DOCK1, INSYN2, NPS and FOX12. A la naissance, le proband presentait des particularites morphologiques faciales avec un front haut, des oreilles mal ourlees, un nez proeminent, et une retrognathie; ainsi que des pieds bots bilateraux et une clinodactylie. A l’âge de 22 mois, il presentait un retard psychomoteur global. Sa mere presentait une taille et un perimetre crânien a la limite inferieure de la normale, un visage allonge avec un front haut et une retraction bitemporale, des sourcils arques et clairsemes, un strabisme, un nez et un menton proeminents, une levre superieure fine et des oreilles larges et decollees. Elle a presente des difficultes d’apprentissage importantes. Cette deletion est la plus petite deletion decrite dans la litterature et permet d’affiner la region minimale critique du syndrome 10q26. Trois genes candidats pourraient participer au phenotype : le gene DOCK1, deja considere comme le gene candidat principal, et les genes INSYN2 and NPS qui pourraient etre impliques dans le phenotype cognitif.

Introduction et objectif Des etudes ont montre que l’incidence du cancer du sein etait plus faible chez les patients trisomiques 21 (T21). Un role protecteur de la matrice extra-cellulaire T21 (MEC-T21) a ete avance. Les tumeurs du sein triple-negatives se caracterisent par un fort potentiel migratoire et invasif pour lequel la transition epithelio-mesenchymateuse, associee a la perte de caracteristiques epitheliales et a l’acquisition d’un phenotype mesenchymateux des cellules, joue un role important. Notre objectif etait d’evaluer la migration des cellules tumorales triple-negatifs (MDA-MB468) sur une MEC-T21 secretee in vitro. Materiels et methodes Une MEC a ete secretee dans des puits de culture par des fibroblastes T21 (MEC-T21) ou des fibroblastes euploides (MEC-EUP). Apres decellularisation, des MDA-MB 468 ont ete cultivees sur les MEC (n = 2 MEC-T21, n = 2 MEC-EUP) ou sur le support plastique de culture (controle, n = 2). Apres 6 jours de contact, un test de blessure a ete realise avec une mesure de l’air de blessure a 0, 2, 4 et 6 heures grâce au logiciel ImageJ®. Les resultats obtenus a H2, H4 et H6 ont ete normalises sur l’aire a H0. L’experience a ete repetee 4 fois. Resultats et discussion Il n’y a pas de difference significative de migration entre les MDA-MB 468 sur MEC-EUP et MEC-T21 quels que soient les temps observes. Cependant, les cellules sur support plastique ont migre plus rapidement que celles sur MEC (p

BackgroundBalanced chromosomal rearrangements associated with abnormal phenotype are rare events, but may be challenging for genetic counselling, since molecular characterisation of breakpoints is not performed routinely. We used next-generation sequencing to characterise breakpoints of balanced chromosomal rearrangements at the molecular level in patients with intellectual disability and/or congenital anomalies.MethodsBreakpoints were characterised by a paired-end low depth whole genome sequencing (WGS) strategy and validated by Sanger sequencing. Expression study of disrupted and neighbouring genes was performed by RT-qPCR from blood or lymphoblastoid cell line RNA.ResultsAmong the 55 patients included (41 reciprocal translocations, 4 inversions, 2 insertions and 8 complex chromosomal rearrangements), we were able to detect 89% of chromosomal rearrangements (49/55). Molecular signatures at the breakpoints suggested that DNA breaks arose randomly and that there was no major influence of repeated elements. Non-homologous end-joining appeared as the main mechanism of repair (55% of rearrangements). A diagnosis could be established in 22/49 patients (44.8%), 15 by gene disruption (KANSL1, FOXP1, SPRED1, TLK2, MBD5, DMD, AUTS2, MEIS2, MEF2C, NRXN1, NFIX, SYNGAP1, GHR, ZMIZ1) and 7 by position effect (DLX5, MEF2C, BCL11B, SATB2, ZMIZ1). In addition, 16 new candidate genes were identified. Systematic gene expression studies further supported these results. We also showed the contribution of topologically associated domain maps to WGS data interpretation.ConclusionPaired-end WGS is a valid strategy and may be used for structural variation characterisation in a clinical setting.

Background Microdeletions encompassing chromosome bands 2q14.1q14.3 are rare. To date, eight reports of relatively large deletions of this region (∼20 Mb) but only two small deletions (

International audience; In the interphase cell nucleus, chromosomes adopt a conserved and non-random arrangement in subnuclear domains called chromosome territories (CTs). Whereas chromosome translocation can affect CT organization in tumor cell nuclei, little is known about how aneuploidies can impact CT organization. Here, we performed 3D-FISH on control and trisomic 21 nuclei to track the patterning of chromosome territories, focusing on the radial distribution of trisomic HSA21 as well as 11 disomic chromosomes. We have established an experimental design based on cultured chorionic villus cells which keep their original mesenchymal features including a characteristic ellipsoid nuclear morphology and a radial CT distribution that correlates with chromosome size. Our study suggests that in trisomy 21 nuclei, the extra HSA21 induces a shift of HSA1 and HSA3 CTs out toward a more peripheral position in nuclear space and a higher compaction of HSA1 and HSA17 CTs. We posit that the presence of a supernumerary chromosome 21 alters chromosome compaction and results in displacement of other chromosome territories from their usual nuclear position.

A, Closed symbols indicate patients affected with cancer. Open symbols indicate healthy individuals. The type of cancer and age at presentation are given in brackets. Blue circle represents c.4471_4474del variant and red circle represents the c.9648 + 1G > A. B, RNA was extracted from blood of patient III-3 and his sisters III-1 and III-4. RT-PCR analysis was performed with primers mapping to exons 25 and 27, and PCR products were separated by Bioanalyzer electrophoresis. The sizes of the DNA marker (M) are indicated to the left. LM, lower marker; UM, upper marker. C, Each RT-PCR product from patient III-3 was gel-purified and analyzed by Sanger sequencing. The 297-bp band corresponds to the reference BRCA2 transcript and the 150-bp band corresponds to a BRCA2 transcript lacking exon 26.

Nous presentons le cas de Camille, petit garcon, ne a terme a 41 + 6 SA par cesarienne pour stagnation du travail. Une hypotonie axiale et peripherique est apparue a la naissance dans un contexte d’anaoxo-ischemie neonatale (APGAR a 0-6-9). La grossesse s’est deroulee sans particularite. Le depistage combine au premier trimestre pour la trisomie 21 a ete calcule a 1/315 (clarte nucale a 0,7 mm). Le depistage sur ADN fœtal libre circulant etait negatif. Devant l’hypotonie majeure de Camille, sans point d’appel particulier, une Analyse sur Puce a ADN (ACPA) a ete prescrite. L’ACPA (Agilent®, puce Oligo 60 K, hg19) a montre des profils de fluorescence complexes au niveau des gonosomes. La realisation d’un caryotype et de FISH a permis de mettre en evidence dans toutes les cellules analysees 47 chromosomes avec presence de deux isodicentriques du bras court du chromosome Y (chromosome X a priori normal), a l’origine d’une tetrasomie de la region du chromosome Y depuis l’extremite du bras court jusqu’a la region Yq11.222 et d’une deletion terminale Yq11.222qter, de novo. Seul un petit nombre d’isodicentrique du bras court du chromosome Y a ete decrit dans la litterature. Les isodicentriques sont des remaniements de structure tres instable liee a la presence des deux centromeres. Ils sont frequemment observes en mosaique associes soit a un contingent 46,XY soit a un clone 45,X. Le tableau phenotypique associe a ces isodicentriques depend donc de la presence ou non d’une mosaique et de la proportion des differents clones mais egalement de la taille des segments concernes. Ici les deux isodicentriques semblent etre retrouves de maniere homogene suggerant une certaine stabilite et laisse envisager des retentissements phenotypiques pour cet enfant assez similaires a ceux dus a la presence d’une polysomie du chromosome Y (susceptibilite accrue au deficit intellectuel, retard des apprentissages, dysmorphie faciale, malformations squelettiques, perturbation de la spermatogenese) [1] , [2] .

Introduction/objectif Devant la faible incidence du cancer du sein chez les patientes trisomiques 21 (T21), un role protecteur de la matrice extracellulaire T21 (MEC-T21) a ete avance. Nos travaux montrent que l’interaction in vitro de cellules d’adenocarcinome mammaire triple-negatif (TNBC) sur une MEC-T21 entraine la deregulation de genes impliques dans la transition epithelio-mesenchymateuse (TEM). Afin de mieux comprendre la regulation de la TEM par les interactions cellule–MEC-T21, nous travaillons a etablir un modele de culture tridimensionnelle (3D) a partir d’une lignee TNBC. Materiels/methodes La production de MEC in vitro a ete realisee a partir de fibroblastes ensemences a 40 000 cellules/cm2. Apres 8 jours de culture, la MEC a ete recuperee par collecte mecanique ou enzymatique (Trypsine-EDTA a 50 μg/mL), suivie ou non par une precipitation (acetone, NaCl) avant dosage proteique (BCA). La culture 3D a partir de MDA-MB-468 a ete realisee par la methode du Liquid Overlay Technique en presence de Geltrex® (Gibco) dans une microplaque « ultra-low-attachment » (Corning® Costar®) (200 a 10 000 cellules/puits, 0 % a 3 % Geltrex®). La morphologie et le diametre des structures cellulaires 3D ont ete suivis de j1 (24 h apres ensemencement) a j14 et la viabilite cellulaire a ete evaluee a j7 et j14 (Live/Dead®, Molecular Probes™). Resultats La quantite de proteines matricielles produites in vitro etait trop faible pour etre utilisee en culture 3D (100–200 μg/mL). Nos resultats montrent que 2000 ou 5000 cellules ensemencees dans 2,5 % ou 3 % de Geltrex® permettent d’obtenir un spheroide qui augmente de diametre entre j1 et j3. Apres j3, les spheroides perdent leur bordure lisse et a j14 un cœur necrotique est observe. Conclusions Les cellules MDA-MB 468 s’organisent en spheroide proliferatif au contact d’un environnement matriciel (Geltrex®). Le protocole actuel de production de MEC in vitro doit etre optimise afin de produire suffisamment de proteines pouvant permettre la formation d’un spheroide en culture 3D.

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.

Research question Is sperm fluorescence in-situ hybridization (FISH) useful to evaluate the risk of chromosomally unbalanced gametes in interchromosomal reciprocal insertion (IRI) carriers? How do these imbalances lead to recurrent miscarriages? Design This study reports a clinical and molecular study of a rare familial balanced IRI resulting in recurrent spontaneous miscarriage. Sperm FISH was performed to estimate the number of unbalanced gametes. Results A 31-year-old healthy male (proband) and his 28-year-old female partner were referred to the Genetics Department for three spontaneous miscarriages occurring during the first trimester of pregnancy. FISH analysis of the proband with the LSI TRA/D (14q11.2) and DiGeorge N25 (22q11.2) break-apart probes showed the presence of a balanced IRI between 14q11.2 and 22q11.2 chromosomal regions. This IRI was also identified in the proband's father. Sperm FISH with the same probes showed that more than 40% of gametes of the proband were unbalanced for either 14q11.2 or 22q11.2, despite normal sperm parameters. FISH analysis of a product of conception indicated that unbalanced gametes result in a non-viable fetus. Conclusions This study shows the value of sperm FISH analysis in improving genetic reproductive advice for IRI carriers. Disruption of critical genes through this rearrangement and their consequent functional impairment could result in recurrent miscarriages. In this case, several genes located in the 14q11.2 region, particularly RNase 3, would be good candidates to explain the lethality of the imbalances.

Microdeletions of 17q12 encompassing TCF2 are associated with maturity-onset of diabetes of the young type 5, cystic renal disease, pancreatic atrophy, Mullerian aplasia in females and variable cognitive impairment. We report on a patient with a de novo 17q12 microdeletion, 1.8 Mb in size, associated with congenital diaphragmatic hernia (CDH). The 5-year-old male patient presented multicystic renal dysplasia kidneys, minor facial dysmorphic features and skeletal anomalies, but neither developmental delay nor behavioral abnormalities. CDH has been previously associated with the 17q12 microdeletion syndrome only in one prenatal case. The present study reinforces the hypothesis that CDH is part of the phenotype for 17q12 microdeletion and that 17q12 encompasses candidate(s) gene(s) involved in diaphragm development. We suggest that PIGW, a gene involved in an early step of GPI biosynthesis, could be a strong candidate gene for CDH. © 2014 Wiley Periodicals, Inc.

High proportion of disease-associated copy number variant maps to chromosome 17. Genomic studies have provided an insight into its complex genomic structure such as relative abundance of segmental duplication and intercepted repetitive elements. 17q21.31, 17q11.2 and 17q12 loci are well known on this chromosome and are associated with microdeletion and microduplication syndrome. No syndrome associated with 17q21.33 locus have been described. We report clinical, cytogenetic and molecular investigations of a 13 years-old girl admitted for evaluation of microcephaly, scoliosis, skeletal defects and learning difficulties. We carried out detailed analysis of the clinical phenotype of this patient and investigated the genetic basis using Agilent 180K Array Comparative Genomic Hybridization. We identified a ∼0.9 Mb de novo microduplication on chromosome 17q21.33. Four genes, COL1A1, SGCA, PPP1R9B and CHAD located within the duplicated region are possible candidates for clinical features present in our patients. Gene expression studies by real-time RT-PCR assay only showed an overexpression of SGCA (P < 0.01), a component of the dystrophin glycoprotein complex. Defect of SGCA was previously shown to lead to severe childhood autosomal recessive muscular dystrophy (LGMD2D) which result in progressive muscle weakness and can also be associated with hyperlordosis or scoliosis. Further cases with similar duplications are expected to be diagnosed. This will contribute to the delineation of this potential new microduplication syndrome and to improve genetic counseling.

Background Interstitial 2q36 deletion is a rare event. Only two previously published cases of 2q36 deletions were characterized using array-CGH. This is the first case diagnosed prenatally. Methods We report on the prenatal diagnosis of a 2q36.1q36.3 interstitial deletion in a fetus with facial dysmorphism, spina bifida, and cleft palate. Results Array-CGH analysis revealed a 5.6 Mb interstitial deletion of the long arm of chromosome 2q36.1q36.3, including the PAX3 and EPHA4 genes. Conclusion The present study reinforces the hypothesis that PAX3 haploinsufficiency may be associated with neural tube defects in humans and suggests that the EPHA4 gene might be implicated during palate development. This report also illustrates the added value of array-CGH to detect cryptic chromosomal imbalances in malformed fetuses and to improve genetic counseling prenatally. Birth Defects Research (Part A) 100:507–511, 2014. © 2014 Wiley Periodicals, Inc.

Introduction/objectif Des etudes epidemiologiques ont montre que l’incidence du cancer du sein etait plus faible chez les patients trisomiques 21 (T21). Un role protecteur de la matrice extracellulaire T21 (MEC-T21) a ete avance. Les tumeurs du sein triple-negatives se caracterisent par un fort potentiel invasif pour lequel la transition epithelio-mesenchymateuse (TEM), associee a la perte de caracteristiques epitheliales et a l’acquisition d’un phenotype mesenchymateux des cellules, joue un role important. Notre objectif etait de determiner si une interaction entre les cellules et une MEC-T21, secretee in vitro, pouvait modifier la TEM d’une lignee d’adenocarcinome mammaire triple-negatif. Materiels et methodes Des cellules MDA-MB 468 ont ete cultivees sur une MEC secretee par des fibroblastes T21 ou euploides (MEC-Eup). L’expression de genes codant des marqueurs epitheliaux (E-Cadherine, occludine, claudin-1) et mesenchymateux (vimentine, fibronectine) a ete evaluee par qRT-PCR en temps reel apres differents temps de culture (j1, j2, j3 et j4). Resultats Le niveau d’expression des genes codant l’occludine et la claudin-1 etait significativement plus eleve a j3 dans les cellules ayant poussees sur une MEC-T21 que dans celles ayant poussees sur une MEC-Eup. Les genes codant la vimentine et la fibronectine etaient significativement moins exprimes sur une MEC-T21 que sur une MEC-Eup a j4. Aucune difference dans l’expression du gene codant la E-cadherine n’a ete observee entre les deux matrices. Conclusion L’expression, au cours du temps, des genes codant les marqueurs de la TEM dans les cellules au contact d’une MEC-T21 est differente de celle observee dans les cellules sur une MEC-Eup, suggerant, d’une part, que les interactions des cellules a la MEC-T21 pourraient modifier la regulation de genes impliques dans la TEM et, d’autre part, que la transdifferenciation des cellules MDA-MB 468 pourrait etre alteree sur une MEC de type trisomie 21.

International audience

International audience; The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMS5), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse.

Microdeletions of 8q21.3–8q22.1 have been identified in all patients with Nablus mask-like facial syndrome (NMLFS). A recent report of a patient without this specific phenotype presented a 1.6 Mb deletion in this region that partially overlapped with previously reported 8q21.3 microdeletions, thus restricting critical region for this syndrome. We report on another case of an 8q21.3 deletion revealed by array comparative genome hybridization (aCGH) in a 4-year-old child with global developmental delay, autism, microcephaly, but without Nablus phenotype. The size of the interstitial deletion was estimated to span 5.2 Mb. By combining the data from previous reports on 8q21.3–8q22.1 deletions and our case, we were able to narrow the critical region of Nablus syndrome to 0.5 Mb. The deleted region includes FAM92A1, which seems to be a potential candidate gene in NMLFS. © 2012 Wiley Periodicals, Inc.

Microduplications 22q11.2 have been recently characterized as a new genomic duplication syndrome showing an extremely variable phenotype ranging from normal or mild learning disability to multiple congenital defects and sharing some overlapping features with DiGeorge/velocardiofacial syndrome (DGS/VCFS), including heart defects, urogenital abnormalities and velopharyngeal insufficiency. We present an atypical and inherited 0.8-Mb duplication at 22q11.2, in the distal segment of the DGS/VCFS syndrome typically deleted region (TDR), in a 3-year-old boy with motor delay, language disorders and mild facial phenotype. This 22q11.2 microduplication was identified by MLPA, designed to detect recurrent microdeletions and microduplications of chromosomal regions frequently involved in mental retardation syndromes and was further characterized by aCGH. The duplicated region encompasses 14 genes, excluding TBX1 but including CRKL, ZNF74, PIK4CA, SNAP29 and PCQAP known to contribute to several aspects of the DGS/VCFS phenotype. To the best of our knowledge, only one case of an isolated duplication in the distal segment of the TDR between chromosome 22-specific low-copy repeats B (LCR22-B) and D (LCR22-D) has been published, but the present report is the first one with a detailed description of physical and developmental features in a patient carrying this kind of atypical 22q11.2 duplication. This case illustrates the importance of reporting unusual 22q11.2 duplications to further evaluate the incidence of these rearrangements in the general population and to improve genotype-phenotype correlations and genetic counseling.

Introduction/Objectifs Les insertions reciproques interchromosomiques sont des rearrangements tres rares. Ils consistent en un echange de segment interstitiel entre deux chromosomes et necessitent donc la presence de quatre points de cassure. Seul 12 cas ont ete publies [1] , [2] , [3] , [4] . Materiels/Patients et methodes Nous rapportons le cas d’un homme de 28 ans adresse dans le service de genetique medicale suite a la survenue de 3 fausses couches spontanees (FCS) chez sa compagne. L’histoire familiale retrouve egalement la notion de 6 FCS chez sa mere. Le caryotype du patient (bandes GTG, RHG ; resolution : 400–550) met en evidence la presence d’un chromosome 14 atypique avec une bande sombre dans la region 14q11 en bandes R. Resultats La FISH de peinture du chromosome 14 montre une absence de marquage sous le centromere d’un des deux chromosome 14 ainsi que la presence de deux signaux sous les centromeres des chromosomes 22 correspondant a l’hybridation croisee des sequences alpha-satellites pericentromeriques. Une FISH LSI DiGeorge N25 (22q11) et LSI TRA/D ba (14q11.2) a confirme la presence d’une insertion reciproque interchromosomique entre les regions chromosomiques 14q11.2 et 22q11.2. L’etude par Sperm FISH montre 40 % des gametes desequilibres malgre des parametres normaux au spermogramme, ainsi qu’un possible effet interchromosomique (EIC). Cette inversion est heritee du pere du patient. Conclusions Les desequilibres de la region 22q11.2 ne semblent pas etre a l’origine des FCS. Les microdeletions de la region 14q11.2 emportant les genes SUPT16H et CDH8 sont rares et associes un retard du developpement, une deficience intellectuelle, des troubles autistiques et une macrocephalie [5] . L’haploinsuffisance des genes APEX1 et PARP2, impliques dans la reparation de l’ADN, et de plusieurs genes de la famille des RNase A pourrait etre impliquee dans la survenue des FCS. La description de cas supplementaire est necessaire pour preciser l’implication d’un desequilibre de la region 14q11.2 dans la survenue de FCS.

Fetuses with increased nuchal translucency thickness (NT) are at increased risk for chromosomal abnormalities. In case of a normal karyotype, a minority of them may present with structural abnormalities or genetic syndromes, which may be related to submicroscopic chromosomal imbalances. The objective of this study was to evaluate whether MLPA screening of 21 syndromic and subtelomeric regions could improve the detection rate of small chromosomal aberrations in fetuses with increased NT and a normal karyotype. A total of 106 prenatal samples from fetuses with NT ≥99th centile and normal R- and G-banding were analyzed by MLPA for subtelomeric imbalances (SALSA P036 and P070) and 21 syndromic regions (SALSA P245). One sample showed a benign CNV (dup(8)pter, FBXO25 gene), and 1 patient was found to have a loss of 18qter and a gain of 5pter as a result of an unbalanced translocation. The incidence of cryptic pathogenic variants was

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Objectif Le cancer du sein triple negatif (TNBC), environ 15 % des cancers du sein, se caracterise par un fort potentiel invasif. Lors de l’invasion tumorale, les cellules epitheliales realisent une transition epithelio-mesenchymateuse (TEM) pour acquerir leur mobilite et disseminer. L’incidence du cancer du sein etant moindre chez les trisomiques 21 [1] , [2] notre objectif etait de determiner si une matrice extracellulaire (MEC) de type trisomie 21 (T21) peut alterer in vitro la TEM de cellules TNBC. Materiel et methode Des cellules d’adenocarcinome mammaire TNBC (MDA-MB468) ont ete cultivees sur une MEC secretee par des fibroblastes T21 (F-T21) ou euploides (F-Eup), ou sur plastique (P), « sans » ou « avec » stimulation de la TEM par du TGFα. L’expression de genes codant des marqueurs epitheliaux (CDH1, OCLN1, CLDN1) et mesenchymateux (VIM, FN1) a ete evaluee a 24 h (j1), 48 h (j2), 72 h (j3) et 96 h (j4) apres stimulation par qRT-PCR en temps reel. Resultats Genes epitheliaux : diminution de leur expression a j4 (tous supports), « sans » et « avec » stimulation. Genes mesenchymateux : faible expression de FN1 rendant difficile l’interpretation des variations d’expression observees. Pour VIM, « sans » stimulation, augmentation progressive de l’expression de j1 a j4 (tous supports) mais plus forte expression sur MEC F-Eup que sur MEC F-T21 a j4. « Avec » stimulation, augmentation de l’expression de j2 a j4 sur MEC F-Eup et a partir de j3 sur MEC-T-21 mais plus forte expression sur MEC F-T21 que sur MEC F-Eup a j3. Conclusion Ces resultats sont en faveur d’un processus « TEM-like » a j4 « sans » et « avec » stimulation. Les differences d’expression observees sur MEC F-Eup et MEC F-T21 suggerent qu’une MEC-T21 pourrait interferer in vitro au niveau transcriptionnel sur la TEM de cellules TNBC.

Le TGNI T21 est realise a partir de l’ADN libre circulant (ADN-LC) present dans le sang. Durant la grossesse, une partie (jusqu’a 20 %) de cet ADN-LC provient des cellules trophoblastiques. Il peut donc etre utilise pour depister une T21 fœtale en faisant appel a du sequencage haut debit. Apres extraction de l’ADN-LC et preparation des librairies, un sequencage parallele massif « tout genome » ou « cible » est realise et, apres alignement sur un genome de reference euploidie, une surrepresentation des sequences du chromosome 21 est statistiquement recherchee. Ce test est indique lorsqu’il existe un risque accru de T21. Le test utilise dans cette etude (Clarigo® – Multiplicom) est une approche ciblee sur 4000 amplicons presents sur les chromosomes 13, 18, 21 et X. Chaque amplicon contient des SNP afin de calculer la fraction fœtale (FF) d’ADN-LC. Le traitement bioinformatique des donnees est assure par un logiciel (Clarigo™ Reporter) accessible sur un cloud prive et securise qui calcule la FF et conclut : – negatif (NEG) ; – positif (POS) ; – FF basse (LFF) ; – non automatiquement calcule (NAC) ; – correlation de l’echantillon basse (ECHEC). A ce jour, pres de 650 gestantes ont beneficie du test. Le test s’est avere POS dans 2 cas, NEG dans pres de 97 % des cas et non-interpretable (LFF et/ou NAC ; ECHEC) dans environ 3 % des cas. Le test, simple d’utilisation, determine la FF d’ADN-LC et permet le TGNI T21 en routine. Le taux d’echec est faible (≈ 3 %). Son utilisation en cas de grossesse gemellaire monochoriale ne pose pas de probleme particulier, contrairement aux bichoriales pour lesquelles la mesure de la FF peut etre prise en defaut. Le surpoids (IMC > 30) s’accompagne d’un taux d’echec superieur. L’incrementation du test dans le Service a permis de diminuer de plus de 55 % le nombre de prelevements invasifs.

Nous presentons le cas d’un enfant porteur d’une hernie diaphragmatique gauche de decouverte antenatale associee a des reins hyperechogenes multikystiques. Devant les malformations renales une recherche de mutation du gene HNF1B/TCF2 avait ete realisee en prenatal et avait permis d’identifier une deletion heterozygote de l’ensemble des exons de TCF2 survenue de novo. A 5 ans et 8 mois l’enfant presente une hyperreactivite bronchique secondaire a de nombreuses infections respiratoires, un astigmatisme, une dysmorphie faciale moderee et des anomalies squelettiques. Il est scolarise normalement et ne presente pas de retard psychomoteur. Une ACPA (Agilent 180K) a permis de mettre en evidence une microdeletion 17q12 de 1,8 Mb emportant 19 genes codants dont TCF2, LHX1 et PIGW. L’enquete familiale a confirme son caractere de novo. La microdeletion 17q12 est associee a la presence de reins dysplasiques multikystiques, d’un diabete MODY5 et d’anomalies pancreatiques, hepatiques et genitales. Un retard mental modere et des troubles neuropsychiatriques ont ete rapportes mais il semble exister une penetrance incomplete pour ces troubles neurocognitifs [1] , [2] . L’haploinsuffisance du gene TCF2 est responsable des anomalies renales et du diabete et le gene LHX1 pourrait etre implique dans les troubles neurologiques. Le gene PIGW, codant pour une proteine du systeme d’ancrage lipidique de certaines proteines, pourrait etre un candidat pour la hernie diaphragmatique congenitale (HDC). A ce jour, l’association microdeletion 17q12 et HDC n’a ete decrite qu’une seule fois [3] . Deux cas de microduplications de cette region ont egalement ete decrits chez des fœtus presentant une HDC [4] . Il semble donc que cette region chromosomique renferme un/des gene(s) implique(s) dans le developpement du diaphragme. L’utilisation de plus en plus repandue de l’ACPA en prenatal devrait permettre de mieux estimer la prevalence de la HDC dans la microdeletion 17q12, probablement sous-evaluee en post-natal en raison de la forte mortalite de cette malformation.

Interstitial 2q36 deletion is a rare event. Only two previously published cases of 2q36 deletions were characterized using array-CGH. This is the first case diagnosed prenatally.We report on the prenatal diagnosis of a 2q36.1q36.3 interstitial deletion in a fetus with facial dysmorphism, spina bifida, and cleft palate.Array-CGH analysis revealed a 5.6 Mb interstitial deletion of the long arm of chromosome 2q36.1q36.3, including the PAX3 and EPHA4 genes.The present study reinforces the hypothesis that PAX3 haploinsufficiency may be associated with neural tube defects in humans and suggests that the EPHA4 gene might be implicated during palate development. This report also illustrates the added value of array-CGH to detect cryptic chromosomal imbalances in malformed fetuses and to improve genetic counseling prenatally.

Les duplications partielles isolees du bras long du chromosome 6 sont peu rapportees dans la litterature. Malgre des regions dupliquees de taille et de localisation variables, un syndrome dup (6q) semble emerger grâce aux differentes descriptions phenotypiques sans qu’aucun gene candidat n’ait ete retenu du fait de l’absence de caracterisation moleculaire. Ce syndrome se caracterise par une dysmorphie cranio-faciale, une deficience intellectuelle, des atteintes articulaires, un retard de croissance, des malformations cardiaques, cerebrales et uro-genitales [1] . Nous rapportons le cas d’un enfant adresse pour avance staturo-ponderale, macrocephalie majeure et retard neuro-moteur. L’analyse chromosomique sur puces a ADN a mis en evidence une duplication de 8 Mb en 6q25.2-q26 comprenant 43 genes Refseq. Parmi ces genes, cinq ont ete retenus comme genes candidats : deux pouvant etre impliques dans le retard neuro-moteur (ARID1, SYNE1) et trois dans l’avance staturo-ponderale (TFB1M, IGF2R et OPRM1). Une etude d’expression sur fibroblastes a permis de reveler une surexpression d’ARID1 et de SYNE1. Aucune expression n’a pu etre quantifiee pour les trois autres genes. Compte tenu du role potentiel dans l’avance staturo-ponderale, l’expression d’IGF2R a egalement ete evaluee a partir d’une lignee lymphoblastoide, mais ne semble pas etre modifiee chez notre patient. L’analyse d’expression a permis de mettre en evidence une surexpression importante des genes ARID1 et SYNE1 impliques tous deux au niveau cerebral. Parmi les trois autres genes, l’absence d’expression des genes TFB1M et OPRM1 au niveau fibroblastique et l’absence de modification d’expression d’IGF2R ne permettent pas de correler directement ces genes a l’avance staturo-ponderale du patient. Cependant, d’autres mecanismes moleculaires pourraient intervenir impliquant en particulier IGF2R soumis a empreinte et identifie comme responsable d’obesite syndromique en cas de duplication d’origine paternelle [2] . Une etude de l’origine parentale de l’allele duplique est en cours afin de preciser le role d’IGF2R dans le phenotype de notre patient.

Objet Clarigo® (Multiplicom) est un nouveau test de DPNI des T13, T18 et T21. Il s’agit d’une approche ciblee reposant sur le sequencage massif parallele de sequences specifiques de ces chromosomes. Nous rapportons les resultats de nos premiers essais pour la validation du test dans notre service. Materiel et methode 36 echantillons (4 T21, 1 T18 et 31 temoins negatifs) ont ete analyses. L’ADN circulant a ete extrait (Blood DNA circulating, Qiagen). Vingt microlitres ont ete utilises pour la PCR multiplexe. Les produits amplifies ont ete purifies pour servir de matrice a la PCR universelle (ajout de MID specifiques a chaque echantillon). Apres purification et controle de la qualite de la PCR, les echantillons ont ete doses et melanges de facon equimolaire. Le pool a ensuite ete sequence (V3 MiSeq, Illumina). Les donnees ont ete traitees avec le logiciel Clarigo Reporter® (Multiplicom). Resultats Pour 3 des 4 echantillons T21, la trisomie 21 a ete detectee ; pour le 4e, le logiciel a conclu a « non automatiquement classe » (NAC). Parmi les 32 autres echantillons (1T18 et 31 temoins), 3 (dont le T18) presentaient une fraction fœtale trop faible pour donner un resultat fiable. Pour les 29 autres, l’analyse s’est revelee negative pour la T21 et « NAC » une fois pour la T18 et trois fois pour la T13. Conclusions Les premiers resultats sont satisfaisants avec une bonne sensibilite pour la trisomie 21 et une tres bonne specificite. Pour les echantillons classes « NAC », l’analyse des graphes a permis de suspecter la trisomie 21 sur le 4e echantillon T21 et d’ecarter une telle T21 pour les 4 temoins. Sur les 3 echantillons a faible fraction fœtale, 2 provenaient de femmes enceintes presentant un indice de masse corporelle ≥ 35, confirmant ce facteur comme limitant a la realisation du test.

Nous presentons le cas d’une deletion 2q14.1q14.3 de 5,8Mb identifiee en prenatal par ACPA chez un fœtus presentant une agenesie partielle du corps calleux associee a un retard de croissance intra-uterin (RCIU) severe. Cette deletion emporte 24 genes codants dont 3 impliques en pathologie : STEAP3, GLI2 et RNU4ATAC. A la naissance, l’enfant etait hypotrophe et dysmorphique. A 6 mois, le developpement psychomoteur etait normal. La meme deletion a ete retrouvee chez la mere qui a presente des difficultes scolaires mais sans anomalie du corps calleux et chez un de ses fils presentant un retard psychomoteur. Dix cas de deletion comprenant la region 2q14.1q14.3 ont ete decrits. Le phenotype est tres variable avec comme traits communs une atteinte cognitive, une dysmorphie faciale et un retard de croissance postnatal. La majorite de ces cas correspond a une deletion de grande taille, de novo et seulement deux des cas rapportes sont des deletions heritees de petites tailles [1] , [2] . Ce cas confirme la variabilite d’expression de la deletion 2q14 et montre qu’une agenesie du corps calleux et un RCIU peuvent faire partie du phenotype. Le gene RNU4ATAC est transcrit en un ARN nucleaire implique dans l’epissage intronique de nombreux genes. Des mutations de ce gene sont a l’origine du nanisme microcephalique osteodysplasique primordial de type 1 (MOPDI) [3] , maladie a transmission autosomique recessive. Cette atteinte du developpement est caracterisee par un RCIU severe et des anomalies cerebrales dont systematiquement une agenesie du corps calleux. Nous emettons l’hypothese qu’une deletion complete du gene RNU4ATAC pourrait participer au phenotype observe chez notre patient par diminution de son activite d’epissage (aucune mutation n’a ete mise en evidence sur son autre allele). Des mutations et perte de fonction du gene GLI2 entrainent un phenotype d’holoprosencepalie associe a un hypopituitarisme [4] . Son haploinsuffisance pourrait aussi participer au phenotype de notre patient.

Purpose: Congenital malformations are a major cause of morbidity and mortality in newborn infants, and genomic imbalances are a significant component of their etiology. The aim of this study was to evaluate the ability of prenatal multiplex ligation probe amplification screening to detect cryptic chromosomal imbalances in fetuses with ultrasound abnormalities of unknown etiology. Methods: Multiplex ligation probe amplification was performed with three separate sets of probes: two for subtelomeric regions and one for mental retardation syndrome loci. Sixty-one fetuses with significant ultrasound anomalies and normal karyotype at a minimum of 400-band resolution were tested between January 2007 and January 2009. Results: We identified four unbalanced rearrangements: one del 18pter/amp 5pter, one del 9pter, one 15q11q13 microdeletion, and one 22q11 microdeletion with atypical presentation. After genetic counseling, two of the pregnancies were terminated. Conclusion: Multiplex ligation probe amplification analysis was able to identify clinically significant rearrangements in 6.5% of fetuses with prenatally identified sonographic abnormalities. This prospective study highlights that multiplex ligation probe amplification screening of fetuses with ultrasound abnormalities in the prenatal period is technically feasible and relevant for diagnosis and prognosis. Genet Med 2010:12(6):376–380.

Objet Les anomalies chromosomiques sont responsables de 50 % des fausses couches spontanees (FCS). Le caryotype standard est jusqu’a maintenant la methode de reference pour l’etude cytogenetique des produits de FCS. Cependant, il est souvent impossible a etablir du fait de la mauvaise qualite de certains prelevements (maceration) a l’origine d’une absence de proliferation des cellules en culture et donne souvent des resultats errones du fait d’une contamination du prelevement par des cellules maternelles. Recemment, une nouvelle approche moleculaire, appelee Bacs-on-Beads, a ete decrite comme un examen fiable pour la detection rapide des aneuploidies en prenatal (Prenatal BoBs™) ou pour les produits de FCS (KaryoLites BoBs™). Dans cette etude, nous rapportons notre experience clinique de l’etude des produits de FCS par le Prenatal BoBs™. Methodes Soixante-dix-sept prelevements (44 villosites choriales issues de produit de curetage, 12 placenta, 9 liquides amniotiques et 37 biopsies cutanees) ont fait l’objet d’une recherche des aneuploidies les plus frequentes et de micro-remaniements pour neuf regions chromosomiques impliquees en pathologie fœtale et postnatale. Quand cela a ete possible, le caryotype standard a ete etabli. Resultats Nous avons diagnostique par Prenatal BoBs™ un cas de trisomie 21 et un cas de deletion 17p13.3 qui, apres caracterisation par analyse chromosomique sur puce a ADN, s’est averee etre une deletion de 2,3 Mb ne comprenant pas les genes responsables du syndrome de Miller-Diecker. Le Prenatal BoBs a ete concluant dans 57 cas pour lesquels le caryotype n’a pu etre realise en raison d’une absence de proliferation cellulaire. Neanmoins, dans 11 % des cas l’examen par Prenatal BoBs™ n’a pas ete concluant. Conclusion Le Prenatal BoBs™ peut etre utilisee dans l’exploration cytogenetique des produits de FCS et constitue une bonne alternative aux techniques conventionnelles comme le caryotype notamment pour les prelevements presentant un etat de maceration avance.

In B-cell chronic lymphocytic leukemia (B-CLL), CD5+CD19+ malignant cells home into the bone marrow (BM) and circulate in the blood. While CLL tumor cells are not susceptible to apoptosis in vivo, they die rapidly in vitro in the absence of specialized non-hematopoietic feeder cells, such as mesenchymal stem cells (MSC). Recent observations have suggested that there is a functional relationship between B cell clone and the stroma. We have thus compared BM-MSC obtained from B-CLL patients and healthy subjects. We first evaluated the influence of in vitro culture conditions on the number of BM-derived CFU-F and the proliferation of MSC and, in parallel, we quantified in unmanipulated normal and malignant BM samples the CD45negCD14negCD73pos cell subset that was previously shown to contain CFU-F (Veyrat-Masson et al., BJH, 2007). Changes in the level of 42 cytokines/chemokines, were then evaluated in MSC-conditioned media (4 CLL vs 4 normal BM-MSCs) using protein-array (RayBio Human Cytokine Antibody Array IIITM, Tebu-bio SA,). In addition, total RNA was extracted (Rneasy MiniKit, Qiagen,) from 9 expanded MSC at passage 1 (P1) in the presence of bFGF (5 untreated B-CLL BM-MSC: 2 Binet stage A, 2 stage B and 1 stage C; 4 normal BM-MSC) and then reverse transcribed (High Capacity cDNA RT Kit, Applied BioSystems). Quantitative PCR reactions, using dedicated microfluid cards screening 384 selected genes, were then performed (TLDAs, Applied Biosystem Courtaboeuf, France). The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize gene expression level. Despite a 16-fold increase in total cell numbers tested, we found that most BM-MSC cultures from B-CLL patients failed under standard culture conditions (IMDM/10%FCS), in contrast with our experience with normal BM (69 % n = 13 vs Conclusions: These results show that the BM-MSC from B-CLL patients were quantitatively and functionally altered and are dependent for their in vitro growth on circulating soluble factors or on growth factors like bFGF. Interestingly, from this small series, we observed 57 differentially expressed genes which could be involved in the B-CLL specific stromal cell alterations previously reported (dysregulation of cytokine secretion, angiogenesis, host-tumor relationships). These findings suggest the possible permissive role of MSC on B-cell clone progression and raise the question as to whether we are dealing with selection of a mesenchymal subset or with alteration of mesenchymal cells induced by malignant B-cells

The purine analogue fludarabine is efficient in treating advanced, chronic B-cell lymphocytic leukemia (CLL). We and others have shown that front-line fludarabine treatment impaired the subsequent mobilization of hematopoietic CD34+ cells by G-CSF. This might imply an effect on hematopoietic and/or mesenchymal compartments. We also previously showed that native mesenchymal cells (MC), like expanded MC, express the 5′-ecto-nucleotidase (CD73) which should aid the entry of fludarabine into the cell. So we investigated the toxicity of fludarabine on NBMMC. We incubated expanded (P1) and fresh NBMMC from femoral heads with increasing doses (0.1 to 6 mMol) of fludarabine for 3 days, and assessed its effect on cell survival (annexin V staining), CFU-F frequency, cell proliferation and immunophenotyping. We used α,β, methylene-adenosine 5 diphosphate (MADP) to inhibit CD73 in order to evaluate its role in the trans-membrane passage of fludarabine. We observed a direct, dose-dependent toxicity of fludarabine on expanded mesenchymal cells, after 3 days incubation, characterized by apoptosis of 48% of MC. The mesenchymal progenitor cells were more sensitive; their frequency amongst the viable cells, being decreased about 5-fold from the 1mMol dose, to 13-fold at the highest dose, where there were far fewer colonies containing>50 cells. Even after fludarabine removal, viable MC showed an impaired ability to proliferate at the next passage. Fresh NBMMC, incubated under same conditions, showed a similar but lower sensitivity than did expanded (P1) mesenchymal cells, as indicated by a shift in the dose-response curve to a 5-fold decrease at the highest dose. Again, we observed a decreasing proportion of large colonies. MADP provided no significant protection against the effect of fludarabine on fresh or expanded cells. The phenotype of progeny was comparable to that of the controls. In conclusion, we have shown for the first time that adult NBMMC are sensitive to fludarabine and could prove to be an unexpected in vivo target. The differences between un-manipulated and expanded cells suggest a biologic alteration during culture. The lack of protective effect of MADP and the absence of lower expression of CD73 on resistant cells suggest another route of trans-membrane passage for fludarabine. The consequences of low dose fludarabine incubation on MC properties are currently being studied.

Genetic studies of intellectual disability and identification of monogenic causes of obesity in humans have made immense contribution toward the understanding of the brain and control of body mass. The leptin > melanocortin > SIM1 pathway is dysregulated in multiple monogenic human obesity syndromes but its downstream targets are still unknown. In ten individuals from six families, with overlapping 6q16.1 deletions, we describe a disorder of variable developmental delay, intellectual disability, and susceptibility to obesity and hyperphagia. The 6q16.1 deletions segregated with the phenotype in multiplex families and were shown to be de novo in four families, and there was dramatic phenotypic overlap among affected individuals who were independently ascertained without bias from clinical features. Analysis of the deletions revealed a ∼350 kb critical region on chromosome 6q16.1 that encompasses a gene for proneuronal transcription factor POU3F2, which is important for hypothalamic development and function. Using morpholino and mutant zebrafish models, we show that POU3F2 lies downstream of SIM1 and controls oxytocin expression in the hypothalamic neuroendocrine preoptic area. We show that this finding is consistent with the expression patterns of POU3F2 and related genes in the human brain. Our work helps to further delineate the neuro-endocrine control of energy balance/body mass and demonstrates that this molecular pathway is conserved across multiple species.