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1.  Extensive clinical, hormonal and genetic screening in a large consecutive series of 46,XY neonates and infants with atypical sexual development 
Background
One in 4500 children is born with ambiguous genitalia, milder phenotypes occur in one in 300 newborns. Conventional time-consuming hormonal and genetic work-up provides a genetic diagnosis in around 20-40% of 46,XY cases with ambiguous genitalia. All others remain without a definitive diagnosis. The investigation of milder cases, as suggested by recent reports remains controversial.
Methods
Integrated clinical, hormonal and genetic screening was performed in a sequential series of 46, XY children, sex-assigned male, who were referred to our pediatric endocrine service for atypical genitalia (2007–2013).
Results
A consecutive cohort of undervirilized 46,XY children with external masculinization score (EMS) 2–12, was extensively investigated. In four patients, a clinical diagnosis of Kallmann syndrome or Mowat-Wilson syndrome was made and genetically supported in 2/3 and 1/1 cases respectively. Hormonal data were suggestive of a (dihydro)testosterone biosynthesis disorder in four cases, however no HSD17B3 or SRD5A2 mutations were found. Array-CGH revealed a causal structural variation in 2/6 syndromic patients. In addition, three novel NR5A1 mutations were found in non-syndromic patients. Interestingly, one mutation was present in a fertile male, underlining the inter- and intrafamilial phenotypic variability of NR5A1-associated phenotypes. No AR, SRY or WT1 mutations were identified.
Conclusion
Overall, a genetic diagnosis could be established in 19% of non-syndromic and 33% of syndromic cases. There is no difference in diagnostic yield between patients with more or less pronounced phenotypes, as expressed by the external masculinisation score (EMS). The clinical utility of array-CGH is high in syndromic cases. Finally, a sequential gene-by-gene approach is time-consuming, expensive and inefficient. Given the low yield and high expense of Sanger sequencing, we anticipate that massively parallel sequencing of gene panels and whole exome sequencing hold promise for genetic diagnosis of 46,XY DSD boys with an undervirilized phenotype.
Electronic supplementary material
The online version of this article (doi:10.1186/s13023-014-0209-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13023-014-0209-2
PMCID: PMC4271496  PMID: 25497574
46,XY DSD; Undervirilization; Integrated screening; Diagnosis; Array-CGH; MLPA; NR5A1 mutations
2.  ViVar: A Comprehensive Platform for the Analysis and Visualization of Structural Genomic Variation 
PLoS ONE  2014;9(12):e113800.
Structural genomic variations play an important role in human disease and phenotypic diversity. With the rise of high-throughput sequencing tools, mate-pair/paired-end/single-read sequencing has become an important technique for the detection and exploration of structural variation. Several analysis tools exist to handle different parts and aspects of such sequencing based structural variation analyses pipelines. A comprehensive analysis platform to handle all steps, from processing the sequencing data, to the discovery and visualization of structural variants, is missing. The ViVar platform is built to handle the discovery of structural variants, from Depth Of Coverage analysis, aberrant read pair clustering to split read analysis. ViVar provides you with powerful visualization options, enables easy reporting of results and better usability and data management. The platform facilitates the processing, analysis and visualization, of structural variation based on massive parallel sequencing data, enabling the rapid identification of disease loci or genes. ViVar allows you to scale your analysis with your work load over multiple (cloud) servers, has user access control to keep your data safe and is easy expandable as analysis techniques advance. URL: https://www.cmgg.be/vivar/
doi:10.1371/journal.pone.0113800
PMCID: PMC4264741  PMID: 25503062
3.  17q24.2 microdeletions: a new syndromal entity with intellectual disability, truncal obesity, mood swings and hallucinations 
Although microdeletions of the long arm of chromosome 17 are being reported with increasing frequency, deletions of chromosome band 17q24.2 are rare. Here we report four patients with a microdeletion encompassing chromosome band 17q24.2 with a smallest region of overlap of 713 kb containing five Refseq genes and one miRNA. The patients share the phenotypic characteristics, such as intellectual disability (4/4), speech delay (4/4), truncal obesity (4/4), seizures (2/4), hearing loss (3/4) and a particular facial gestalt. Hallucinations and mood swings were also noted in two patients. The PRKCA gene is a very interesting candidate gene for many of the observed phenotypic features, as this gene plays an important role in many cellular processes. Deletion of this gene might explain the observed truncal obesity and could also account for the hallucinations and mood swings seen in two patients, whereas deletion of a CACNG gene cluster might be responsible for the seizures observed in two patients. In one of the patients, the PRKAR1A gene responsible for Carney Complex and the KCNJ2 gene causal for Andersen syndrome are deleted. This is the first report of a patient with a whole gene deletion of the KCNJ2 gene.
doi:10.1038/ejhg.2011.239
PMCID: PMC3330218  PMID: 22166941
17q24.2 deletion; array CGH; PRKCA; mood swings; hallucinations
5.  Microhomology-Mediated Mechanisms Underlie Non-Recurrent Disease-Causing Microdeletions of the FOXL2 Gene or Its Regulatory Domain 
PLoS Genetics  2013;9(3):e1003358.
Genomic disorders are often caused by recurrent copy number variations (CNVs), with nonallelic homologous recombination (NAHR) as the underlying mechanism. Recently, several microhomology-mediated repair mechanisms—such as microhomology-mediated end-joining (MMEJ), fork stalling and template switching (FoSTeS), microhomology-mediated break-induced replication (MMBIR), serial replication slippage (SRS), and break-induced SRS (BISRS)—were described in the etiology of non-recurrent CNVs in human disease. In addition, their formation may be stimulated by genomic architectural features. It is, however, largely unexplored to what extent these mechanisms contribute to rare, locus-specific pathogenic CNVs. Here, fine-mapping of 42 microdeletions of the FOXL2 locus, encompassing FOXL2 (32) or its regulatory domain (10), serves as a model for rare, locus-specific CNVs implicated in genetic disease. These deletions lead to blepharophimosis syndrome (BPES), a developmental condition affecting the eyelids and the ovary. For breakpoint mapping we used targeted array-based comparative genomic hybridization (aCGH), quantitative PCR (qPCR), long-range PCR, and Sanger sequencing of the junction products. Microhomology, ranging from 1 bp to 66 bp, was found in 91.7% of 24 characterized breakpoint junctions, being significantly enriched in comparison with a random control sample. Our results show that microhomology-mediated repair mechanisms underlie at least 50% of these microdeletions. Moreover, genomic architectural features, like sequence motifs, non-B DNA conformations, and repetitive elements, were found in all breakpoint regions. In conclusion, the majority of these microdeletions result from microhomology-mediated mechanisms like MMEJ, FoSTeS, MMBIR, SRS, or BISRS. Moreover, we hypothesize that the genomic architecture might drive their formation by increasing the susceptibility for DNA breakage or promote replication fork stalling. Finally, our locus-centered study, elucidating the etiology of a large set of rare microdeletions involved in a monogenic disorder, can serve as a model for other clustered, non-recurrent microdeletions in genetic disease.
Author Summary
Genomic disorder is a general term describing conditions caused by genomic aberrations leading to a copy number change of one or more genes. Copy number changes with the same length and clustered breakpoints for a group of patients with the same disorder are named recurrent rearrangements. These originate mostly from a well-studied mechanism, namely nonallelic homologous recombination (NAHR). In contrast, non-recurrent rearrangements vary in size, have scattered breakpoints, and can originate from several different mechanisms that are not fully understood. Here we tried to gain further insight into the extent to which these mechanisms contribute to non-recurrent rearrangements and into the possible role of the surrounding genomic architecture. To this end, we investigated a unique group of patients with non-recurrent deletions of the FOXL2 region causing blepharophimosis syndrome. We observed that the majority of these deletions can result from several mechanisms mediated by microhomology. Furthermore, our data suggest that rare pathogenic microdeletions do not occur at random genome sequences, but are possibly guided by the surrounding genomic architecture. Finally, our study, elucidating the etiology of a unique cohort of locus-specific microdeletions implicated in genetic disease, can serve as a model for the formation of genomic aberrations in other genetic disorders.
doi:10.1371/journal.pgen.1003358
PMCID: PMC3597517  PMID: 23516377
6.  Focal DNA Copy Number Changes in Neuroblastoma Target MYCN Regulated Genes 
PLoS ONE  2013;8(1):e52321.
Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.
doi:10.1371/journal.pone.0052321
PMCID: PMC3537730  PMID: 23308108
7.  LNCipedia: a database for annotated human lncRNA transcript sequences and structures 
Nucleic Acids Research  2012;41(Database issue):D246-D251.
Here, we present LNCipedia (http://www.lncipedia.org), a novel database for human long non-coding RNA (lncRNA) transcripts and genes. LncRNAs constitute a large and diverse class of non-coding RNA genes. Although several lncRNAs have been functionally annotated, the majority remains to be characterized. Different high-throughput methods to identify new lncRNAs (including RNA sequencing and annotation of chromatin-state maps) have been applied in various studies resulting in multiple unrelated lncRNA data sets. LNCipedia offers 21 488 annotated human lncRNA transcripts obtained from different sources. In addition to basic transcript information and gene structure, several statistics are determined for each entry in the database, such as secondary structure information, protein coding potential and microRNA binding sites. Our analyses suggest that, much like microRNAs, many lncRNAs have a significant secondary structure, in-line with their presumed association with proteins or protein complexes. Available literature on specific lncRNAs is linked, and users or authors can submit articles through a web interface. Protein coding potential is assessed by two different prediction algorithms: Coding Potential Calculator and HMMER. In addition, a novel strategy has been integrated for detecting potentially coding lncRNAs by automatically re-analysing the large body of publicly available mass spectrometry data in the PRIDE database. LNCipedia is publicly available and allows users to query and download lncRNA sequences and structures based on different search criteria. The database may serve as a resource to initiate small- and large-scale lncRNA studies. As an example, the LNCipedia content was used to develop a custom microarray for expression profiling of all available lncRNAs.
doi:10.1093/nar/gks915
PMCID: PMC3531107  PMID: 23042674
8.  Nasal speech and hypothyroidism are common hallmarks of 12q15 microdeletions 
European Journal of Human Genetics  2011;19(10):1032-1037.
The introduction of array CGH in clinical diagnostics has led to the discovery of many new microdeletion/microduplication syndromes. Most of them are rare and often present with a variable range of clinical anomalies. In this study we report three patients with a de novo overlapping microdeletion of chromosome bands 12q15q21.1. The deletions are ∼2.5 Mb in size, with a 1.34-Mb common deleted region containing six RefSeq genes. All three patients present with learning disability or developmental delay, nasal speech and hypothyroidism. In this paper we will further elaborate on the genotype–phenotype correlation associated with this deletion and compare our patients with previously reported cases.
doi:10.1038/ejhg.2011.67
PMCID: PMC3190250  PMID: 21505450
12q15 microdeletion; nasal speech; hypothyroidism; developmental delay
9.  Anti-NMDA-receptor encephalitis in a 3 year old patient with chromosome 6p21.32 microdeletion including the HLA cluster 
Anti-NMDA-receptor encephalitis was initially described as a paraneoplastic disorder in young women with ovarian teratoma. We report on a 3-year-old boy who developed anti-NMDA-receptor encephalitis one month after a respiratory infection. Moreover, array-comparative genomic hybridization in this patient revealed an inherited microdeletion in chromosomeband 6p21.32, including the HLA-DPB1 and HLA-DPB2 genes. The clinical relevance of this microdeletion is discussed.
doi:10.1016/j.ejpn.2010.07.004
PMCID: PMC3086678  PMID: 20692195
NMDAR encephalitis; Childhood; Post-infectious; Microdeletion; HLA cluster
10.  Nonsyndromic bilateral and unilateral optic nerve aplasia: first familial occurrence and potential implication of CYP26A1 and CYP26C1 genes 
Molecular Vision  2011;17:2072-2079.
Purpose
Optic nerve aplasia (ONA, OMIM 165550) is a very rare unilateral or bilateral condition that leads to blindness in the affected eye, and is usually associated with other ocular abnormalities. Although bilateral ONA often occurs in association with severe congenital anomalies of the brain, nonsyndromic sporadic forms with bilateral ONA have been described. So far, no autosomal-dominant nonsyndromic ONA has been reported. The genetic basis of this condition remains largely unknown, as no developmental genes other than paired box gene 6 (PAX6) are known to be implicated in sporadic bilateral ONA.
Methods
The individuals reported underwent extensive ophthalmological, endocrinological, and neurologic evaluation, including neuroimaging of the visual pathways. In addition genomewide copy number screening was performed.
Results
Here we report an autosomal-dominant form of nonsyndromic ONA in a Belgian pedigree, with unilateral microphthalmia and ONA in the second generation (II:1), and bilateral ONA in two sibs of the third generation (III:1; III:2). No PAX6 mutation was found. Genome wide copy number screening revealed a microdeletion of maximal 363 kb of chromosome 10q23.33q23.33 in all affected individuals (II:1, III:1; III:2) and in unaffected I:1, containing three genes: exocyst complex component 6 (EXOC6), cytochrome p450, subfamily XXVIA, polypeptide 1 (CYP26A1), and cytochrome p450, subfamily XXVIC, polypeptide 1 (CYP26C1). The latter two encode retinoic acid-degrading enzymes.
Conclusions
This is the first study reporting an autosomal-dominant form of nonsyndromic ONA. The diagnostic value of neuroimaging in uncovering ONA in microphthalmic patients is demonstrated. Although involvement of other genetic factors cannot be ruled out, our study might point to a role of CYP26A1 and CYP26C1 in the pathogenesis of nonsyndromic ONA.
PMCID: PMC3156792  PMID: 21850183
11.  Osteopoikilosis, short stature and mental retardation as key features of a new microdeletion syndrome on 12q14 
Journal of Medical Genetics  2007;44(4):264-268.
This report presents the detection of a heterozygous deletion at chromosome 12q14 in three unrelated patients with a similar phenotype consisting of mild mental retardation, failure to thrive in infancy, proportionate short stature and osteopoikilosis as the most characteristic features. In each case, this interstitial deletion was found using molecular karyotyping. The deletion occurred as a de novo event and varied between 3.44 and 6 megabases (Mb) in size with a 3.44 Mb common deleted region. The deleted interval was not flanked by low‐copy repeats or segmental duplications. It contains 13 RefSeq genes, including LEMD3, which was previously shown to be the causal gene for osteopoikilosis. The observation of osteopoikilosis lesions should facilitate recognition of this new microdeletion syndrome among children with failure to thrive, short stature and learning disabilities.
doi:10.1136/jmg.2006.047860
PMCID: PMC2598049  PMID: 17220210
osteopoikilosis; short stature; mental retardation;  HMGA2 ;  GRIP1
12.  Identification of Two Critically Deleted Regions within Chromosome Segment 7q35-q36 in EVI1 Deregulated Myeloid Leukemia Cell Lines 
PLoS ONE  2010;5(1):e8676.
Chromosomal rearrangements involving the EVI1 proto-oncogene are a recurrent finding in myeloid leukemias and are indicative of a poor prognosis. Rearrangements of the EVI1 locus are often associated with monosomy 7 or cytogenetic detectable deletions of part of 7q. As EVI1 overexpression alone is not sufficient to induce leukemia, loss of a 7q tumour suppressor gene might be a required cooperating event. To test this hypothesis, we performed high-resolution array comparative genomic hybridization analysis of twelve EVI1 overexpressing patients and three EVI1 deregulated cell lines to search for 7q submicroscopic deletions. This analysis lead to the delineation of two critical regions, one of 0.39 Mb on 7q35 containing the CNTNAP2 gene and one of 1.33 Mb on chromosome bands 7q35–q36 comprising nine genes in EVI1 deregulated cell lines. These findings open the way to further studies aimed at identifying the culprit EVI1 implicated tumour suppressor genes on 7q.
doi:10.1371/journal.pone.0008676
PMCID: PMC2800774  PMID: 20084277
13.  Array comparative genomic hybridization and flow cytometry analysis of spontaneous abortions and mors in utero samples 
BMC Medical Genetics  2009;10:89.
Background
It is estimated that 10-15% of all clinically recognised pregnancies result in a spontaneous abortion or miscarriage. Previous studies have indicated that in up to 50% of first trimester miscarriages, chromosomal abnormalities can be identified. For several decades chromosome analysis has been the golden standard to detect these genomic imbalances. A major drawback of this method is the requirement of short term cultures of fetal cells. In this study we evaluated the combined use of array CGH and flow cytometry (FCM), for detection of chromosomal abnormalities, as an alternative for karyotyping.
Methods
In total 100 spontaneous abortions and mors in utero samples were investigated by karyotyping and array CGH in combination with FCM in order to compare the results for both methods.
Results
Chromosome analysis revealed 17 abnormal karyotypes whereas array CGH in combination with FCM identified 26 aberrations due to the increased test success rate. Karyotyping was unsuccessful in 28% of cases as compared to only two out of hundred samples with inconclusive results for combined array CGH and FCM analysis.
Conclusion
This study convincingly shows that array CGH analysis for detection of numerical and segmental imbalances in combination with flow cytometry for detection of ploidy status has a significant higher detection rate for chromosomal abnormalities as compared to karyotyping of miscarriages samples.
doi:10.1186/1471-2350-10-89
PMCID: PMC2753309  PMID: 19751515
14.  Recurrent Rearrangements of Chromosome 1q21.1 and Variable Pediatric Phenotypes 
Mefford, Heather C. | Sharp, Andrew J. | Baker, Carl | Itsara, Andy | Jiang, Zhaoshi | Buysse, Karen | Huang, Shuwen | Maloney, Viv K. | Crolla, John A. | Baralle, Diana | Collins, Amanda | Mercer, Catherine | Norga, Koen | de Ravel, Thomy | Devriendt, Koen | Bongers, Ernie M.H.F. | de Leeuw, Nicole | Reardon, William | Gimelli, Stefania | Bena, Frederique | Hennekam, Raoul C. | Male, Alison | Gaunt, Lorraine | Clayton-Smith, Jill | Simonic, Ingrid | Park, Soo Mi | Mehta, Sarju G. | Nik-Zainal, Serena | Woods, C. Geoffrey | Firth, Helen V. | Parkin, Georgina | Fichera, Marco | Reitano, Santina | Giudice, Mariangela Lo | Li, Kelly E. | Casuga, Iris | Broomer, Adam | Conrad, Bernard | Schwerzmann, Markus | Räber, Lorenz | Gallati, Sabina | Striano, Pasquale | Coppola, Antonietta | Tolmie, John L. | Tobias, Edward S. | Lilley, Chris | Armengol, Lluis | Spysschaert, Yves | Verloo, Patrick | De Coene, Anja | Goossens, Linde | Mortier, Geert | Speleman, Frank | van Binsbergen, Ellen | Nelen, Marcel R. | Hochstenbach, Ron | Poot, Martin | Gallagher, Louise | Gill, Michael | McClellan, Jon | King, Mary-Claire | Regan, Regina | Skinner, Cindy | Stevenson, Roger E. | Antonarakis, Stylianos E. | Chen, Caifu | Estivill, Xavier | Menten, Björn | Gimelli, Giorgio | Gribble, Susan | Schwartz, Stuart | Sutcliffe, James S. | Walsh, Tom | Knight, Samantha J.L. | Sebat, Jonathan | Romano, Corrado | Schwartz, Charles E. | Veltman, Joris A. | de Vries, Bert B.A. | Vermeesch, Joris R. | Barber, John C.K. | Willatt, Lionel | Tassabehji, May | Eichler, Evan E.
The New England journal of medicine  2008;359(16):1685-1699.
BACKGROUND
Duplications and deletions in the human genome can cause disease or predispose persons to disease. Advances in technologies to detect these changes allow for the routine identification of submicroscopic imbalances in large numbers of patients.
METHODS
We tested for the presence of microdeletions and microduplications at a specific region of chromosome 1q21.1 in two groups of patients with unexplained mental retardation, autism, or congenital anomalies and in unaffected persons.
RESULTS
We identified 25 persons with a recurrent 1.35-Mb deletion within 1q21.1 from screening 5218 patients. The microdeletions had arisen de novo in eight patients, were inherited from a mildly affected parent in three patients, were inherited from an apparently unaffected parent in six patients, and were of unknown inheritance in eight patients. The deletion was absent in a series of 4737 control persons (P = 1.1×10−7). We found considerable variability in the level of phenotypic expression of the microdeletion; phenotypes included mild-to-moderate mental retardation, microcephaly, cardiac abnormalities, and cataracts. The reciprocal duplication was enriched in the nine children with mental retardation or autism spectrum disorder and other variable features (P = 0.02). We identified three deletions and three duplications of the 1q21.1 region in an independent sample of 788 patients with mental retardation and congenital anomalies.
CONCLUSIONS
We have identified recurrent molecular lesions that elude syndromic classification and whose disease manifestations must be considered in a broader context of development as opposed to being assigned to a specific disease. Clinical diagnosis in patients with these lesions may be most readily achieved on the basis of genotype rather than phenotype.
doi:10.1056/NEJMoa0805384
PMCID: PMC2703742  PMID: 18784092
15.  Disease-Causing 7.4 kb Cis-Regulatory Deletion Disrupting Conserved Non-Coding Sequences and Their Interaction with the FOXL2 Promotor: Implications for Mutation Screening 
PLoS Genetics  2009;5(6):e1000522.
To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5′ to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular.
Author Summary
Long-range genetic control is an inherent feature of genes harbouring a highly complex spatiotemporal expression pattern, requiring a combined action of multiple cis-regulatory elements such as promoters, enhancers, and silencers. Consequently, disruption of the long-range genetic control of a target gene by genomic rearrangements of regulatory elements may lead to aberrant gene transcription and disease. To date, the contribution of mutated regulatory elements to human disease has not been studied frequently. Here, we explored the contribution of genetic changes in potentially cis-regulatory elements of the FOXL2 gene in blepharophimosis syndrome (BPES), a developmental monogenic condition of the eyelids and ovaries. We identified a de novo very subtle deletion of 7.4 kb causing BPES. Moreover, we studied the functional capacities and chromosome conformation of the deleted region in FOXL2 expressing cellular systems. Interestingly, the chromosome conformation analysis demonstrated the close proximity of the 7.4 kb deleted fragment and two other conserved regions with the FOXL2 core promoter, and the necessity of their integrity for correct FOXL2 expression. Finally, our study revealed the smallest distant deletion causing monogenic disease and emphasized the importance of mutation screening of cis-regulatory elements in human genetic disease.
doi:10.1371/journal.pgen.1000522
PMCID: PMC2689649  PMID: 19543368
16.  Report of a female patient with mental retardation and tall stature due to a chromosomal rearrangement disrupting the OPHN1 gene on Xq12 
We report on a patient with mental retardation, seizures and tall stature with advanced bone age in whom a de novo apparently balanced chromosomal rearrangement 46,XX,t(X;9)(q12;p13.3) was identified. Using array CGH on flow-sorted derivative chromosomes (array painting) and subsequent FISH and qPCR analysis, we mapped and sequenced both breakpoints. The Xq12 breakpoint was located within the gene coding for oligophrenin 1 (OPHN1) whereas the 9p13.3 breakpoint was assigned to a non-coding segment within a gene dense region. Disruption of OPHN1 by the Xq12 breakpoint was considered the major cause of the abnormal phenotype observed in the proband.
doi:10.1016/j.ejmg.2007.07.003
PMCID: PMC2688819  PMID: 17845870
Translocation; OPHN1; Tall stature; Mental retardation
17.  Mapping biomedical concepts onto the human genome by mining literature on chromosomal aberrations 
Nucleic Acids Research  2007;35(8):2533-2543.
Biomedical literature provides a rich but unstructured source of associations between chromosomal regions and biomedical concepts. By mining MEDLINE abstracts, we annotate the human genome at the level of cytogenetic bands. Our method creates a set of chromosomal aberration maps that associate cytogenetic bands to biomedical concepts from a variety of controlled vocabularies, including disease, dysmorphology, anatomy, development and Gene Ontology branches. The association between a band (e.g. 4p16.3) and a concept (e.g. microcephaly) is assessed by the statistical overrepresentation of this concept in the abstracts relating to this band. Our method is validated using existing genome annotation resources and known chromosomal aberration maps and is further illustrated through a case study on heart disease. Our chromosomal aberration maps provide diagnostics support to clinical geneticists, aid cytogeneticists to interpret and report cytogenetic findings and support researchers interested in human gene function. The method is available as a web application, aBandApart, at http://www.esat.kuleuven.be/abandapart/.
doi:10.1093/nar/gkm054
PMCID: PMC1885641  PMID: 17403693
18.  Positional and functional mapping of a neuroblastoma differentiation gene on chromosome 11 
BMC Genomics  2005;6:97.
Background
Loss of chromosome 11q defines a subset of high-stage aggressive neuroblastomas. Deletions are typically large and mapping efforts have thus far not lead to a well defined consensus region, which hampers the identification of positional candidate tumour suppressor genes. In a previous study, functional evidence for a neuroblastoma suppressor gene on chromosome 11 was obtained through microcell mediated chromosome transfer, indicated by differentiation of neuroblastoma cells with loss of distal 11q upon introduction of chromosome 11. Interestingly, some of these microcell hybrid clones were shown to harbour deletions in the transferred chromosome 11. We decided to further exploit this model system as a means to identify candidate tumour suppressor or differentiation genes located on chromosome 11.
Results
In a first step, we performed high-resolution arrayCGH DNA copy-number analysis in order to evaluate the chromosome 11 status in the hybrids. Several deletions in both parental and transferred chromosomes in the investigated microcell hybrids were observed. Subsequent correlation of these deletion events with the observed morphological changes lead to the delineation of three putative regions on chromosome 11: 11q25, 11p13->11p15.1 and 11p15.3, that may harbour the responsible differentiation gene.
Conclusion
Using an available model system, we were able to put forward some candidate regions that may be involved in neuroblastoma. Additional studies will be required to clarify the putative role of the genes located in these chromosomal segments in the observed differentiation phenotype specifically or in neuroblastoma pathogenesis in general.
doi:10.1186/1471-2164-6-97
PMCID: PMC1185534  PMID: 16000168
19.  arrayCGHbase: an analysis platform for comparative genomic hybridization microarrays 
BMC Bioinformatics  2005;6:124.
Background
The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH). One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment.
Results
We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment) supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser.
Conclusion
ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at .
doi:10.1186/1471-2105-6-124
PMCID: PMC1173083  PMID: 15910681
20.  Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons 
BMC Genomics  2004;5:11.
Background
Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes.
Results
As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences.
Conclusions
The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.
doi:10.1186/1471-2164-5-11
PMCID: PMC365025  PMID: 15018647
Amplification; Overexpression; Oncogene; SSH; Array CGH; Neuroblastoma

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