Available Assays

The mutations in the 9.8kb genomic DNA of the MEN1 gene that are associated with the MEN1 syndrome are diverse in both type and distribution. This creates a considerable challenge in the design of a DNA diagnostic test. The initial molecular pathology screening of affected members of MEN1 families should include analysis of the entire gene. Most approaches incorporate polymerase chain reaction (PCR) amplification of each exon with a survey for sequence variants by dideoxy fingerprinting,4 heteroduplex analysis (HA), single-strand conformation polymorphism (SSCP), or direct DNA sequencing.3,10,12 PCR products from exons showing sequence variants by conformational changes are usually sequenced to identify the specific mutation. These strategies will identify mutations in 75% to 80% of MEN1 families. The majority of mutations are nonsense or frameshift mutations that predict expression of a truncated menin protein with loss of function. Missense mutations in MEN1 are primarily clustered in the domains that interact with the transcription factors, JUND (codons 1-40, 139-242, 323-428), SMAD3 (distal to codon 478), and NFKB (codons 276-479), of key cell growth pathways (Figure 20-1).13 Protein truncation testing (PTT) could be useful in detecting the effects of such mutations, but PTT has been complicated for clinical molecular laboratory implementation. Newer versions of PTT technology may yield more promising approaches for a comprehensive MEN1 mutation detection strategy.14,15 As in most PCR-based screening procedures, some types of mutations can be missed, including large deletions or insertions, point mutations in the 5' regulatory or untranslated regions, nucleotide changes in the introns, or at the sites of PCR primer annealing, which may adversely affect PCR amplification efficiency. Large germline DNA deletions can be identified by Southern blot analysis and have been described by several investigators16,17 as abnormal restriction endonuclease fragments. These were observed in MEN1 families following negative findings using dideoxy fingerprinting and direct DNA sequencing approaches. Linkage analysis also may be used to define familial haplotypes that confer genetic predisposition to develop MEN1.

0 0

Post a comment