Novel and recurrent mutations in lamin A/C in patients with Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by slowly progressive muscle wasting and weakness; early contractures of the elbows, Achilles tendons, and spine; and cardiomyopathy associated with cardiac conduction defects. Clinically indistinguishable X-linked and autosomal forms of EDMD have been described. Mutations in the STA gene, encoding the nuclear envelope protein emerin, are responsible for X-linked EDMD, while mutations in the LMNA gene encoding lamins A and C by alternative splicing have been found in patients with autosomal dominant, autosomal recessive, and sporadic forms of EDMD. We report mutations in LMNA found in four familial and seven sporadic cases of EDMD, including seven novel mutations. Nine missense mutations and two small in-frame deletions were detected distributed throughout the gene. Most mutations (7/11) were detected within the LMNA exons encoding the central rod domain common to both lamins A/C. All of these missense mutations alter residues in the lamin A/C proteins conserved throughout evolution, implying an essential structural and/or functional role of these residues. One severely affected patient possesed two mutations, one specific to lamin A that may modify the phenotype of this patient. Mutations in LMNA were frequently identified among patients with sporadic and familial forms of EDMD. Further studies are needed to identify the factors modifying disease phenotype among patients harboring mutations within lamin A/C and to determine the effect of various mutations on lamin A/C structure and function.

An improved methodology for the detection of the common mutation in the FGFR3 gene responsible for achondroplasia.

Homozygous achondroplasia is a neonatal lethal condition which can only be diagnosed in the first trimester of pregnancy by molecular analysis. The vast majority of patients with achondroplasia have a G-->A substitution at position 1138 of the fibroblast growth factor receptor (FGFR3) cDNA sequence, resulting in the substitution of an arginine for a glycine residue at position 380 of the FGFR3 protein. This mutation has typically been detected by SfcI digestion of amplified genomic DNA. We have demonstrated that the SfcI digestion protocol does not consistently distinguish between DNA samples heterozygous and homozygous for the G1138A substitution, and illustrates how the misdiagnosis of a homozygous affected fetus for one carrying only one copy of the G1138A mutation could occur. We report here an improved, simple nonradioactive technique which can reliably and consistently detect the presence of the G1138A mutation both in the heterozygous and homozygous state.

DNA fingerprinting of crude bacterial lysates using degenerate RAPD primers.

Methods for identifying isolates of various pathogenic bacteria by DNA fingerprinting with random primers (RAPD) have been described recently. In these methods many primers are screened and the primers that generate the most informative DNA pattern are selected. A new strategy that simplifies the primer selection process for RAPD fingerprinting has been developed in our laboratory. In this approach, one or more degenerate nucleotides is introduced into the core RAPD primer sequence at various nucleotide positions. Results show that a single degenerate nucleotide in the primer sequence can significantly change the DNA profile obtained for the same template. The more removed the degenerate nucleotide is from the 3' end of the primer, the less dramatic is its effect on banding pattern. This method utilizing degenerate RAPD (D-RAPD) primers was tested on clinical isolates of Legionella pneumoniae, and results were confirmed with nondegenerate RAPD primers. Results obtained with D-RAPD primers were in total agreement with those obtained with nondegenerate RAPD primers. We propose that the use of a core RAPD primer sequence with one or more degenerate nucleotide(s) at various positions can expedite the generation of unique DNA fingerprints individual organisms. A general method for selecting the most useful fingerprinting RAPD primers is discussed.