Available Assays

Screening for RET protooncogene mutations to confirm MEN2 diagnosis or to determine predisposition for MEN2 in asymptomatic relatives may be accomplished by a focused survey of a few exons of the RET gene. The majority of pathogenic mutations have been characterized in exons 10,11,13,14, and 16, including codons 609,611,618, 620,634, 768, 804, and 918, with few exceptions (Table 201). In addition, all mutations of these RET codons associated with MEN2 are missense substitutions.20,21,23,24,26,27 Nonsense and frameshift mutations in RET have not been observed in MEN2, and small, in-frame deletions or insertions are rarely identified.32 These cysteine codons are not mutated with equal frequency,with the majority of patients with MEN2A having alterations of codon C634 (Figure 203). Nearly every base position of these TGC cysteine codons has been a point of allelic variation, generating more than 20 distinct alleles; thus, mutation detection methods must be designed with the capability to interrogate each nucleotide position of the codon. In clinical laboratory practice, the preferred approach first is to characterize the causative (pathogenic) mutation in an affected individual using a comprehensive PCR plus direct DNA sequencing approach (Table 20-2) either of the commonly altered exons (Table 20-1) or of the entire RET gene. A variety of physical detection methods for rapid discovery of DNA sequence variation such as denaturing gradient gel

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Figure 20-3. Distribution of RET mutations in MEN2A and FMTC. Summarized from the literature. Codon 634 in exon 11 represents a mutation hotspot in MEN2A.

electrophoresis (DGGE),33 SSCP,32,34,35 HA,36 and denaturing high-performance liquid chromatography (DHPLC)36 have been used extensively in research studies and are very useful for initial localization of a single-nucleotide sequence variation. Most of these methods identify a single-nucleotide sequence change by the altered mobility caused by the DNA conformational change using elec-trophoretic or melting profile shift analysis in comparison to the wild-type nucleotide sequence for the same DNA fragment. Sensitivity in the detection of the mobility shift is optimal in fragments of 200 to 400 base pairs (bp) for these approaches, and may include 50 to 600 bp targets or up to 1.5 kb with DHPLC. Determination of the exact position of the base change within these fragments (for example, TGC to TAC or TGC to GGC) can sometimes be deduced by comparison with an extensive panel of controls of defined mutant alleles previously confirmed by DNA sequencing.33'36 The high GC content of some RET

sequences additionally can confound physical detection methods by reducing the sensitivity of resolution between homoduplex and heteroduplex DNA molecules. Confirmation by direct DNA sequencing identifies both the exact position and nature of the base change localized by conformational screening methods. One major advantage of many of these conformational techniques is that they are frequently performed with automated instrumentation.

Following the identification of the specific pathogenic mutation in an affected relative, other family members may be tested for just this allele with a more targeted, follow-up test of a single codon within one exon of RET. The molecular methodology may not be limited to direct DNA sequencing.37 For example, many missense mutations identified in the comprehensive screening also may be directly detected by PCR-restriction fragment length polymorphism (RFLP) using a variety of enzymes,37 DGGE, DHPLC, SSCP, HA, or single nucleotide polymorphism (SNP) testing methods.38 Site-specific, single-base extension assays, such as those used for single-nucleotide polymorphism detection, have been described38 for detection of many of the RET mutations. Linkage analysis is feasible39 when direct mutation analysis fails to identify one of the common mutations in a family with a clear pattern of heritable disease.

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