Search

Duplication of the Xq28 region, involving MECP2 (dupMECP2), has been primarily described in males with severe developmental delay, spasticity, epilepsy, stereotyped movements and recurrent infections. Carrier mothers are usually asymptomatic with an extremely skewed X chromosome inactivation (XCI) pattern. We report a series of six novel symptomatic females carrying a de novo interstitial dupMECP2, and review the 14 symptomatic females reported to date, with the aim to further delineate their phenotype and give clues for genetic counselling. One patient was adopted and among the other 19 patients, seven (37%) had inherited their duplication from their mother, including three mildly (XCI: 70/30, 63/37, 100/0 in blood and random in saliva), one moderately (XCI: random) and three severely (XCI: uninformative and 88/12) affected patients. After combining our data with data from the literature, we could not show a correlation between XCI in the blood or duplication size and the severity of the phenotype, or explain the presence of a phenotype in these females. These findings confirm that an abnormal phenotype, even severe, can be a rare event in females born to asymptomatic carrier mothers, making genetic counselling difficult in couples at risk in terms of prognosis, in particular in prenatal cases.

Frontonasal dysplasia (FND) is a genetically heterogeneous malformation spectrum with marked hypertelorism, broad nasal tip and bifid nose. Only a small number of genes have been associated with FND phenotypes until now, the first gene being EFNB1, related to craniofrontonasal syndrome (CFNS) with craniosynostosis in addition, and more recently the aristaless-like homeobox genes ALX3, ALX4, and ALX1, which have been related with distinct phenotypes named FND1, FND2, and FND3 respectively. We here report on a female patient presenting with severe FND features along with partial alopecia, hypogonadism and intellectual disability. While molecular investigations did not reveal mutations in any of the known genes, ALX4, ALX3, ALX1 and EFNB1, comparative genomic hybridization (array CGH) techniques showed a large heterozygous de novo deletion at 11p11.12p12, encompassing the ALX4 gene. Deletions in this region have been described in patients with Potocki–Shaffer syndrome (PSS), characterized by biparietal foramina, multiple exostoses, and intellectual disability. Although the patient reported herein manifests some overlapping features of FND and PPS, it is likely that the observed phenotype maybe due to a second unidentified mutation in the ALX4 gene. The phenotype will be discussed in view of the deleted region encompassing the ALX4 gene. © 2013 Wiley Periodicals, Inc.

International audience; Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions.

Genomic rearrangements at chromosome 13q31.3q32.1 have been associated with digital anomalies, dysmorphic features, and variable degree of mental disability. Microdeletions leading to haploinsufficiency of miR17∼92, a cluster of micro RNA genes closely linked to GPC5 in both mouse and human genomes, has recently been associated with digital anomalies in the Feingold like syndrome. Here, we report on a boy with familial dominant post-axial polydactyly (PAP) type A, overgrowth, significant facial dysmorphisms and autistic traits who carries the smallest germline microduplication known so far in that region. The microduplication encompasses the whole miR17∼92 cluster and the first 5 exons of GPC5. This report supports the newly recognized role of miR17∼92 gene dosage in digital developmental anomalies, and suggests a possible role of GPC5 in growth regulation and in cognitive development.

Background The recurrent ∼600 kb 16p11.2 BP4-BP5 deletion is among the most frequent known genetic aetiologies of autism spectrum disorder (ASD) and related neurodevelopmental disorders. Objective To define the medical, neuropsychological, and behavioural phenotypes in carriers of this deletion. Methods We collected clinical data on 285 deletion carriers and performed detailed evaluations on 72 carriers and 68 intrafamilial non-carrier controls. Results When compared to intrafamilial controls, full scale intelligence quotient (FSIQ) is two standard deviations lower in carriers, and there is no difference between carriers referred for neurodevelopmental disorders and carriers identified through cascade family testing. Verbal IQ (mean 74) is lower than non-verbal IQ (mean 83) and a majority of carriers require speech therapy. Over 80% of individuals exhibit psychiatric disorders including ASD, which is present in 15% of the paediatric carriers. Increase in head circumference (HC) during infancy is similar to the HC and brain growth patterns observed in idiopathic ASD. Obesity, a major comorbidity present in 50% of the carriers by the age of 7 years, does not correlate with FSIQ or any behavioural trait. Seizures are present in 24% of carriers and occur independently of other symptoms. Malformations are infrequently found, confirming only a few of the previously reported associations. Conclusions The 16p11.2 deletion impacts in a quantitative and independent manner FSIQ, behaviour and body mass index, possibly through direct influences on neural circuitry. Although non-specific, these features are clinically significant and reproducible. Lastly, this study demonstrates the necessity of studying large patient cohorts ascertained through multiple methods to characterise the clinical consequences of rare variants involved in common diseases.

The development of chemoresistance represents a major obstacle in the successful treatment of cancers such as neuroblastoma (NB), a particularly aggressive childhood solid tumour. The mechanisms underlying the chemoresistant phenotype in NB were addressed by gene expression profiling of two doxorubicin (DoxR)-resistant vs sensitive parental cell lines. Not surprisingly, the MDR1 gene was included in the identified upregulated genes, although the highest overexpressed transcript in both cell lines was the frizzled-1 Wnt receptor (FZD1) gene, an essential component of the Wnt/beta-catenin pathway. FZD1 upregulation in resistant variants was shown to mediate sustained activation of the Wnt/beta-catenin pathway as revealed by nuclear beta-catenin translocation and target genes transactivation. Interestingly, specific micro-adapted short hairpin RNA (shRNAmir)-mediated FZD1 silencing induced parallel strong decrease in the expression of MDR1, another beta-catenin target gene, revealing a complex, Wnt/beta-catenin-mediated implication of FZD1 in chemoresistance. The significant restoration of drug sensitivity in FZD1-silenced cells confirmed the FZD1-associated chemoresistance. RNA samples from 21 patient tumours (diagnosis and postchemotherapy), showed a highly significant FZD1 and/or MDR1 overexpression after treatment, underlining a role for FZD1-mediated Wnt/beta-catenin pathway in clinical chemoresistance. Our data represent the first implication of the Wnt/beta-catenin pathway in NB chemoresistance and identify potential new targets to treat aggressive and resistant NB.

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

We report on two patients with de novo subtelomeric terminal deletion of chromosome 6p. Patient 1 is an 8-month-old female born with normal growth parameters, typical facial features of 6pter deletion, bilateral corectopia, and protruding tongue. She has severe developmental delay, profound bilateral neurosensory deafness, poor visual contact, and hypsarrhythmia since the age of 6 months. Patient 2 is a 5-year-old male born with normal growth parameters and unilateral hip dysplasia; he has a characteristic facial phenotype, bilateral embryotoxon, and moderate mental retardation. Further characterization of the deletion, using high-resolution array comparative genomic hybridization (array-CGH; Agilent Human Genome kit 244 K), revealed that Patient 1 has a 8.1 Mb 6pter-6p24.3 deletion associated with a contiguous 5.8 Mb 6p24.3-6p24.1 duplication and Patient 2 a 5.7 Mb 6pter-6p25.1 deletion partially overlapping with that of Patient 1. Complementary FISH and array analysis showed that the inv del dup(6) in Patient 1 originated de novo. Our results demonstrate that simple rearrangements are often more complex than defined by standard techniques. We also discuss genotype-phenotype correlations including previously reported cases of deletion 6p.

Gene transfer in eukaryotic cells and organisms suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. Use of epigenetic regulators such as matrix attachment regions (MARs) is a promising approach to alleviate such unwanted effects. Dissection of a known MAR allowed the identification of sequence motifs that mediate elevated transgene expression. Bioinformatics analysis implied that these motifs adopt a curved DNA structure that positions nucleosomes and binds specific transcription factors. From these observations, we computed putative MARs from the human genome. Cloning of several predicted MARs indicated that they are much more potent than the previously known element, boosting the expression of recombinant proteins from cultured cells as well as mediating high and sustained expression in mice. Thus we computationally identified potent epigenetic regulators, opening new strategies toward high and stable transgene expression for research, therapeutic production or gene-based therapies.

We report on a 24-week fetus with multiple organ anomalies secondary to biparental inheritance of an apparently balanced t(17;20) reciprocal translocation. The pregnancy was terminated following the discovery by ultrasound of an abnormal heart and micrognathia. At autopsy, the following anomalies were found: Pierre–Robin sequence, hypoplasia of the right ventricle with muscular hypertrophy, and endocardial fibroelastosis, hypoplastic lungs, dysplastic left kidney, bilateral pelvicalyceal dilatation, central nervous system periventricular heterotopias and right sided club foot. Given the endocardial fibroelastosis and cleft palate, Eastman–Bixler syndrome (Facio-cardio-renal) is a possible diagnosis. The parents were first cousins and each had an identical t(17;20)(q21.1;p11.21) translocation. The fetal karyotype was 46,XX,t(17;20)(q21.1;p11.21)mat,t(17;20)(q21.1;p11.21)pat. While there are a few reports of consanguineous families where both the mother and father had the same reciprocal translocation and offspring with unbalanced karyotypes, we were unable to find any reports of a fetus/child with double identical reciprocal translocations. We propose that although the fetus had an apparently balanced karyotype, inheriting only the translocated chromosomes led to the unmasking of a recessive syndrome. It seems most likely that a gene (or genes) was disrupted by the breaks but the parents might also be heterozygous carriers of a recessive gene mutation since the fetus must be homozygous by descent for many loci on both chromosomes 17 and 20 (as well as on other chromosomal segments). It was not possible to totally exclude segmental uniparental disomy as a cause of the anomalies as no recombinations were detected for chromosome 17. However, there is no evidence to suggest that chromosome 17 is imprinted and UPD 20 was excluded thus making an imprinting error unlikely. © 2006 Wiley-Liss, Inc.

We have identified and characterized a spontaneous Brown Norway from Janvier rat strain (BN-J) presenting a progressive retinal degeneration associated with early retinal telangiectasia, neuronal alterations, and loss of retinal Müller glial cells resembling human macular telangiectasia type 2 (MacTel 2), which is a retinal disease of unknown cause. Genetic analyses showed that the BN-J phenotype results from an autosomal recessive indel novel mutation in theCrb1gene, causing dislocalization of the protein from the retinal Müller glia (RMG)/photoreceptor cell junction. The transcriptomic analyses of primary RMG cultures allowed identification of the dysregulated pathways in BN-J rats compared with wild-type BN rats. Among those pathways, TGF-β and Kit Receptor Signaling, MAPK Cascade, Growth Factors and Inflammatory Pathways, G-Protein Signaling Pathways, Regulation of Actin Cytoskeleton, and Cardiovascular Signaling were found. Potential molecular targets linking RMG/photoreceptor interaction with the development of retinal telangiectasia are identified. This model can help us to better understand the physiopathologic mechanisms of MacTel 2 and other retinal diseases associated with telangiectasia.

International audience; There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.

Item does not contain fulltext The 16p11.2 600 kb copy-number variants (CNVs) are associated with mirror phenotypes on BMI, head circumference, and brain volume and represent frequent genetic lesions in autism spectrum disorders (ASDs) and schizophrenia. Here we interrogated the transcriptome of individuals carrying reciprocal 16p11.2 CNVs. Transcript perturbations correlated with clinical endophenotypes and were enriched for genes associated with ASDs, abnormalities of head size, and ciliopathies. Ciliary gene expression was also perturbed in orthologous mouse models, raising the possibility that ciliary dysfunction contributes to 16p11.2 pathologies. In support of this hypothesis, we found structural ciliary defects in the CA1 hippocampal region of 16p11.2 duplication mice. Moreover, by using an established zebrafish model, we show genetic interaction between KCTD13, a key driver of the mirrored neuroanatomical phenotypes of the 16p11.2 CNV, and ciliopathy-associated genes. Overexpression of BBS7 rescues head size and neuroanatomical defects of kctd13 morphants, whereas suppression or overexpression of CEP290 rescues phenotypes induced by KCTD13 under- or overexpression, respectively. Our data suggest that dysregulation of ciliopathy genes contributes to the clinical phenotypes of these CNVs.

The limited ability of common variants to account for the genetic contribution to complex disease has prompted searches for rare variants of large effect, to partly explain the 'missing heritability'. Analyses of genome-wide genotyping data have identified genomic structural variants (GSVs) as a source of such rare causal variants. Recent studies have reported multiple GSV loci associated with risk of obesity. We attempted to replicate these associations by similar analysis of two familial-obesity case-control cohorts and a population cohort, and detected GSVs at 11 out of 18 loci, at frequencies similar to those previously reported. Based on their reported frequencies and effect sizes (OR≥25), we had sufficient statistical power to detect the large majority (80%) of genuine associations at these loci. However, only one obesity association was replicated. Deletion of a 220 kb region on chromosome 16p11.2 has a carrier population frequency of 2×10(-4) (95% confidence interval [9.6×10(-5)-3.1×10(-4)]); accounts overall for 0.5% [0.19%-0.82%] of severe childhood obesity cases (P = 3.8×10(-10); odds ratio = 25.0 [9.9-60.6]); and results in a mean body mass index (BMI) increase of 5.8 kg.m(-2) [1.8-10.3] in adults from the general population. We also attempted replication using BMI as a quantitative trait in our population cohort; associations with BMI at or near nominal significance were detected at two further loci near KIF2B and within FOXP2, but these did not survive correction for multiple testing. These findings emphasise several issues of importance when conducting rare GSV association, including the need for careful cohort selection and replication strategy, accurate GSV identification, and appropriate correction for multiple testing and/or control of false discovery rate. Moreover, they highlight the potential difficulty in replicating rare CNV associations across different populations. Nevertheless, we show that such studies are potentially valuable for the identification of variants making an appreciable contribution to complex disease.

Small supernumerary marker chromosomes (sSMCs) are structurally abnormal chromosomes that cannot be characterized by karyotype. In many prenatal cases of de novo sSMC, the outcome of pregnancy is difficult to predict because the euchromatin content is unclear. This study aimed to determine the presence or absence of euchromatin material of 39 de novo prenatally ascertained sSMC by array-comparative genomic hybridization (array-CGH) or single nucleotide polymorphism (SNP) array. Cases were prospectively ascertained from the study of 65,000 prenatal samples [0.060%; 95% confidence interval (CI), 0.042-0.082]. Array-CGH showed that 22 markers were derived from non-acrocentric markers (56.4%) and 7 from acrocentic markers (18%). The 10 additional cases remained unidentified (25.6%), but 7 of 10 could be further identified using fluorescence in situ hybridization; 69% of de novo sSMC contained euchromatin material, 95.4% of which for non-acrocentric markers. Some sSMC containing euchromatin had a normal phenotype (31% for non-acrocentric and 75% for acrocentric markers). Statistical differences between normal and abnormal phenotypes were shown for the size of the euchromatin material (more or less than 1 Mb, p = 0.0006) and number of genes (more or less than 10, p = 0.0009). This study is the largest to date and shows the utility of array-CGH or SNP array in the detection and characterization of de novo sSMC in a prenatal context.

We describe 19 unrelated individuals with submicroscopic deletions involving 10p15.3 characterized by chromosomal microarray (CMA). Interestingly, to our knowledge, only two individuals with isolated, submicroscopic 10p15.3 deletion have been reported to date; however, only limited clinical information is available for these probands and the deleted region has not been molecularly mapped. Comprehensive clinical history was obtained for 12 of the 19 individuals described in this study. Common features among these 12 individuals include: cognitive/behavioral/developmental differences (11/11), speech delay/language disorder (10/10), motor delay (10/10), craniofacial dysmorphism (9/12), hypotonia (7/11,), brain anomalies (4/6) and seizures (3/7). Parental studies were performed for nine of the 19 individuals; the 10p15.3 deletion was de novo in seven of the probands, not maternally inherited in one proband and inherited from an apparently affected mother in one proband. Molecular mapping of the 19 individuals reported in this study has identified two genes, ZMYND11 (OMIM# 608668) and DIP2C (OMIM# 611380) (UCSC Genome Browser), mapping within 10p15.3 which are most commonly deleted. Although no single gene has been identified which is deleted in all 19 individuals studied, the deleted region in all but one individual includes ZMYND11 and the deleted region in all but one other individual includes DIP2C. There is not a clearly identifiable phenotypic difference between these two individuals and the size of the deleted region does not generally predict clinical features. Little is currently known about these genes complicating a direct genotype/phenotype correlation at this time. These data however, suggest that ZMYND11 and/or DIP2C haploinsufficiency contributes to the clinical features associated with 10p15 deletions in probands described in this study.

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers.

Item does not contain fulltext The genomic architecture of the 10q22q23 region is characterised by two low-copy repeats (LCRs3 and 4), and deletions in this region appear to be rare. We report the clinical and molecular characterisation of eight novel deletions and six duplications within the 10q22.3q23.3 region. Five deletions and three duplications occur between LCRs3 and 4, whereas three deletions and three duplications have unique breakpoints. Most of the individuals with the LCR3-4 deletion had developmental delay, mainly affecting speech. In addition, macrocephaly, mild facial dysmorphisms, cerebellar anomalies, cardiac defects and congenital breast aplasia were observed. For congenital breast aplasia, the NRG3 gene, known to be involved in early mammary gland development in mice, is a putative candidate gene. For cardiac defects, BMPR1A and GRID1 are putative candidate genes because of their association with cardiac structure and function. Duplications between LCRs3 and 4 are associated with variable phenotypic penetrance. Probands had speech and/or motor delays and dysmorphisms including a broad forehead, deep-set eyes, upslanting palpebral fissures, a smooth philtrum and a thin upper lip. In conclusion, duplications between LCRs3 and 4 on 10q22.3q23.2 may lead to a distinct facial appearance and delays in speech and motor development. However, the phenotypic spectrum is broad, and duplications have also been found in healthy family members of a proband. Reciprocal deletions lead to speech and language delay, mild facial dysmorphisms and, in some individuals, to cerebellar, breast developmental and cardiac defects.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field. Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance. Leenaards Foundation Jerome Lejeune Foundation Telethon Action Suisse Foundation Swiss National Science Foundation European Commission 037627 QLG1-CT-2000-01643 Ludwig Institute for Cancer Research Swiss Institute of Bioinformatics Imperial College Department of Medicine Comprehensive Biomedical Research Centre Imperial College Healthcare NHS Trust National Institute for Health Research Wellcome Trust Medical Research Council Instituto de Salud Carlos III (ISCIII)-FIS German Mental Retardation Network German Federal Ministry of Education and Research NGFNplus 01GS08160 European Union PI081714 PS09/01778 201413 245536 info:eu-repo/grantAgreement/EC/FP7/223423 Belgian National Fund for Scientific Research, Flanders Dutch Organisation for Health Research and Development (ZON-MW) 917-86-319 Hersenstichting Nederland (B.B.A.d.V.) Chinese National Natural Science Foundation 81000346 Simons Foundation Autism Research Initiative Autism Speaks NIH GM061354 MH071425 Oesterreichische Nationalbank (OENB) 13059 Children's Tumor Foundation Harvard Medical School Fudan University Chinese National '973' project on Population and Health 2010CB529601 Science and Technology Council of Shanghai 09JC1402400 Michael Smith Foundation for Health CIHR MOP 74502 Estonian Government SF0180142s08 SF0180026s09 SF0180027s10 Helmholtz Zentrum Munich State of Bavaria German National Genome Research Network 01GS0823 German Federal Ministry of Education and Research (BMBF) Munich Center of Health Sciences (MC Health, LMUinnovativ) Genome Canada McLaughlin Centre Academy of Finland 104781 120315 129269 1114194 University Hospital Oulu Biocenter University of Oulu, Finland 75617 NHLBI 5R01HL087679-02 1RL1MH083268-01 NIH/NIMH 5R01MH63706:02 ENGAGE project Medical Research Council, UK G0500539 G0600705 Academy of Finland Biocentrum Helsinki SAF2008-02278 HEALTH-F4-2007-201413

journal homepage: http: / /www.elsevier .com/locate/ejmg 56 57 58 59 60 61 62 63 64 Corrigendum 65 66 67 68 69 70 71 72 73 74 75 76 77 Corrigendum to “Post-axial polydactyly type A2, overgrowth and autistic traits associated with a chromosome 13q31.3 microduplication encompassing miR-17-92 and GPC5” [Eur J Med Genet 56 (8) (2013) 452e457] P. Kannu *, A.B. Campos-Xavier , D. Hull , D. Martinet , D. Ballhausen , L. Bonafe b

Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception ( Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255–66. ©2010 AACR.

Obesity has become a major worldwide challenge to public health, owing to an interaction between the Western 'obesogenic' environment and a strong genetic contribution. Recent extensive genome-wide association studies (GWASs) have identified numerous single nucleotide polymorphisms associated with obesity, but these loci together account for only a small fraction of the known heritable component. Thus, the 'common disease, common variant' hypothesis is increasingly coming under challenge. Here we report a highly penetrant form of obesity, initially observed in 31 subjects who were heterozygous for deletions of at least 593 kilobases at 16p11.2 and whose ascertainment included cognitive deficits. Nineteen similar deletions were identified from GWAS data in 16,053 individuals from eight European cohorts. These deletions were absent from healthy non-obese controls and accounted for 0.7% of our morbid obesity cases (body mass index (BMI) >or= 40 kg m(-2) or BMI standard deviation score >or= 4; P = 6.4 x 10(-8), odds ratio 43.0), demonstrating the potential importance in common disease of rare variants with strong effects. This highlights a promising strategy for identifying missing heritability in obesity and other complex traits: cohorts with extreme phenotypes are likely to be enriched for rare variants, thereby improving power for their discovery. Subsequent analysis of the loci so identified may well reveal additional rare variants that further contribute to the missing heritability, as recently reported for SIM1 (ref. 3). The most productive approach may therefore be to combine the 'power of the extreme' in small, well-phenotyped cohorts, with targeted follow-up in case-control and population cohorts.

Telomeres are associated with chromatin-mediated silencing of genes in their vicinity. However, how epigenetic markers mediate mammalian telomeric silencing and whether specific proteins may counteract this effect are not known. We evaluated the ability of CTF1, a DNA- and histone-binding transcription factor, to prevent transgene silencing at human telomeres. CTF1 was found to protect a gene from silencing when its DNA-binding sites were interposed between the gene and the telomeric extremity, while it did not affect a gene adjacent to the telomere. Protein fusions containing the CTF1 histone-binding domain displayed similar activities, while mutants impaired in their ability to interact with the histone did not. Chromatin immunoprecipitation indicated the propagation of a hypoacetylated histone structure to various extents depending on the telomere. The CTF1 fusion protein was found to recruit the H2A.Z histone variant at the telomeric locus and to restore high histone acetylation levels to the insulated telomeric transgene. Histone lysine trimethylations were also increased on the insulated transgene, indicating that these modifications may mediate expression rather than silencing at human telomeres. Overall, these results indicate that transcription factors can act to delimit chromatin domain boundaries at mammalian telomeres, thereby blocking the propagation of a silent chromatin structure.

Background Data on patients affected by chronic mucocutaneous candidiasis underscore the preponderant role of IL-17 receptor A (IL-17RA) in preserving mucocutaneous immunity. Little is known about the role of adenosine deaminase (ADA) 2 in regulation of immune responses, although recent reports linked ADA2 deficiency with inflammation and vasculitis. Objective We sought to investigate the mechanisms of chronic inflammation and vasculitis in a child lacking IL-17RA and ADA2 to identify therapeutic targets. Methods We report a family with 2 siblings who have had recurrent mucocutaneous infections with Candida albicans and Staphylococcus aureus and chronic inflammatory disease and vasculitis since early childhood, which were refractory to classical treatments. Array-based comparative genomic hybridization analysis showed that both siblings are homozygous for a 770-kb deletion on chr22q11.1 encompassing both IL17RA and cat eye critical region 1 ( CECR1 ). Immunologic studies were carried out by means of flow cytometry, ELISA, and RIA. Results As expected, in the affected child we found a lack of IL-17RA expression, which implies a severe malfunction in the IL-17 signaling pathway, conferring susceptibility to recurrent mucocutaneous infections. Surprisingly, we detected an in vitro and in vivo upregulation of proinflammatory cytokines, notably IL-1β and TNF-α, which is consistent with the persistent systemic inflammation. Conclusions This work emphasizes the utility of whole-genome analyses combined with immunologic investigation in patients with unresolved immunodeficiency. This approach is likely to provide an insight into immunologic pathways and mechanisms of disease. It also provides molecular evidence for more targeted therapies. In addition, our report further corroborates a potential role of ADA2 in modulating immunity and inflammation.

Chinese hamster ovary cells (CHO) are widely used for the stable production of recombinant proteins. Typically, recombinant cell lines are characterized for the stability of protein expression over a period corresponding to the time needed to scale-up the culture and harvest the product (e.g. 2 to 3 months), for the number of plasmid copies integrated into the host genome, and for the quality and quantity of the recombinant protein. In this study we extended the characterization to the cytogenetic level. Sixteen recombinant CHO cell lines were established using calcium phosphate transfection and microinjection as DNA transfer methods. For each cell line we observed by fluorescence in situ hybridization a single integration site regardless of the gene delivery method, the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 50). Integration was not targeted to a specific chromosome. Chromosomal rearrangements were observed in about half of these cell lines. This phenomenon occurred independently of the gene transfer method. Interestingly the rearrangements were not on the chromosome where the plasmid integrated. We observed rearrangements between chromosomes and chromosomal imbalances.

Neuroblastoma is a heterogeneous neural crest–derived embryonic childhood neoplasm that is the second most common solid tumor found in children. Despite recent advances in combined therapy, the overall survival of patients with high-stage disease has not improved in the last decades. Treatment failure is in part attributed to multidrug resistance. To address the mechanisms involved in the development of multidrug resistance, we have generated two doxorubicin-resistant neuroblastoma cell lines (IGRN-91R and LAN-1R). These cells were shown to overexpress the MDR1 gene coding for the P-glycoprotein and were resistant to other MDR1- and non-MDR1-substrate drugs. Indeed, the MDR1 inhibitor verapamil only partially restored sensitivity to drugs, confirming that P-glycoprotein-mediated drug efflux was not responsible for 100% resistance. High-resolution and array-based comparative genomic hybridization analyses revealed the presence of an amplicon in the 7q21 region as the unique genomic alteration common to both doxorubicin-resistant cell lines. In addition to the MDR1 locus, this large amplified region is likely to harbor additional genes potentially involved in the development of drug resistance. This study represents the first molecular cytogenetic and genomic approach to identifying genomic regions involved in the multidrug-resistant phenotype of neuroblastoma. These results could lead to the identification of relevant target genes for the development of new therapeutic modalities. © 2006 Wiley-Liss, Inc.

International audience; The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.

Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expression.

Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression ('SCNA-genes') in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.

Epigenetic silencing of the O6-methylaguanine DNA methyl transferase (MGMT) gene by promoter hypermethylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Quantitative methylation specific PCR (Q-MSP) suggests that not all cells in a glioblastoma exhibiting MGMT methylation carry a methylated MGMT allele, even after adjustment for tumor cell content. This raises the question of the clinically relevant methylation threshold for predicting response to therapy for treatment stratification. There are several potential reasons: heterogeneity of glioblastoma; contaminating normal tissue; a heterogenous methylation pattern not correctly detectable by the assay, or presence of MGMT methylation in distinct subpopulations of cells, namely tumor initiating cells. In order to address the question we investigated the degree and pattern of MGMT promoter methylation in 10 paired samples of glioblastoma and spheres derived thereof using Q-MSP and methylation specific clone sequencing. We observed that in MGMT methylated glioblastoma, the percentage of methylated alleles ranged from 10 to 90%. In contrast, in most of the respective glioblastoma derived spheres, cultured under stem cell conditions, methylated alleles were highly enriched (10/10 clones), even if both MGMT-alleles were retained. The methylation pattern was conserved in the spheres, even when passing them in nude mice. Most importantly, this hypermethylation was associated with complete loss of MGMT expression. For one case we observed only 50% methylated clones in the sphere fraction, associated with low expression of MGMT. Taken together, our data suggest that in MGMT methylated glioblastoma, cells with MGMT promoter hypermethylation are enriched in the sphere fraction which supposedly comprises cancer initiating cells. Thus, even a low percentage of MGMT methylation in a glioblastoma could be predictive marker for a benefit from alkylating agent therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4927.