Fikret Erdogan

High frequency of submicroscopic genomic aberrations detected by tiling path array comparative genome hybridisation in patients with isolated congenital heart disease

Background: Congenital heart disease (CHD) is the most common birth defect and affects nearly 1% of newborns. The aetiology of CHD is largely unknown and only a small percentage can be assigned to environmental risk factors such as maternal diseases or exposure to mutagenic agents during pregnancy. Chromosomal imbalances have been identified in many forms of syndromic CHD, but very little is known about the impact of DNA copy number changes in non-syndromic CHD. Method: A sub-megabase resolution array comparative genome hybridisation (CGH) screen was carried out on 105 patients with CHD as the sole abnormality at the time of diagnosis. Results: There were 18 chromosomal changes detected, which do not coincide with common DNA copy number variants, including one de novo deletion, two de novo duplications and eight familial copy number variations (one deletion and seven duplications). Conclusions: Our data show that submicroscopic deletions and duplications play an important role in the aetiology of this condition, either as direct causes or as genetic risk factors for CHD. These findings have immediate consequences for genetic counselling and should pave the way for the elucidation of the pathogenetic mechanisms underlying CHD.

: CGHPRO – a comprehensive data analysis tool for array CGH

BackgroundArray CGH (Comparative Genomic Hybridisation) is a molecular cytogenetic technique for the genome wide detection of chromosomal imbalances. It is based on the co-hybridisation of differentially labelled test and reference DNA onto arrays of genomic BAC clones, cDNAs or oligonucleotides, and after correction for various intervening variables, loss or gain in the test DNA can be indicated from spots showing aberrant signal intensity ratios.Now that this technique is no longer confined to highly specialized laboratories and is entering the realm of clinical application, there is a need for a user-friendly software package that facilitates estimates of DNA dosage from raw signal intensities obtained by array CGH experiments, and which does not depend on a sophisticated computational environment.ResultsWe have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection and comparative analysis of array-CGH data. CGHPRO is a stand-alone JAVA application that guides the user through the whole process of data analysis. The import option for image analysis data covers several data formats, but users can also customize their own data formats. Several graphical representation tools assist in the selection of the appropriate normalization method. Intensity ratios of each clone can be plotted in a size-dependent manner along the chromosome ideograms. The interactive graphical interface offers the chance to explore the characteristics of each clone, such as the involvement of the clones sequence in segmental duplications. Circular Binary Segmentation and unsupervised Hidden Markov Model algorithms facilitate objective detection of chromosomal breakpoints. The storage of all essential data in a back-end database allows the simultaneously comparative analysis of different cases. The various display options facilitate also the definition of shortest regions of overlap and simplify the identification of odd clones.ConclusionCGHPRO is a comprehensive and easy-to-use data analysis tool for array CGH. Since all of its features are available offline, CGHPRO may be especially suitable in situations where protection of sensitive patient data is an issue. It is distributed under GNU GPL licence and runs on Linux and Windows.

Mutations in Autism Susceptibility Candidate 2 (AUTS2) in patients with mental retardation

We report on three unrelated mentally disabled patients, each carrying a de novo balanced translocation that truncates the autism susceptibility candidate 2 (AUTS2) gene at 7q11.2. One of our patients shows relatively mild mental retardation; the other two display more profound disorders. One patient is also physically disabled, exhibiting urogenital and limb malformations in addition to severe mental retardation. The function of AUTS2 is presently unknown, but it has been shown to be disrupted in monozygotic twins with autism and mental retardation, both carrying a translocation t(7;20)(q11.2;p11.2) (de la Barra et al. in Rev Chil Pediatr 57:549–554, 1986; Sultana et al. in Genomics 80:129–134, 2002). Given the overlap of this autism/mental retardation (MR) phenotype and the MR-associated disorders in our patients, together with the fact that mapping of the additional autosomal breakpoints involved did not disclose obvious candidate disease genes, we ascertain with this study that AUTS2 mutations are clearly linked to autosomal dominant mental retardation.

Impact of low copy repeats on the generation of balanced and unbalanced chromosomal aberrations in mental retardation

Low copy repeats (LCRs) are stretches of duplicated DNA that are more than 1 kb in size and share a sequence similarity that exceeds 90%. Non-allelic homologous recombination (NAHR) between highly similar LCRs has been implicated in numerous genomic disorders. This study aimed at defining the impact of LCRs on the generation of balanced and unbalanced chromosomal rearrangements in mentally retarded patients. A cohort of 22 patients, preselected for the presence of submicroscopic imbalances, was analysed using submegabase resolution tiling path array CGH and the results were compared with a set of 41 patients with balanced translocations and breakpoints that were mapped to the BAC level by FISH. Our data indicate an accumulation of LCRs at breakpoints of both balanced and unbalanced rearrangements. LCRs with high sequence similarity in both breakpoint regions, suggesting NAHR as the most likely cause of rearrangement, were observed in 6/22 patients with chromosomal imbalances, but not in any of the balanced translocation cases studied. In case of chromosomal imbalances, the likelihood of NAHR seems to be inversely related to the size of the aberration. Our data also suggest the presence of additional mechanisms coinciding with or dependent on the presence of LCRs that may induce an increased instability at these chromosomal sites.

Chromosome deletions in 13q33–34: Report of four patients and review of the literature

Deletions of chromosome bands 13q33–34 are rare. Patients with such deletions have mental retardation, microcephaly, and distinct facial features. Male patients frequently also have genital malformations. We report on four patients with three overlapping deletions of 13q33–34 that have been characterized by tiling‐path array‐CGH. Patient 1 had mental retardation and microcephaly with an interstitial 4.7 Mb deletion and a translocation t(12;13)(q13.3;q32.3). His mother (Patient 2), who also had mental retardation and microcephaly, carried the identical chromosome aberration. Patient 3 was a girl with a de novo insertion ins(7;13)(p15.1;q22q31) and interstitial 4.5 Mb deletion in 13q33–34. She had mental retardation and microcephaly. Patient 4 was a newborn boy with severe genital malformation (penoscrotal transposition and hypospadias) and microcephaly. He had a de novo ring chromosome 13 lacking the terminal 9.3 Mb of 13q. Karyotype–phenotype comparisons of these and eight previously published del13q33–34 patients suggest EFNB2 as a candidate gene for genital malformations in males. Molecular cytogenetic definition of a common deleted region in all patients suggests ARHGEF7 as a candidate gene for mental retardation and microcephaly.

Characterization of a 5.3 Mb deletion in 15q14 by comparative genomic hybridization using a whole genome “tiling path” BAC array in a girl with heart defect, cleft palate, and developmental delay†

High‐resolution array CGH utilizing sets of overlapping BAC and PAC clones (“tiling path”) covering the whole genome is a powerful novel tool for fast detection of submicroscopic chromosome deletions or duplications. We describe the successful application of a submegabase resolution whole genome “tiling path” BAC array to confirm and characterize a de novo interstitial deletion of chromosome 15. The deletion has a size of 5.3 Mb and is located within chromosome band 15q14, distal to the Prader–Willi/Angelman region. The affected girl had a heart defect, cleft palate, recurrent infections, and developmental delay. In contrast to GTG banding, array CGH determined the exact number of deleted genes and thus allowed the identification of candidate genes for cleft palate (GREM1, CX36, MEIS2), congenital heart defect (ACTC, GREM1, CX36, MEIS2), and mental retardation (ARHGAP11A, CHRNA7, CHRM5).

High frequency of rare copy number variants affecting functionally related genes in patients with structural brain malformations

During the past years, significant advances have been made in our understanding of the development of the human brain, and much of this knowledge comes from genetic studies of disorders associated with abnormal brain development. We employed array‐comparative genomic hybridization (CGH) to investigate copy number variants (CNVs) in a cohort of 169 patients with various structural brain malformations including lissencephaly, polymicrogyria, focal cortical dysplasia, and corpus callosum agenesis. The majority of the patients had intellectual disabilities (ID) and suffered from symptomatic epilepsy. We detected at least one rare CNV in 38 patients (22.5%). All genes located within the rare CNVs were subjected to enrichment analysis for specific Gene Ontology Terms or Kyoto Encyclopedia of Genes and Genomes pathways and to protein–protein network analysis. Based on these analyses, we propose that genes involved in “axonal transport,” “cation transmembrane transporter activity,” and the “c‐Jun N‐terminal kinase (JNK) cascade” play a significant role in the etiology of brain malformations. This is to the best of our knowledge the first systematic study of CNVs in patients with structural brain malformations and our data show that CNVs play an important role in the etiology of these malformations, either as direct causes or as genetic risk factors. 32:1427–1435, 2011. ©2011 Wiley Periodicals, Inc.

Delineation of a 2.2 Mb microdeletion at 5q35 associated with microcephaly and congenital heart disease

Fine mapping of chromosomal deletions and genotype–phenotype comparisons of clinically well‐defined patients can be used to confirm or reveal loci and genes associated with human disorders. Eleven patients with cytogenetically visible deletions involving the terminal region of chromosome 5q have been described, but the extent of the deletion was determined only in one case. In this study we describe a 15‐year‐old boy with Ebstein anomaly, atrial septal defect (ASD), atrioventricular (AV) conduction defect, and microcephaly. He had an apparently balanced paracentric inversion of chromosome 5, with the karyotype 46, XY,inv(5)(q13q35) de novo. Further mapping of the chromosome breakpoints using fluorescence in situ hybridization (FISH) revealed a 2.2 Mb microdeletion at the 5q35 breakpoint, which spans 16 genes, including the cardiac homeobox transcription factor gene NKX2‐5. The current data suggest that haploinsufficiency of NKX2‐5 cause Ebstein anomaly and support previous results showing that NKX2‐5 mutations cause ASD and AV conduction defect. Furthermore, we suggest presence of a new microcephaly locus within a 2.2 Mb region at 5q35.1–q35.2.

Characterization of interstitial Xp duplications in two families by tiling path array CGH

Duplications of the short arm of the X chromosome in male patients are rare. We report on the clinical features of mentally retarded patients in two families with different interstitial duplications of Xp and their characterization by tiling path array comparative genomic hybridization (array CGH). In Family A, we detected a duplication of 9.3 Mb in Xp11p21 in a male with severe mental retardation [karyotype 46,XY,dup(X)(p11.3p21.1)] and his healthy mother. The clinical features of this patient—severe mental retardation, obesity, macrocephaly—are in accordance with those of a previously reported patient with a similar duplication. In Family B, a duplication of 8.5 Mb was diagnosed in Xp22 in three male patients with mental retardation [karyotype 46,XY,dup(X)(p22.11p22.2)] and two healthy females. Characterization of the duplications by array CGH enabled the identification of the genes within these intervals. These comprise known mental retardation genes such as MAOA, NDP, TM4SF2, NDP, RSK2, and CDKL5. Duplication of MAOA will be discussed as a possible cause of obesity.

A novel 8 Mb interstitial deletion of chromosome 8p12‐p21.2

We report on a girl with delayed mental and motor development, ophthalmological abnormalities, and peripheral neuropathy. Chromosome analysis suggested a deletion within chromosome 8p. Further investigation by array‐based comparative genomic hybridization (array‐CGH) delineated an 8 Mb interstitial deletion on the short arm of chromosome 8. The breakpoints are located at chromosome bands 8p12 and 8p21.2. Forty‐two known genes including gonadotropin‐releasing hormone 1 (GNRH1), transcription factor EBF2, exostosin‐like 3 (EXTL3), glutathione reductase (GSR), and neuregulin 1 (NRG1), are located within the deleted region on chromosome 8p. A comparison of our patient with the cases described in the literature is presented, and we discuss the genotype–phenotype correlation in our patient. This is the first report of array‐CGH analysis of an interstitial deletion at chromosome 8p.