Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach

Abstract

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases caused by dystrophin gene mutations. Deletions, or more rarely duplications, of single or multiple exons within the dystrophin gene can be detected by current molecular methods in approximately 65% of DMD patients. Mothers of affected males have a two-thirds chance of carrying a dystrophin mutation, whilst approximately one-third of affected males have de novo mutations. Currently, Southern blot analysis and multiplex PCR directed against exons in deletion hot spots are used to determine female carrier status. However, both of these assays depend on dosage assessment to accurately identify carriers since, in females, the normal X chromosome is also present. To obviate quantitation of gene dosage, we have developed exon-specific probes from the dystrophin gene and applied them to a screen for potential carrier females using fluorescence in situ hybridization (FISH). Cosmid clones, representing 16 exons, were identified and used in FISH analysis of DMD/BMD families. Our preliminary work has identified multiple, informative probes for several families with dystrophin deletions and has shown that a FISH-based assay can be an effective and direct method for establishing the DMD/BMD carrier status of females.

Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA)

“…The above described PCR approach is not useful in detecting female carriers in whom deletions are masked by the amplification of the normal X chromosome. Thus, identification of female carriers must be carried out by different strategies, such as linkage analysis with intragenic polymorphic markers (Darras et al 1987;Clemens et al 1991), quantitative analysis of gene dosage (Prior et al 1990;Abbs and Bobrow 1993) and fluorescence in situ hybridisation (FISH) analysis (Voskova-Goldman et al 1997;Ligon et al 2000). However, linkage analysis cannot be used in families with a single affected male or performed if DNA of the affected members of the family is not available.…”

Identification of Duchenne Muscular Dystrophy Female Carriers by Fluorescence In Situ Hybridization and RT-PCR

“…Thus, while confirming the results of other groups, FISH using DMD exon-specific probes represents an effective, highly accurate, direct, qualitative approach for establishing DMD carrier status of females (Ligon et al, 2000;Hermanova et al, 2002). Even when there is no cure for DMD patients, providing an accurate diagnosis for female carriers allows bringing the family to a precise genetic counseling.…”

“…These molecular tools are very useful for detecting DMD gene deletions in males, but for females the detection of deletions can be problematic since women possess two X chromosomes (Ligon et al, 2000). More recently, FISH detecting chromosomal deletions has been introduced to identify specific DMD exon deletions (Voskova-Goldman et al, 1997;Ligon et al, 2000;Hermanova et al, 2002).…”

“…However, some groups have used the fluorescence in situ hybridization (FISH) assay to identify DMD female carriers since it provides an alternative approach to current molecular methods for carrier detection (Voskova-Goldman et al, 1997;Ligon et al, 2000;Hermanova et al, 2002). Importantly, this method is very useful to detect deletions since it is directed at exon-specific hot spots of the dystrophin gene, which are responsible for the majority of deletions.…”

Detection of deletions and duplications in the Duchenne muscular dystrophy gene by the molecular method MLPA in the first Argentine affected families

“…The above described PCR approach is unable to detect either duplications or female carriers in whom deletions are masked by the amplification of the normal X chromosome. Thus, identification of female carriers requires different strategies, such as segregation analysis with intragenic polymorphic markers (Darras et al, 1987;Clemens et al, 1991), quantitative analysis of gene dosage (Prior et al, 1990;Abbs and Bobrow, 1993) and fluorescence in situ hybridization (FISH) analysis (Voskova-Goldman et al, 1997;Ligon et al, 2000). However, segregation analysis cannot be used in families with a single affected male or performed if DNA of the affected members of the family is not available.…”