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Case Reports
. 2013 Dec;161A(12):3182-6.
doi: 10.1002/ajmg.a.36178. Epub 2013 Aug 16.

Evidence for Replicative Mechanism in a CHD7 Rearrangement in a Patient With CHARGE Syndrome

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Case Reports

Evidence for Replicative Mechanism in a CHD7 Rearrangement in a Patient With CHARGE Syndrome

Matteo Vatta et al. Am J Med Genet A. .
Free PMC article

Abstract

Haploinsufficiency of CHD7 (OMIM# 608892) is known to cause CHARGE syndrome (OMIM# 214800). Molecular testing supports a definitive diagnosis in approximately 65-70% of cases. Most CHD7 mutations arise de novo, and no mutations affecting exon-7 have been reported to date. We report on an 8-year-old girl diagnosed with CHARGE syndrome that was referred to our laboratory for comprehensive CHD7 gene screening. Genomic DNA from the subject with a suspected diagnosis of CHARGE was isolated from peripheral blood lymphocytes and comprehensive Sanger sequencing, along with deletion/duplication analysis of the CHD7 gene using multiplex ligation-dependent probe amplification (MLPA), was performed. MLPA analysis identified a reduced single probe signal for exon-7 of the CHD7 gene consistent with potential heterozygous deletion. Long-range PCR breakpoint analysis identified a complex genomic rearrangement (CGR) leading to the deletion of exon-7 and breakpoints consistent with a replicative mechanism such as fork stalling and template switching (FoSTeS) or microhomology-mediated break-induced replication (MMBIR). Taken together this represents the first evidence for a CHD7 intragenic CGR in a patient with CHARGE syndrome leading to what appears to be also the first report of a mutation specifically disrupting exon-7. Although likely rare, CGR may represent an overlooked mechanism in subjects with CHARGE syndrome that can be missed by current sequencing and dosage assays.

Keywords: CHARGE syndrome; CHD7; FoSTeS/MMBIR; MLPA.

Conflict of interest statement

DISCLOSURE

MV, ZN, PF, CME and ASW at the time of performing the project were at the Medical Genetics Laboratories, a Diagnostic Laboratory at Baylor College of Medicine. JRL is a paid consultant for Athena Diagnostics, has stock ownership in 23 and Me and Ion Torrent Systems, and is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from the chromosomal microarray analysis (CMA) and clinical exome sequencing offered in the Medical Genetics Laboratory (MGL; http://www.bcm.edu/geneticlabs/). At the best of our knowledge there are no other conflicts of interest to disclose and all procedures have been compliant with ethical regulations.

Figures

Figure 1
Figure 1. MLPA analysis and genomic structure encompassing exon 7
A) MLPA analysis of the proband and available first degree relatives. Filled symbol and black arrow indicates the proband. Unfilled symbols indicate unaffected relatives. The red arrow indicates the single probe with reduced signal in proband DNA. B) Genomic structure of CHD7 encompassing exon 7. Filled in black are the exons, in light blue the sequence composing the junctional fragment, while in red are the repetitive elements. Arrows indicate the position of the primers employed for the long range PCR analysis.
Figure 1
Figure 1. MLPA analysis and genomic structure encompassing exon 7
A) MLPA analysis of the proband and available first degree relatives. Filled symbol and black arrow indicates the proband. Unfilled symbols indicate unaffected relatives. The red arrow indicates the single probe with reduced signal in proband DNA. B) Genomic structure of CHD7 encompassing exon 7. Filled in black are the exons, in light blue the sequence composing the junctional fragment, while in red are the repetitive elements. Arrows indicate the position of the primers employed for the long range PCR analysis.
Figure 2
Figure 2. Multiplex PCR analysis of the wild type and mutant CHD7 alleles
A) Relative position of the primers designed for the amplification of the wild type CHD7 allele fragments (a) and for mutant CHD7 allele fragments (b). B) Electrophoretic analysis of the multiplex PCR amplification along with the family pedigree. PCR fragment size is indicated. C) Computational analysis and electropherograms of the sequences composing the junctional fragment indicating the microhomology of the two breakpoints consistent with FoSTeS/MMBIR (FoSTeS 1 and 2). The position numbering of the nucleotide sequence refers to the genomic coordinates.
Figure 2
Figure 2. Multiplex PCR analysis of the wild type and mutant CHD7 alleles
A) Relative position of the primers designed for the amplification of the wild type CHD7 allele fragments (a) and for mutant CHD7 allele fragments (b). B) Electrophoretic analysis of the multiplex PCR amplification along with the family pedigree. PCR fragment size is indicated. C) Computational analysis and electropherograms of the sequences composing the junctional fragment indicating the microhomology of the two breakpoints consistent with FoSTeS/MMBIR (FoSTeS 1 and 2). The position numbering of the nucleotide sequence refers to the genomic coordinates.
Figure 2
Figure 2. Multiplex PCR analysis of the wild type and mutant CHD7 alleles
A) Relative position of the primers designed for the amplification of the wild type CHD7 allele fragments (a) and for mutant CHD7 allele fragments (b). B) Electrophoretic analysis of the multiplex PCR amplification along with the family pedigree. PCR fragment size is indicated. C) Computational analysis and electropherograms of the sequences composing the junctional fragment indicating the microhomology of the two breakpoints consistent with FoSTeS/MMBIR (FoSTeS 1 and 2). The position numbering of the nucleotide sequence refers to the genomic coordinates.

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