Complex chromosome 17p rearrangements associated with low-copy repeats in two patients with congenital anomalies

Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Human Genetics (Impact Factor: 4.52). 08/2007; 121(6):697-709. DOI: 10.1007/s00439-007-0359-6
Source: PubMed

ABSTRACT Recent molecular cytogenetic data have shown that the constitution of complex chromosome rearrangements (CCRs) may be more complicated than previously thought. The complicated nature of these rearrangements challenges the accurate delineation of the chromosomal breakpoints and mechanisms involved. Here, we report a molecular cytogenetic analysis of two patients with congenital anomalies and unbalanced de novo CCRs involving chromosome 17p using high-resolution array-based comparative genomic hybridization (array CGH) and fluorescent in situ hybridization (FISH). In the first patient, a 4-month-old boy with developmental delay, hypotonia, growth retardation, coronal synostosis, mild hypertelorism, and bilateral club feet, we found a duplication of the Charcot-Marie-Tooth disease type 1A and Smith-Magenis syndrome (SMS) chromosome regions, inverted insertion of the Miller-Dieker lissencephaly syndrome region into the SMS region, and two microdeletions including a terminal deletion of 17p. The latter, together with a duplication of 21q22.3-qter detected by array CGH, are likely the unbalanced product of a translocation t(17;21)(p13.3;q22.3). In the second patient, an 8-year-old girl with mental retardation, short stature, microcephaly and mild dysmorphic features, we identified four submicroscopic interspersed 17p duplications. All 17 breakpoints were examined in detail by FISH analysis. We found that four of the breakpoints mapped within known low-copy repeats (LCRs), including LCR17pA, middle SMS-REP/LCR17pB block, and LCR17pC. Our findings suggest that the LCR burden in proximal 17p may have stimulated the formation of these CCRs and, thus, that genome architectural features such as LCRs may have been instrumental in the generation of these CCRs.

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Available from: Lisenka E L M Vissers, Jul 29, 2015
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    • "In addition to this, simple rearrangements account for the vast majority of RAF gene fusions (95%), which is similar to studies of BIR in yeast, where template switching was observed in only 20% of cases (Smith et al. 2007). Unlike other studies that have reported large interspersed duplications and deletions (Lee et al. 2007; Vissers et al. 2007), the complex rearrangements we detected were <2 kb in length and directly adjacent to the breakpoint. Although it is possible that the interspersed copy number changes observed by other groups could occur by "
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    • "LIS1 has been identified as the specific gene responsible for the classical brain and facial abnormalities of MDLS, but other genes in the region are thought to contribute to associated features often observed in the syndrome, including somatic growth retardation. Also of interest is the study by Vissers et al. (2007), who recently reported a number of complex chromosomal rearrangements in proximal 17p (including the suggestive QTL at 17p13.2 reported here for the midchildhood growth spurt) in patients with several development abnormalities, including significantly reduced growth. Thus it is possible that the proximal region of chromosome 17p contains as yet unidentified genes that might influence features of normal growth such as the midchildhood growth spurt. "
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    ABSTRACT: Het chromosomenonderzoek vormt nog steeds een van de hoekstenen van het genetisch onderzoek bij kinderen met een aangeboren hartafwijking. In dit onderzoek worden de chromosomen in delende bloedcellen bekeken onder de microscoop. Op die manier wordt onderzocht of hele chromosomen of delen van chromosomen aanwezig zijn in te veel of te weinig kopijen. In deze studie hebben we onderzocht of chromosoomafwijkingen die niet zichtbaar zijn met de microscoop een frequente oorzaak voor aangeboren hartafwijkingen zijn. We hebben een nieuwe technologie geïntroduceerd (“array CGH”) die toelaat om een profiel van het kopijaantal op te stellen van alle chromosomen. We toonden aan dat deze technologie een betrouwbare detectie van chromosoomafwijkingen mogelijk maakt, en dit met een resolutie die veel beter is dan het klassieke chromosomenonderzoek. Het toepassen van deze array CGH techniek in patiënten met een syndromale hartafwijking verhoogt de kans om de onderliggende genetische oorzaak te identificeren sterk: in 20% van deze patiënten wordt een chromosoomafwijking gevonden die onzichtbaar was met de klassieke technieken. Bovendien wordt op deze manier het genetische defect zeer precies afgelijnd waardoor de patiënt een meer specifieke en gepersonaliseerde diagnose krijgt. Een diagnose is van zeer groot belang voor de verdere follow-up van patiënten en hun families, vermits dit toelaat patiënt en ouders accuraat voor te lichten wat betreft herhalingsrisico en de mentale en fysieke ontwikkeling die verwacht kan worden. In bepaalde gevallen kan ook de behandeling van de patiënten verbeterd worden: complicaties die frequent voorkomen bij bepaalde genetische aandoeningen kunnen verhinderd of behandeld worden voordat ze optreden (bijvoorbeeld gehoorsverlies of complicaties aan het hart). We tonen dat de toepassing van deze hoge-resolutie technologieën de identificatie mogelijk maakt van kleine deletie- of duplicatiemutaties in enkelvoudige genen (bijvoorbeeld in het FOXC1 of het ATRX gen). Deze verhoogde resolutie bemoeilijkt echter ook de interpretatie in verband met de oorzakelijkheid van de genetische afwijkingen die aangetroffen worden. Chromosoomafwijkingen in het DNA van syndromale CHD patiënten wijzen ons de chromosoomregio’s waar genen liggen die verantwoordelijk zijn voor de normale ontwikkeling van het hart. Soms weten we reeds welk gen in deze regio hartafwijkingen veroorzaakt. In andere gevallen was nog geen gen bekend en zijn alle genen die in deze regio gelegen zijn in theorie mogelijk verantwoordelijk voor de hartafwijking. Onze doelstelling was om uit deze verschillende kandidaatgenen het gen te identificeren dat verantwoordelijk is voor de hartafwijking. We hebben geraffineerde strategieën ontwikkeld om de beste kandidaatgenen te selecteren. Hierbij werd gebruik gemaakt van de vele grote gegevensbanken die recent publiekelijk beschikbaar zijn gesteld. De 24 beste kandidaatgenen (afkomstig uit in totaal 6 chromosoomregio’s) die via deze computeralgoritmes geïdentificeerd werden hebben we verder onderzocht. Ons uitgangspunt was dat het oorzakelijk gen voor een hartafwijking in het hart actief moet zijn tijdens de ontwikkeling van het embryo. Daarom werd de activiteit van alle genen onderzocht in verschillende stadia van de ontwikkeling van de zebravis. Slecht 2 van de 24 geselecteerde kandidaatgenen waren specifiek in het hart actief: BMP4 and HAND2. Aangezien studies in de muis voor beide genen reeds aantoonden dat ze essentieel zijn voor de hartontwikkeling, zijn dit inderdaad excellente kandidaten om de hartafwijkingen van de patiënten te verklaren. Bij één persoon met een hartafwijking werd op chromosoom 6 een bijkomende interessante regio geïdentificeerd. TAB2 was daar het beste kandidaatgen volgens het computeralgoritme. Dit gen is bovendien afwijkend bij verschillende hartpatiënten die een afwijking op chromosoom 6 dragen. Wanneer we de activiteit van dit gen onderzochten in zebravis embryo’s, bleek ook dat TAB2 actief is in het ontwikkelende hart. Studies in muizen toonden nog geen betrokkenheid van dit gen in de hartontwikkeling, hoewel een verstoring van dit gen een hoge sterfte veroorzaakt vlak na de geboorte, gelijkaardig aan de verstoring in de patiënten. We hebben dit gen op een gelijkaardige manier uitgeschakeld in de zebravis, wat ook daar leidde tot ontwikkelingsafwijkingen. Bij 100 andere hartpatiënten konden we geen fouten in dit gen terugvinden, maar een Deense onderzoeksgroep waar we mee samenwerken vond wel een verstoring van dit gen in een familie met hartafwijkingen. Dit toont aan dat verstoring van TAB2 een zeldzame oorzaak is voor hartafwijkingen. Chromosome investigations are still an important part of the genetic investigations in children with congenital heart defects (CHDs). For this, chromosomes from dividing white blood cells are investigated under a microscope to check if certain chromosomes or parts of chromosomes are present in too many or too little copies. In the present work we have investigated whether submicroscopic chromosome imbalances are a frequent cause for CHDs. We introduced a novel genome-wide copy number profiling technique (aCGH) and showed that it enables a reliable detection of such imbalances at a resolution far surpassing the resolution of microscopic chromosome investigations. The application of this technique in patients with a syndromic CHD greatly enhances the chance of finding an etiological diagnosis. More precisely, in 20% of them, a disease-causing submicroscopic chromosome imbalance can be demonstrated. The correct delineation of chromosome aberrations by aCGH also entails a more accurate characterization of the genotype of the patient, permitting a more personalized, specific genetic diagnosis. A diagnosis is of the utmost importance for the follow-up of the patients and their families, as it allows more correct counseling of patient and parents regarding recurrence risks and the mental and physical development that can be expected. In some cases it also impacts treatment of the patients as complications associated with certain genetic conditions can be prevented or managed from a subclinical stage (e.g. hearing loss or cardiac complications). We showed that the application of higher-resolution platforms enables the genome-wide identification of indel mutations of single genes (e.g. in FOXC1 or ATRX), but that this increased resolution is accompanied by an unexpected complexity in the evaluation of their causality. The identification of submicroscopic indels in the DNA of syndromic CHD patients pinpoints regions that contain a gene responsible for heart development. We detected many imbalances that affect genes known to cause CHDs. Accordingly, imbalances identified in this way that do not affect known genes for CHDs pinpoint novel candidate regions. The use of advanced database mining strategies like ENDEAVOUR aids in ranking and selecting valuable candidate genes from these loci, and we showed that there is room for improvement by tailoring these tools to the needs of the underlying clinical or scientific question. We have used expression analyses in zebrafish embryos to identify the most valuable candidates from a group of high-ranked candidate genes. Genes that showed a specific expression in the developing zebrafish heart were considered good candidate genes. Only 2 out of 24 candidate genes displayed such a pattern: BMP4 and HAND2. Both genes are excellent candidates as they were already known to be involved in mammalian heart development through studies in mice. In one person with a CHD we detected a deletion on the long arm of chromosome 6. In this region, our algorithm identified TAB2 as the best candidate gene for causing heart defects. This gene is deleted in multiple patients with CHDs, is located in the critical deletion region and is ranked first as a candidate gene amongst over 100 genes from the region. Loss of a copy of this gene is described to be associated with a high mortality in newborn mice, and we have shown that it is associated with developmental defects in zebrafish. Although we could not identify pathogenic mutations in a group of 100 patients with isolated heart defects, others did find a disruption of this gene in 3 members of a small family that have heart defects. This shows that loss of a copy of TAB2 is a rare cause of CHDs. Doctor of Medical Sciences Afdeling CME-UZ Departement Menselijke Erfelijkheid Faculteit Geneeskunde Doctoral thesis Doctoraatsthesis
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