Quality Control and Laboratory Issues

Complete analysis of the MEN1 gene sequence by PCR amplification of each exon and direct DNA sequencing fails to identify mutations in 20% to 25% of patients with MEN1. Some MEN1 mutations have been identified repeatedly in apparently unrelated families, suggesting some tendency for mutational hot spots within the gene. Caution must be used in the interpretation of the pathogenic consequences of DNA sequence variants. Predicting abnormal protein function from a DNA sequence change is imperfect. Several MEN1 polymorphisms have been reported (Arg171Gln and Ala54lThr) that produce amino acid substitutions featuring different polar side chains but apparently are not associated with a disease phenotype.10 Missense mutations in amino acids surrounding these same codons are consistent with MEN1 incidence in affected families.3 Classification of polymorphisms as benign may change with long-term follow-up or ascertainment of additional MEN1 kindreds. Some frameshift or nonsense mutations do not completely abolish normal protein function. The existence of pheno-copies (clinical presentations that may mimic familial disorders) also should be a consideration due to the relatively high frequency of sporadic parathyroid and pituitary ade-nomas,10 particularly in the absence of a family history of MEN1. Genotype-phenotype prediction may be further complicated by variable expression among family members or different families with the same sequence change, or by slow growth of an MEN1 tumor even after a gene mutation has been characterized. Once a sequence change has been documented as present in individuals with clinical features of MEN1 and absent in unaffected individuals, one can be reasonably certain of the significance of the finding as a true disease-associated mutation.

Of mutations reported in the literature, approximately 70% are nonsense, frameshift, or splice-site mutations that predict the expression of a truncated menin protein product. We investigated the feasibility and utility of an in vitro protein truncation test (PTT) for diagnostic MEN1 screening. This proved to be too problematic for routine clinical laboratory implementation, given the problems inherent in (a) the instability of abnormal mRNA in peripheral blood lymphocytes of patients with MEN1 and (b) the scarcity of methionine residues for 35S-methionine substitution in labeling the amino terminus polypeptides of the menin protein. Newer approaches with noniso-topic PTT14,15 may be more successful in applying this strategy to MEN1 mutation detection. Identification of large deletions by Southern blot or fluorescence in situ hybridization (FISH) analysis may be useful for mutation screening because deletion of an entire exon or exons will result in amplification and sequencing of the remaining, normal allele and a false-negative test result. Genetic variants in the regulatory regions of MEN1, including promoter mutations or methylation defects, may indicate abnormal gene expression but have not yet been documented.


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