Normalized Southern Hybridization to Enhance Testing for Charcot-Marie-Tooth Disease, Type 1A.

Background: Charcot-Marie-Tooth disease, type 1A (CMT,1A) is a common autosomal dominant neuromuscular disorder, which affects both motor and sensory function and is characterized usually by duplication of a region on chromosome 17 through unequal crossover. As a result, affected patients carry three copies of this region. Individuals inheriting the other deleted chromosome involved in the crossover have one copy of the region and manifest herditary neuropathy with susceptibility to pressure palsies (HNPP). One diagnostic approach to CMT,1A exploits Southern blot hybridization and the relative intensity for three polymorphic MspI restriction fragment lenth polymorphism bands within the duplicated area to judge whether patients have two or three copies of this region using a probe such as VAW409R3A. This is usually straightfoward and works well for the majority of samples that display polymorphisms. However, it is difficult to judge dosage for this region in patients who do not demonstrate polymorphic bands. Methods and Results: An assay has been developed in which a simultaneously hybridized probe (pH15), which detects a nonpolymorphic band on chromosome 22 generated by the same restriction enzyme used to digest genomic DNA, is used to normalize the signal from the CMT,1A probe after phosphorimager analysis. Normalized ratios for VAW409R3A-hybridizing Southern bands fell within discrete ranges for patients with three copies (2.72-3.69), two copies (1.60-2.40) and one copy (0.75-1.30) of this region in over 45 patient and control samples studied. Conclusions: This assay appears to provide a reliable and consistent method of analysis.

A Novel Missense Mutation, R1283S, of the Cystic Fibrosis Transmembrane Conductance Regulator Gene in a 47-Year-Old African-American Patient.

Background: More than 600 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described; however, at least 50% of the disease-associated mutations in the African-American population remain unknown. Reported here is a novel missense mutation, R1283S, in a 47-year-old African-American patient with mild cystic fibrosis. Methods and Results: The patient was screened for 27 common and less common CFTR mutations and 2 mutations were detected. Direct sequencing confirmed the presence of the DeltaF508 mutation and revealed the presence of a novel missense mutation, R1283S. Conclusions: R1283S appears to be a cystic fibrosis mutation associated with mild disease, and adds to the number of known mutations in African-Americans. R1283S can be confused with the more common mutation, W1282X, when polymerase chain reaction-restriction fragment length polymorphism analysis is used for detection.