Interpretation

The sensitivity of RET mutation screening requires concise clinical description of the patient's phenotype. At least 95% of patients with MEN2 will have one of a limited number of well-characterized mutations in the RET gene leading to a gain of function. There is some genotype-phenotype

Table 20-2. Oligonucleotide Primer Sequences in Use for Clinical Diagnostic PCR and Direct DNA Sequencing of RET Exons 10,11,13,14, and 16

Primer Name

10NF2 10R2 10F 10R3 11F2 11NR 11F3 11R CRT4F CRT4E CRT4EN 2

14F 14R

14RINT

16LC 16RC 16LH 16RH

RET Exon

14 14 14

16 16 16 16

Forward or Reverse

Primer Sequence

Nested R F R

Nested

5'-TCAGGGGGCAGCATTGTTGGG-3'

5'-ATGGGGCCCACTCGCCTCCCAGC-3'

5'-GCGCCCCAGGAGGCTGAGTG-3'

5'-CCTCCCTTGTTGGGACCTCAGATGT-3'

5'-AGAGCATACGCAGCCTGTACCC-3'

5'-GTCATCTCAGCTGAGGAG-3'

5'-ACACGGCAGGCTGGAGAGCCATGA-3'

5'-CACCGGAAGAGGAGTAGCTG-3'

5'-GCAGGCCTCTCTGTCTGAACTT-3'

5'-GGAGAACAGGGCTGTATGGA-3' (PCR only)

5'-AGGCCCCATACAATTTGATG-3' (for Seq. only)*

5'-TGGCTCCTAGAAGACCCA-3' 5'-AGAGCCATATGCACGCAC-3' (PCR only) 5'-TCTCGGCCAGATACTGCATC-3' (for Seq. only)*

5'-AGAGAGTTAGAGTAACTTCAATGTC-3' 5'-CTACATGTATAAGGGTGTTT-3' 5'-AGGGATAGGGCCTGGGCTTC-3' 5'-TAACCTCCACCCCAAGAGAG-3'

*Nested reverse primers (internal to the PCR reverse primer) are used for the cycle sequencing of exons 13 and 14 PCR products.

specificity for the separate MEN2 subtypes, as summarized in Table 20-1. There is significant overlap of clinical symptoms and missense variations at any of the five conserved cysteine codons (609,611,618,620,634) that may be diagnostic for either MEN2A (95%) or FMTC (80%) (Figure 203). Mutations at codon 768 or 804 in exons 13 and 14, respectively, are consistent with the diagnosis of FMTC alone.26 The substitution of methionine at codon 918 with threonine, M918T, is diagnostic for the MEN2B variant and is observed in 95% to 98% of MEN2B patients. The specificity of RET mutation detection for these disorders is nearly 100%.40

Interpretation should be conducted within the context of family history and the clinical presentation of the types of endocrine tumors in an affected family member, when available. Specific classification of a patient's MEN2 subtype is essential for the most accurate prognosis and effective clinical management. Distinguishing FMTC or MEN2A may be difficult in small kindreds, with limited or relatively young affected family members, or when scant family medical history is documented because pheochro-mocytomas or parathyroid hyperplasia may primarily manifest in older relatives.

Identical point mutations have been observed in families with only FMTC and in those with MEN2A as well as in kindreds that have MEN2A plus HSCR. Thus, the geno-type-phenotype associations of MEN2 expressivity are not clearly defined by RET mutations alone. Within particular kindreds, the clinical presentation is fairly consistent, considering the variable expression of tumor types in MEN2A. As a membrane tyrosine kinase molecule, the RET protein interacts with several coreceptor molecules and ligands including GDNF, neurturin, persephin, and artemin.41 Base substitutions at the key conserved cysteine codons affect intermolecular associations, disrupt normal signaling, and change the activity or specificity of the kinase, or both.25 It is hoped that clearer elucidation of concomitant variations in these other "modifier" genes will aid in the definition of more precise genotype-phenotype effects.

MEN2 syndromes account for approximately 25% of MTC cases, based on epidemiological studies of new index cases. The remainder of MTC cases are sporadic, unilateral, or manifest in older adults with negative genetic findings of germline RET mutations. A significant proportion of the sporadic tumors (30% to 67%) have somatic M918T mutations of RET in the absence of a constitutive germline mutation in leukocyte DNA of the same patient.42,43 Sporadic MTC also may feature a much more diverse type and distribution of RET mutations than MEN2A and FMTC. Disease-causing germline RET mutations also have been reported in 1% to 24% of individuals with apparently sporadic MTC, but the lack of previous family history of MEN2 may be attributed to incomplete penetrance of some genetic variations. Somatic RET mutations have not been detected in sporadic pheochromocytoma,43 but this tumor may also present in von Hippel Lindau (VHL) disease and neurofibromatosis type 1 (NF1). Neumann and colleagues showed the utility of additional mutation screening of the

VHL gene associated with VHL disease in individuals with pheochromocytoma, but no other MEN2 symptoms and no identifiable RET gene mutation.44 Screening for mutations in VHL and NF1 genes should be considered in familial cases of pheochromocytoma with no detectable germline mutation in RET. HSCR is generally characterized by a broader spectrum of mutations throughout RET than those typical of MEN2, in both the familial and sporadic HSCR presentations.29,30 The rare occurrence of an MEN2 family with no identifiable RET mutation presents the possibility of a clinical misdiagnosis.

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