Michael L. Peacock

Mutational analysis of multiple endocrine neoplasia type 2A associated with Hirschsprung's disease†

BACKGROUND The clinical association of multiple endocrine neoplasia type 2A (MEN 2A) and Hirschsprungs disease (HD), although rare, has been previously observed. Recently, germline mutations in the RET proto-oncogene, a transmembrane receptor with tyrosine kinase activity, have been detected in patients with familial HD. RET is also the predisposition gene for the inherited cancer syndrome MEN 2A. METHODS We describe a DNA sequence variation within the coding region of RET in two large unrelated kindreds with MEN 2A (with 83 and 42 persons affected) in which HD cosegregated with MEN 2A in seven patients. Mutational analysis was performed with a highly sensitive polymerase chain reaction-based denaturing gradient gel electrophoresis technique followed by direct sequencing of mutants. RESULTS Genetic analysis by denaturing gradient gel electrophoresis detected mutant bands in RET exon 10 in patients with MEN 2A from both kindreds. Direct DNA sequencing of mutants revealed a thymine-to-adenine base change in codon 618, resulting in a cysteine-to-serine substitution. The identical mutation was present in all seven children with HD. Of these children five underwent thyroidectomy for C-cell abnormalities; one 3-year-old child is awaiting thyroid surgery, and the remaining patient died at age of 12 weeks. CONCLUSIONS The RET codon 618 Ser mutation could predispose patients with MEN 2A to HD. RET may assume a critical role in embryologic enteric nerve migration and tumorigenesis of cells from neural crest lineage.

Progress in genetic screening of multiple endocrine neoplasia type 2A: Is calcitonin testing obsolete?

BACKGROUND Recent identification of RET mutations in multiple endocrine neoplasia type 2A (MEN 2A) allows a DNA-based approach to diagnosis in lieu of calcitonin sampling. To prospectively evaluate the efficacy of mutational analysis, genetic screening was performed in 124 patients (53 male, 71 female; age, 1 month to 80 years) at risk for MEN 2A referred over 3 months. METHODS Analysis used genomic DNA and a polymerase chain reaction-based denaturing gradient gel electrophoresis strategy for mutation detection at RET codons 609, 611, 618, 620, and 634. Ninety-three of 124 patients were from established MEN 2A kindreds (group A), and screening replaced calcitonin testing. Twenty-one of 124 patients (group B) represented index cases of medullary thyroid carcinoma (MTC), and DNA analysis was performed to distinguish sporadic from hereditary disease. Ten patients (group C) had modest calcitonin elevations or had undergone thyroidectomy without confirming pathologic results, and testing was undertaken to clarify status. RESULTS Group A: RET mutations occurred in 29 (median age, 10 years) of 93 patients, 14 of whom underwent thyroidectomy. No false-positive results were observed. Group B: five (24%) of 21 patients with seemingly sporadic MTC had RET mutations at codons 618 (one), 620 (one), or 634 (three). Group C: Nine of 10 patients with alleged MEN 2A had genetically negative results. CONCLUSIONS Denaturing gradient gel electrophoresis reliably detects MEN 2A. Modest calcitonin elevations may lead to a false-positive diagnosis of MTC. DNA testing is the optimal approach to evaluating MEN 2A. Index cases of sporadic MTC should also undergo DNA analysis.

Prophylactic Surgery for Multiple Endocrine Neoplasia Type IIa after Genetic Diagnosis: Is Parathyroid Transplantation Indicated?

Abstract. Identification of germline mutations in theRET proto-oncogene predisposing to multiple endocrine neoplasia type IIa (MEN-IIa) has allowed a DNA-based approach to diagnosis and treatment by prophylactic thyroidectomy in children testing genetically positive. Although total thyroidectomy is the accepted operation for C cell disease, the necessity of routine total parathyroidectomy and autotransplantation as previously described in these asymptomatic children is questionable, particularly given the low occurrence of hyperparathyroidism in MEN-IIa (10–20%). Thirty-six children (ages 1 month to 12 years) from four MEN-IIa kindreds at risk for disease underwent genetic testing. Mutational analysis was done using a highly sensitive PCR-based denaturing gradient gel electrophoresis technique. Parathyroid or serum calcium concentrations were determined preoperatively. Of the 36 children at risk, 18 were found to have a MEN-IIa mutation; 11 have undergone prophylactic thyroidectomy at ages ranging from 2 to 12 years (mean 7.5 years). In each case, there was no biochemical evidence of hypercalcemia preoperatively, and all parathyroid glands were identified and were found to be grossly normal at exploration. Glands were carefully dissected and left in situ. Postoperatively, 10 of the 11 children maintained normocalcemia, allowing discharge within 24 to 36 hours. Resected thyroid glands contained C cell hyperplasia in nine, medullary carcinoma in one, and normal histology in one. We conclude that an alternative to routine parathyroidectomy may be desirable for prophylactic treatment of MEN-IIa. In situ parathyroid preservation can be safely achieved without compromising the completeness of the thyroid resection. This conservative approach obviates the potential morbidity associated with total parathyroidectomy and autotransplantation.

ApoE ϵ4 allelic association with Alzheimer's disease Independent confirmation using denaturing gradient gel electrophoresis

There is intriguing evidence associating apolipoprotein E (ApoE) with Alzheimers disease (AD). ApoE is deposited with (3-amyloid in senile plaques and binds to (β-amyloid in vitro. We used denaturing gradient gel electrophoresis to identify ApoE ϵ2, ϵ3, and ϵ 4 alleles in 135 control subjects and 57 AD patients. We observed a marked increase in ApoE ϵ4 allele frequency (0.40) in AD patients compared with control subjects (0.14) (p < 0.0001). Our independent finding of a marked association of ApoE t4 allele with AD further supports a possible role of ApoE in the pathogenesis of AD and confirms the study of Saunders et al (Neurology 1993;43:1467-1472).

Novel polymorphism in the A4 region of the amyloid precursor protein gene in a patient without Alzheimer's disease

We found a novel polymorphism in the amyloid precursor protein (APP) gene in a patient with ischemic cerebrovascular disease who had no evidence of Alzheimers disease (AD). This polymorphism deletes a Fok I restriction enzyme site and causes the substitution of threonine for alanine at codon 673. This is adjacent to the site at which APP is thought to undergo cleavage in AD. Analysis of this polymorphism may provide insight into the basis of APP processing.

Occurrence of MEN 2a in familial hirschsprung's disease: A new indication for genetic testing of the RET proto-oncogene☆☆☆

PURPOSE The association of the rare hereditary cancer syndrome, multiple endocrine neoplasia type 2a (MEN 2a) with Hirschsprungs disease, both linked to germline mutations in the RET proto-oncogene, has been reported recently. With the widespread availability of genetic screening for MEN 2a, it is necessary to define the indications for genetic testing of MEN 2a and population subgroups at high risk for inheriting the disease. The purpose of this study was to assess the prevalence of Hirschsprungs disease in MEN 2a and investigate the value of genetic analysis for MEN 2a in children with familial Hirschsprungs disease. METHODS The ethnically diverse study group consisted of unselected consecutive patients (n=426) at risk for hereditary medullary thyroid cancer (MTC) referred to a single laboratory for genetic testing. Analysis used genomic DNA and a polymerase chain reaction-based heteroduplex mutation detection strategy for exons 10, 11, 13, and 14 of the RET proto-oncogene followed by direct DNA sequencing. Significance of RET genotype-phenotype correlation was determined by Fishers two-tailed Exact test and a 2 x 2 contingency table. RESULTS Thirty-six distinctly new MEN 2a kindreds were identified. Hirschsprungs disease cosegregated among siblings with MEN 2a in 15 patients from 6 of the 36 (17%) families. The extent of aganglionosis in the 15 patients ranged from midrectum to duodenum. Of the 15 patients with Hirschsprungs disease, 10 (six boys, four girls) underwent thyroidectomy for MTC (n=5) or C-cell hyperplasia (n = 5) at ages 2 to 47 years (mean, 15.6 years), and the remaining five patients died in childhood of complications related to the aganglionosis. In retrospect, Hirschsprungs disease was the presenting feature of MEN 2a in five of the six families rather than MTC or pheochromocytoma. In all six MEN 2a families expressing Hirschsprungs disease, the RET mutation predisposing to the combined phenotype occurred in exon 10 at codons 609 (n=2), 618 (n=3), or 620 (n = 1). By contrast, the MEN 2a with Hirschsprungs phenotype was not found in any of the 22 families with a RETexon 11, 13, or 14 mutation (P=.0007). CONCLUSIONS The authors conclude that Hirschsprungs disease is a phenotypic marker for MEN 2a and possibly more common than originally appreciated. The expression of Hirschsprungs disease with MEN 2a may be uniquely linked to RETexon 10 mutations. The authors recommend that (1) patients affected with MEN 2a may be counseled regarding the potential risk of Hirschsprungs disease in offspring and (2) a family history of MTC be explored in children with familial Hirschsprungs disease and genetic screening for MEN 2a be considered.

Autosomal dominant, familial spastic paraplegia, type I Clinical and genetic analysis of a large North American family

Article abstract—“Familial spastic paraplegia” (FSP) refers to clinically and genetically diverse syndromes characterized by insidiously progressive lower extremity spasticity. We evaluated 126 members of a large kindred, including 31 affected subjects, in which FSP was transmitted as a stereotyped, autosomal dominant disorder that showed complete genetic penetrance. Affected subjects developed insidiously progressive gait disturbance between ages 12 and thirty-five. Neurologic examination revealed hyperreflexia and spasticity in the lower extremities, weakness of hip flexion and ankle dorsiflexion, extensor plantar response, diminished vibratory sense in the feet, and pes cavus. Using genetic linkage analysis, we excluded the FSPl locus on chromosome 14q 11.2 as the disease locus in this family. We present the clinical and genetic features of FSP type I, including the age-adjusted risk of developing the disorder in this family.

Allele-specific sequencing confirms novel prion gene polymorphism in creutzfeldt-jakob disease

We analyzed the prion protein coding sequence in a familial Creutzfeldt-Jakob disease patient who did not have any of the currently recognized prion protein mutations. Denaturing gradient gel electrophoresis indicated that the prion protein coding sequence was heterozygous at at least one location. We isolated each allele by denaturing gradient gel electrophoresis and directly sequenced. We found a DNA polymorphism at codon 178 that predicted the amino acid substitution, aspartate → asparagine. Whether this represents a benign polymorphism or pathogenic mutation will depend on analysis of the functional consequences of this change. Denaturing gradient gel electrophoresis and allele-specific sequencing proved to be efficient means of analyzing sequence polymorphisms in this gene.

Detection of RET mutations in multiple endocrine neoplasia type 2a and familial medullary thyroid carcinoma by denaturing gradient gel electrophoresis

Germline missense mutations within the coding region of the RET proto‐oncogene have recently been described in patients with the dominantly inherited cancer syndromes, multiple endocrine neoplasia type 2a (MEN 2a) and familial medullary thyroid carcinoma (FMTC). To date, the sequence variations occur in RET exons 10 and 11 and alter highly conserved cysteine residues in the proposed extracellular domain at codons 609, 611, 618, 620, and 634. To expedite rapid screening of populations at risk of MEN 2a or FMTC, we developed a PCR‐based denaturing gradient gel electrophoresis (DGGE) strategy that detects polymorphisms occurring at all five Cys codons in both RET exons using identical gel conditions. In this report, the screening results from DGGE analysis of 15 distinct MEN 2a and FMTC mutations are shown. Each mutation generated a clearly distinguishable and unique homo‐ and heteroduplex band pattern. Given the highly efficient, reproducible, and sensitive nature of this approach, DGGE is particularly appropriate for rapid, large‐scale screening of patients. Since prior knowledge of the RET mutation is unnecessary for analysis, DGGE is potentially valuable for distinguishing germline from seemingly sporadic medullary thyroid cancer as well as identifying novel sequence changes.