Long-term calcitonin after thyroidectomy for medullary thyroid cancer in MEN2A.

Medullary Thyroid Cancer in a Patient with Hirschsprung Disease with a C609Y Germline RET-mutation

Objective To discuss clinical characteristics,treatment and clinical significance for RET screening in 3 multiple endocrine neoplasia type 2A (MEN2A) pedigrees.Methods The clinical data of 10 MEN2A patients from 3 unrelated MEN2A pedigrees from April 1990 to December 2011 were analyzed.There were 4 males and 6 females.5 patients with symptomatic palpable neck masses underwent bilateral total thyroidectomy + bilateral neck lymph node dissection.Other 5 RET screening detected asymptomatic thyroid cancer patients underwent bilateral total thyroidectomy + bilateral level Ⅵ lymph node dissection.6 adrenal pheochromocytoma (PHEO) patients received bilateral adrenalectomy (5 cases) or unilateral adrenalectomy (1 case).23 members from 3 families agreed to participate in biochemical testing,image examinations and RET screening.Results Histopathology revealed bilateral medullary thyroid carcinoma (MTC) in all 10 patients (100％).The two groups (symptomatic and asymptomatic) have significantly differences from the first diagnostic age [39.0 (31-64) years vs.18.2 (5.5-36) years],tumor maximum diameter [2.8 (1.2-5.6) cm vs.0.7 (0.2-1.3) cm] and positive lymph node metastatic ratio [100％ (5/5) vs.20％ (1/5)] (P ＜ 0.05).These MTC cases were followed-up for 7-66 months,postoperative calcitonin still positive in all 5 preoperatively symptomatic patients,while in only 1 preoperatively asymptomatic patient (P ＜ 0.05).PHEO was bilateral,multiple in 5 patients and unilateral multiple in 1 patient.Postoperatively 3 patients need lifelong steroid substitution.19-104 months follow-up found no recurrence or metastasis.RET screening showed a missense mutations of TGC to TAC (p.C634Y)at codon 634 on exon 11 in all 10 patients,and a patient was diagnosed as having de novo MEN2A.Conclusions Based on RET screening and serum calcitonin monitoring,early and radical surgery can cure MEN2A related MTC; One stage bilateral laparoscopic cortical-sparing adrenalectomy is recommended for bilateral PHEO in MEN2A patients. Key words: Multiple endocrine neoplasia type 2a; Thyroid neoplasms ; Pheochromocytoma; Proto-oncogene ; Point mutation

Germline mutations in the RET proto-oncogene (RET gene) are well documented as the genetic causes of multiple endocrine neoplasia type 2A (MEN2A). We performed genetic analysis by direct RET gene mutation analysis in a Chinese MEN2A family and compared these results with biochemical screening tests and pathological examinations. Twenty-one exons and flanking introns of the RET gene were amplified using polymerase chain reaction (PCR). The PCR products were subjected to sequencing directly, or cloned into pGEM-T plasmids and sequenced. Restriction fragment length polymorphism (RFLP) was employed to confirm the mutation on the RET sequence. A novel heterozygous mutation of a 3-bp (GAC) deletion at codon 631 (D631del) of exon 11, resulting in the deletion of an aspartic acid at the locus, was identified in four MEN2A patients and one phenotypically normal family member. The average clinical onset-age of four MEN2A patients was 33.7 years, no cervical lymph node metastasis was found in MEN2A patients with medullary thyroid carcinoma in the family. The study indicated that the novel heterozygous deletion mutation at D631 of RET gene was co-segregated with MEN2A phenotype and promoted the development of MEN2A. This report is the first description of the D631del mutation in the family with MEN2A.

Multiple endocrine neoplasia type 2 is caused by autosomal dominant gain-of-function mutations in the RET proto-oncogene, which includes multiple endocrine neoplasia type 2A (MEN2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC). In this paper we present the phenotype-genotype correlation of 20 unrelated Chinese families with 15 cases of MEN2A and five cases of MEN2B. Cross-sectional study. A total of 147 members from the 20 families were included. Among them, 119 family members were from MEN2A pedigrees (including 15 MEN2A probands) and 28 members from MEN2B pedigrees (including five MEN2B probands). Genomic DNA was isolated from peripheral blood leucocytes and was amplified using polymerase chain reaction (PCR). DNA analysis for RET mutations in exons 8, 10, 11, 13, 14, 15 and 16 was performed with specific primers. Thirty-seven MEN2A and five MEN2B patients were identified as having RET mutations. The incidence of medullary thyroid carcinoma (MTC), pheochromocytoma (PCC) and hyperparathyroidism (HPT) in the 37 MEN2A patients was 91.9, 56.8 and 10.8%, respectively; the onset of MTC in MEN2A patients was earlier than that of PCC and HPT. Five germline mutations, all located at codon 634 of exon11 in the RET proto-oncogene, were detected in all of the 37 MEN2A patients. The highest frequency of the five germline mutations was C634Y (46.7%), followed by C634R (26.7%), C634W (13.3%), C634F (6.7%) and C634S (6.7%). No statistical significance was found between the incidence of PCC and different genotypes of codon 634 in MEN2A patients, whereas the incidence of HPT was closely associated with C634R and C634Y. The gene mutation (M918T) at exon16 of the RET proto-oncogene was present in five MEN2B probands. RET proto-oncogene mutations were restricted to codon 634 and 918 in Chinese families with MEN2A and MEN2B. In general the genetic characteristics of these patients with MEN2A and MEN2B reflect the general pattern around the world and it remains to be determined with larger studies in China whether Chinese patients have a different genetic pattern of mutations.

To detect RET mutations in a rare Chinese big family with Multiple endocrine neoplasia type 2A (MEN2A). One MEN2A family, including the proband, have 22 members of two generations, it is a rare big family in modern Chinese families. The DNAs of the 22 members from the family including 4 patients were extracted from blood leukocytes, PCR and gene sequencing of PCR products by an automated DNA sequencer were applied to scan the exon10 and 11 of RET proto-oncogene. Sequencing results were compared with the Pubmed's. Clone sequencing was adopt to further confirm the results, then verifying the novel mutation through the human gene mutation database at the institute of medical genetics in cardiff. Invitrogen biotechnology company (Shanghai) provided the technology of clone sequencing. A novel deletion mutation of D631 (GAC) (del D631) was detected in exon11 of the RET proto-oncogene in 4 MEN2A patients of the family, this rare deletion mutation of D631 (GAC) lead base sequence of TGC(angle)GACGAGCTG change to TGCGAGCTG. Besides 4 MEN2A patients, the son of II6 (the first class relative) was found to be a carrier of delD631 mutation. A novel deletion mutation (del D631) of RET proto-oncogene was detected in the family with MEN2A and it has never been reported before in the world. DelD631 may be related to the late onset of MEN2A compared to the cysteine mutations and pheochromocytoma might be the first manifestation prior to the development of MTC.

P23-8 Multiple endocrine neoplasia type 2a syndrome: A tertiary care center experience in Pakistan

IntroductionMultiple endocrine neoplasia 2A (MEN2A) is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism (HPT). RET polymorphisms have been associated with susceptibility and prognosis of MTC.ObjectiveTo evaluate the frequencies of RET polymorphisms (G691S, L769L, S836S, and S904S) in MEN2A patients and verify their association with susceptibility and age-dependent penetrance of HPT.MethodsRET variants G691S, L769L, S836S, and S904S were evaluated in a cohort of 157 MEN2A patients. RET variant frequencies were compared between patients with and without HPT. Kaplan–Meier curves and Cox regression analysis estimated the effect of RET polymorphisms on age-dependent penetrance.ResultsSixteen percent of the patients presented MEN2A-associated HPT. The mean age at diagnosis was 35.3 ± 12.7 years; 71% were women. Female subjects had a higher risk of HPT (OR = 2.61; 95% CI: 1.04–6.55). Ninety percent of the patients had RET pathogenic variants at codon 634, and 60% had some RET polymorphisms. The frequencies of RET polymorphisms were similar between patients with or without HPT (P = 0.63). RET variant frequencies were as follows: 33.7% G691S/S904S, 33.1% L769L, 12.7% S836S, and no association was found between these frequencies and HPT development. Kaplan–Meier estimates of cumulative HPT diagnosis showed similar curves for patients harboring no polymorphism, one polymorphism, and two or more polymorphisms (P = 0.066). Two or more RET variants compared to none or one polymorphism grouped exhibited an increased risk for early HPT development (P = 0.015; OR 3.03; 95% CI: 1.24–7.39).ConclusionsRET polymorphism alleles have an additive effect on the estimated risk of age-related HPT development in MEN2A patients.Significance statementGermline pathogenic variants in the RET proto-oncogene are a well-known genetic cause of multiple endocrine neoplasia type 2A (MEN2A). This rare hereditary cancer syndrome predisposes individuals to the development of MTC, pheochromocytoma, and primary hyperparathyroidism (HPT). This study investigated the association between RET polymorphisms and MEN2A-associated HPT, a common and often asymptomatic manifestation of the disease. Our findings suggest that cumulative RET polymorphisms may be associated with an increased risk of HPT in MEN2A patients, and this risk may be influenced by age and gender. These findings may contribute to the refinement of risk stratification and the personalization of surveillance protocols in individuals carrying RET pathogenic variants.

Multiple endocrine neoplasia type 2A (MEN2A) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. The gene responsible for MEN2A was localized by linkage analysis to chromosome 10q11.2 in 1987, and recently mutations in RET, a proto-oncogene in the candidate region, were discovered in patients with MEN. The majority of mutations found so far in MEN2A patients have been located in nucleotide sequences encoding cysteine residues in the extracellular domain of RET. To characterize MEN2A germline alterations in the Japanese population, we screened DNA from eight unrelated patients for mutations in exons 10 and 11 of the RET proto-oncogene and found mutations in all eight patients, at codons 618, 620, or 634; each of these sites encodes a cysteine residue in the extracellular domain of RET. The mutations were confirmed in other affected individuals in the respective families by digestion of polymerase chain reaction (PCR) products containing the mutated codons with restriction enzymes (Rs alpha I, CfoI, or AluI) for which cleavage sites had been generated by the specific genetic alteration. These PCR-restriction enzyme systems will be useful for genetic diagnosis in members of families carrying these mutations.

Medullary thyroid carcinoma (MTC) is observed in nearly 100 % of patients with multiple endocrine neoplasia type 2A (MEN2A). The gene responsible for MEN2A is the RET proto-oncogene and about 95 % of MEN2A patients have germline mutations in five specific cysteine codons (609, 611, 618, 620 and 634). A retrospective study of children from families with MEN2A in our geographic area was performed. Variables analyzed included demographic data, kinship relations, age at genetic screening, age at prophylactic thyroidectomy, genetic mutation subtype and histological findings. The genetic study consisted in direct molecular analysis by automatic sequencing of RET mutated exon in the studied family. We performed 13 prophylactic total thyroidectomies from 1997 to 2013, 8 females and 5 males. The mean age at genetic diagnosis was 3.8 years (range 2-5.9). All children belonged to four interconnected families living in the same geographic area and presenting C634Y mutation in all the cases. The mean age at prophylactic thyroidectomy was 5.6 years (range 4-8.5). Histopathological findings demonstrated seven cases of C-cells nodular hyperplasia, one lymphocytic thyroiditis, two without evidence of disease, two micro-carcinomas and one multicentric carcinoma. The mutation found in the RET proto-oncogene responsible for MEN2A in pediatric patients in the south of Spain is the C635Y. It is considered a high-risk mutation, associated with an earlier malignant transformation and development of MTC.

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

BackgroundMultiple endocrine neoplasia type 2A (MEN2A) is a rare syndrome that presents as medullary thyroid carcinoma, pheochromocytoma, and hyperparathyroidism. Experience is lacking in the anesthetic management of patients with this syndrome, particularly in those who present with pheochromocytoma receiving nonpheochromocytoma resection. We aimed to share our experience with the anesthetic management of MEN2A patients.MethodWe retrospectively enrolled 24 MEN2A patients who had received different types of surgery at Peking Union Medical College Hospital from January 1, 2015, to December 31, 2021. All the medical records were reviewed and analyzed.ResultIn total, 33 surgeries were performed in 24 MEN2A patients, with 20 surgeries comprising pheochromocytoma resection in 17 patients. Most of these patients who had received pheochromocytoma resection had typical hemodynamic changes during surgery and anesthesia. Regarding the other 13 nonpheochromocytoma resections in 13 patients, 10 were performed in patients without pheochromocytoma, and 3 surgeries were performed with either functional primary (1, bilateral tumor whose patient refused adrenalectomy) or metastatic pheochromocytoma (2, unresectable and malign tumors developed years after bilateral adrenalectomy). Regarding the latter 3 patients, 1 showed hypertension and tachycardia during anesthesia induction, 1 showed tachycardia during surgery and the other showed stability during surgery. Patients who had received pheochromocytoma resection (n=17) required longer postoperative hospital stays than those who had received nonpheochromocytoma resection without pheochromocytoma (n=10) (5.8 ± 1.8 vs. 4.3 ± 1.6; P = 0.031).ConclusionsWhenever MEN2A patients are diagnosed with pheochromocytoma, surgical resection of the pheochromocytoma remains the primary choice for MEN2A treatment. Nonpheochromocytoma surgeries performed with existing pheochromocytoma could be risky and require full caution and preparation.

Following the recent identification of specific germline mutations of the RET proto-oncogene in Multiple Endocrine Neoplasia type 2A (MEN2A) patients, we looked for mutations of this gene in a pedigree showing recurrence of MEN2A and localized Cutaneous Lichen Amyloidosis (CLA). Basal calcitonin and/or pentagastrin test performed in all the 10 available members of this pedigree confirmed the clinical diagnosis and allowed the presymptomatic identification of an additional carrier. A cys634-->tyr missense mutation, already reported as causative in MEN2A patients, was identified after SSCP analysis and direct sequencing of exon 11 of the RET protooncogene in one individual affected with both MEN2A and CLA, thus suggesting a common etiology for the two disorders. Taking advantage of the observation of an RsaI restriction site in the sequence surrounding the mutated codon, we could demonstrate that the same mutation is present in three other affected members, in the presymptomatic carrier and in one additional 25 years old healthy member who shows a mildly positive pentagastrin test.

Familial PHEOs (pheochromocytomas) are inherited as an autosomal dominant trait, and inherited PHEOs can be one clinical phenotype of clinical syndromes, such as multiple endocrine neoplasia type 2A (MEN2A). In recent years, there has been a lot of controversy about the factors affecting the penetrance of PHEOs in MEN2A, of which the effects of RET (rearranged during transfection) proto-oncogene mutations are the primary concern. In this report, we performed genetic screening of patients in one family presenting with PHEOs and found they carried a RET c.1901G>A mutation. They were ultimately diagnosed with familial MEN2A. We found that MEN2A patients with the RET c.1901G>A mutation tended to have bilateral PHEOs that appeared earlier than medullary thyroid carcinoma. Genetic analysis showed that the patients also carried novel SLC12A3 (solute carrier family 12 member 3) variants, which are highly associated with Giteman syndrome. The results of protein structure prediction models suggest this SLC12A3 mutant has altered both the protein structure and the interaction with surrounding amino acids. Further studies of the phenotypes and related mechanisms of the gene mutations are required to guide individual assessment and treatment.

While there is no doubt that total thyroidectomy is necessary for medullary thyroid carcinoma (MTC) in multiple endocrine neoplasia type 2A (MEN2A) patients, there is still controversy regarding the management of the parathyroid glands. Although most, but not all, endocrine surgeons leave normal-appearing parathyroid glands in situ during thyroid surgery for MEN2A, we have employed total parathyroidectomy with autotransplantation. Between 1994 and 2006, 12 MEN2A patients underwent therapeutic total or completion thyroidectomy and lymph nodes dissection at least in the central compartment for MTC. Total or completion parathyroidectomy with autotransplantation was performed concurrently with above-mentioned surgery. All patients were over 25 years old, and the median age was 48.5 years. There were 5 males and 7 females from 8 families. The average number of transplanted parathyroid glands was 3. Serum calcium and intact PTH levels have been maintained during the median follow up of 107 months in all patients except for one who of died of advanced MTC one year after surgery. Total parathyroidectomy with autotransplantation at the time of primary surgery for MTC, i.e. total thyroidectomy with bilateral central neck dissection, is a feasible approach for managing the risk of hyperparathyroidism.

Medullary thyroid carcinoma is the most common cause of death in patients with multiple endocrine neoplasia (MEN) type 2A (MEN-2A) or type 2B or familial medullary thyroid carcinoma. We sought to determine whether total thyroidectomy in asymptomatic young members of kindreds with MEN-2A who had a mutated allele of the RET proto-oncogene could prevent or cure medullary thyroid carcinoma. A total of 50 patients 19 years of age or younger who were consecutively identified through a genetic screening program as carriers of a RET mutation characteristic of MEN-2A underwent total thyroidectomy. Five to 10 years after the surgery, each patient was evaluated by physical examination and by determination of plasma calcitonin levels after stimulation with provocative agents. In 44 of the 50 patients, basal and stimulated plasma calcitonin levels were at or below the limits of detection of the assay (proportion, 0.88; 95 percent confidence interval, 0.76 to 0.95). Two patients had basal and stimulated plasma calcitonin levels above the normal range. Stimulated plasma calcitonin levels had increased but remained within the normal range in four patients. The data suggest that there was a lower incidence of persistent or recurrent disease in children who underwent total thyroidectomy before eight years of age and in children in whom there were no metastases to cervical lymph nodes. In this study, young patients identified by direct DNA analysis as carriers of a RET mutation characteristic of MEN-2A had no evidence of persistent or recurrent medullary thyroid carcinoma five or more years after total thyroidectomy. A longer period of evaluation will be necessary to confirm that they are cured.