Mary S. Sedra

Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2

Purpose: This study describes SMN1 deletion frequency, carrier studies, and the effect of the modifying SMN2 gene on the spinal muscular atrophy (SMA) phenotype. A novel allele-specific intragenic mutation panel increases the sensitivity of SMN1 testing.Methods: From 1995 to 2001, 610 patients were tested for SMN1 deletions and 399 relatives of probands have been tested for carrier status. SMN2 copy number was compared between 52 type I and 90 type III patients, and between type I and type III patients with chimeric SMN genes. A fluorescent allele-specific polymerase chain reaction (PCR) -based strategy detected intragenic mutations in potential compound heterozygotes and was used on 366 patients.Results: Less than half of the patients tested were homozygously deleted for SMN1. A PCR-based panel detected the seven most common intragenic mutations. SMN2 copy number was significantly different between mild and severely affected patients.Conclusions: SMN1 molecular testing is essential for the diagnosis of SMA and allows for accurate carrier testing. Screening for intragenic mutations in SMN1 increases the sensitivity of diagnostic testing. Finally, SMN2 copy number is conclusively shown to ameliorate the phenotype and provide valuable prognostic information.

Strategies for Amplification of Trinucleotide Repeats: Optimization of Fragile X and Androgen Receptor PCR.

Background: Trinucleotide repeat regions are heritable unstable elements that change in copy number from generation to generation. Amplification of these triplet repeats is an important diagnostic tool for molecular medicine. However, these repeats are often difficult to amplify and may require the use of different cosolvents or amplification strategies. Methods and Results: We used the fragile X and androgen receptor triplet repeat regions to demonstrate a series of conditions that may be used to optimize the amplification of repeat sequences. Conclusions: For androgen receptor, we show that predigestion of the template DNA was sufficient to generate consistent amplification. In the case of fragile X we found that predigestion, when combined with use of betaine as a destabilizing additive, was superior to other methods and yielded consistent amplification of normal and premutation alleles in both isotopic and nonisotopic reactions.

Detection of an exon 53 polymorphism in the dystrophin gene

We utilized a heteroduplex method to screen for small mutations in Duchenne muscular dystrophy patients who did not have deletions or duplications. A dystrophin exon 53 heteroduplex band was identified in 14.4% of the affected patients. Direct sequencing of the amplified product from DNA producing the heteroduplex revealed the presence of a polymorphism in the coding region. The codon for asparagine was converted from AAT to AAC.

Identification of a deletion in the mismatch repair gene, MSH2, using mouse-human cell hybrids monosomal for chromosome 2

Hereditary non‐polyposis colorectal cancer is characterized by mutations in one of the DNA mismatch repair genes, primarily MLH1, MSH2, or MSH6. We report here the identification of a genomic deletion of ≈ 11.4 kb encompassing the first two exons of the MSH2 gene in two generations of an Ohio family. By Southern blot analysis, using a cDNA probe spanning the first seven exons of MSH2, an alteration in each of three different enzyme digests (including a unique 13‐kb band on HindIII digests) was observed, which suggested the presence of a large alteration in the 5′ region of this gene. Mouse–human cell hybrids from a mutation carrier were then generated which contained a single copy each of human chromosome 2 on which the MSH2 gene resides. Southern blots on DNA from the cell hybrids demonstrated the same, unique 13‐kb band from one MSH2 allele, as seen in the diploid DNA. DNA from this same monosomal cell hybrid failed to amplify in polymerase chain reactions (PCRs) using primers to exons 1 and 2, demonstrating the deletion of these sequences in one MSH2 allele, and the breakpoints involving Alu repeats were identified by PCR amplification and sequence analysis.