Rearranged During Transfection Mutations Research Articles

A cohort study on 10-year survival of sporadic medullary thyroid carcinoma with somatic RET mutation

Somatic rearranged during transfection (RET) mutations are reported in 40–50% of sporadic medullary thyroid carcinoma (sMTC) patients with prognostic significance. As there is a lack of somatic RET mutations reported previously for the Taiwanese population, we tried to assess the presence of somatic RET mutations and evaluate the potential outcome predictors for our sMTC patients. We collected data from seven sMTC patients from the years 1997 to 2005 and analyzed their clinic-pathological features up to 2015. All patients were still alive to follow up for 11∼18 years. Tumor DNAs were extracted to assess exons 10–11 and 13–16, and the intron-exon boundaries of the RET gene. Six cases (86%) were screened positive of somatic RET gene mutations in hotspot regions, one at M918T, one at C620R, and three at C634S, with another two rare mutations at L629Q and V642I. Comparing the current tumor, node, metastases staging system, the 10-year survival outcomes for our sMTC patients was not predicted by serum calcitonin and/or carcinoembryonic antigen, surgical extent, and presence of the somatic RET gene mutations. The small cohort demonstrated a relatively good outcome of sMTC patients to survive >10 years. In addition, intensive treatment with total thyroidectomy with extensive neck lymph node dissection seemed to be the critical determinant of better survival outcome for sMTC patients.

Expression of Tenascin C, EGFR, E-Cadherin, and TTF-1 in Medullary Thyroid Carcinoma and the Correlation with RET Mutation Status.

Tenascin C expression correlates with tumor grade and indicates worse prognosis in several tumors. Epidermal growth factor receptor (EGFR) plays an important role in driving proliferation in many tumors. Loss of E-cadherin function is associated with tumor invasion and metastasis. Thyroid transcription factor-1 (TTF-1) is involved in rearranged during transfection (RET) transcription in Hirschsprung’s disease. Tenascin C, EGFR, E-cadherin, TTF-1-expression, and their correlations with RET mutation status were investigated in 30 patients with medullary thyroid carcinoma (MTC) (n = 26) or C-cell hyperplasia (n = 4). Tenascin C was found in all, EGFR in 4/26, E-cadherin in 23/26, and TTF-1 in 25/26 MTC. Tenascin C correlated significantly with tumor proliferation (overall, r = 0.61, p < 0.005; RET-mutated, r = 0.81, p < 0.01). E-cadherin showed weak correlation, whereas EGFR and TTF-1 showed no significant correlation with tumor proliferation. EGFR, E-cadherin, and TTF-1 showed weak correlation with proliferation of RET-mutated tumors. Correlation between TTF-1 and tenascin C, E-cadherin, and EGFR was r = −0.10, 0.37, and 0.21, respectively. In conclusion, MTC express tenascin C, E-cadherin, and TTF-1. Tenascin C correlates significantly with tumor proliferation, especially in RET-mutated tumors. EGFR is low, and tumors expressing EGFR do not exhibit higher proliferation. TTF-1 does not correlate with RET mutation status and has a weak correlation with tenascin C, E-cadherin, and EGFR expression.

Detection of Molecular Alterations in Medullary Thyroid Carcinoma Using Next-Generation Sequencing: an Institutional Experience.

Medullary thyroid carcinoma (MTC) harbors rearranged during transfection (RET) gene and rarely RAS gene mutations. The knowledge of the type of gene mutation in MTC is important to determine the treatment of the patients and the management of their family members. Targeted next-generation sequencing with a panel of 47 genes was performed in a total of 12 cases of sporadic (9/12) and hereditary MTC (3/12). Two of three hereditary MTCs had RET/C634R mutation, while the other one harbored two RET mutations (L790F and S649L). All the sporadic MTC had RET/M918T mutation except one case with HRAS mutation. Next-generation sequencing (NGS) can provide comprehensive analysis of molecular alterations in MTC in a routine clinical setting, which facilitate the management of the patient and the family members.

How to Assess the Clinical Relevance of Novel RET Missense Variants in the Absence of Functional Studies?

Introduction and Background: Familial medullary thyroid cancer (FMTC) is caused by gain of function mutations in the proto-oncogene RET (rearranged during transfection). Missense mutations within exon 14 including p.Val804Met are known to cause FMTC and multiple endocrine neoplasia type 2a/b. The clinical significance of other novel missense variants within this hotspot region of exon 14 is not delineated. Case Description: A three-generation pedigree of FMTC is presented with the co-occurrence of two missense variants within exon 14 of the RET gene, the known variant p.Val804Met and the novel variant p.Val826Met. The female index patient developed medullary thyroid cancer at the age of 42 years and was heterozygous for both missense variants. Her younger sister was also tested to be compound heterozygous for both mutations, and five further relatives were heterozygous for only one of both sequence variants. Prophylactic thyroidectomy was recommended for the two carriers of the RET mutation p.Val804Met, revealing a C-cell hyperplasia for one of them at the age of 19 years. Medical surveillance of 6 heterozygous carriers including repeated neck ultrasound examination as well as basal and calcium (pentagastrin)-stimulated calcitonin levels were recommended. Conclusion: Our data emphasize the importance of an interdisciplinary approach to assess the functional and clinical significance of novel RET variants. In the absence of functional studies, the plausibility of the pathologic significance of a detected endocrine genetic variant can be estimated by in silico methods such as computational analysis of protein structure and biophysical differences or comparative database search for evolutionary conservation.

Multiple endocrine neoplasia type 2 kindred with novel tandem RET mutations: Case report with an applied in silico mutational tolerance analysis

The American Thyroid Association (ATA) has established guidelines for prophylactic thyroidectomy in multiple endocrine neoplasia type 2A (MEN2A) based on rearranged during transfection (RET) mutations. In silico analysis, which uses computer modeling to predict alterations in protein structure, is a new method for studying these mutations. We describe a kindred with MEN2A, all sharing a well-documented RET mutation, p.C634Y, as well as a mutation of undetermined significance, p.I852M, which we analyzed via in silico analysis. The p.C634Y mutation resulted in severe predicted RET alterations, whereas the p.I852M resulted in only modest changes. Both mutations together resulted in only a small additional disruptive effect in protein structure beyond that which occurred with p.C634Y alone. Although in silico analysis may be helpful in quantitating changes in protein structure that occur in patients who have novel RET mutations (single or multiple), additional factors must account for the highly variable aggressiveness of the disease (C-cell hyperplasia/medullary thyroid carcinoma [MTC]) noted in our kindred. © 2016 Wiley Periodicals, Inc. Head Neck 38: E1881-E1885, 2016.

A comprehensive overview of the role of the RET proto-oncogene in thyroid carcinoma.

The rearranged during transfection (RET) proto-oncogene was identified in 1985 and, very soon thereafter, a rearrangement named RET/PTC was discovered in papillary thyroid carcinoma (PTC). After this discovery, other RET rearrangements were found in PTCs, particularly in those induced by radiation. For many years, it was thought that these genetic alterations only occurred in PTC, but, in the past couple of years, some RET/PTC rearrangements have been found in other human tumours. 5 years after the discovery of RET/PTC rearrangements in PTC, activating point mutations in the RET proto-oncogene were discovered in both hereditary and sporadic forms of medullary thyroid carcinoma (MTC). In contrast to the alterations found in PTC, the activation of RET in MTC is mainly due to activating point mutations. Interestingly, in the past year, RET rearrangements that were different to those described in PTC were observed in sporadic MTC. The identification of RET mutations is relevant to the early diagnosis of hereditary MTC and the prognosis of sporadic MTC. The diagnostic and prognostic role of the RET/PTC rearrangements in PTC is less relevant but still important in patient management, particularly for deciding if a targeted therapy should be initiated. In this Review, we discuss the pathogenic, diagnostic and prognostic roles of the RET proto-oncogene in both PTC and MTC.

RET Gene Analysis in Patients with Medullary Thyroid Carcinoma.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor from the para follicular C cells of the thyroid gland. It occurs either sporadically or as part of an inherited syndrome. It is caused by an autosomal dominant mutation in the RET (Rearranged during Transfection) proto-oncogene. The studied population consisted of 47 patients diagnosed with MTC in a specific population of northwest Iran along with their three children. Blood samples were collected from all subjects, genomic DNA was extracted and RET exons 10, 11, 13, 14, 15, and 16 were analyzed using PCR and direct sequencing. 32 missense mutations were identified in exons 10 (6.25%) and 11 (84.4%). Moreover, two novel mutations in codon 595 in exon 10 (E595D and E595A) and a new mutation in codon 689 exon 11 (S689T) were detected, and a new nucleotide change was found in exon 11 (T675T). Four different polymorphisms were also identified in exons 11, 13, 14, and 15. Based on our data, the frequency profile of RET mutations in the Azari population of Iran with MTC is 61.7%. The most frequent mutation in our population was C364G, whereas in most populations it is C634R. These results underline the importance of the genetic background of family members of any patient with MTC.

Variability in penetrance of multiple endocrine neoplasia 2A with amino acid substitutions in RET codon 634.

Controversy surrounds the importance of the different amino acids substituting for cysteine in REarranged during Transfection (RET) codon 634 for multiple endocrine neoplasia 2A (MEN 2A). This study aimed to clarify the relevance of these amino acid substitutions for the development of MEN 2A. Cross-sectional study at surgical referral centres in Germany. Included were 184 carriers of RET mutations in codon 634. Arginine (79 carriers) and tyrosine (50 carriers) substitutions in codon 634 were compared with each other and, for the first time, gauged against a common reference standard comprising all other amino acid substitutions (phenylalanine, 35 carriers; serine, 12 carriers; glycine, 7 carriers; tryptophan, 1 carrier). Arginine substitutions in codon 634 were associated with higher penetrance of medullary thyroid cancer (MTC; 82% vs 62%; P=0·010), any phaeochromocytoma (44% vs 15%, P<0·001), bilateral phaeochromocytoma (32% vs 5%; P<0·001) and primary hyperparathyroidism (18% vs 5%; P=0·039) relative to the reference standard. The penetrance rates of any phaeochromocytoma (44% vs 26%; P=0·041) and bilateral phaeochromocytoma (32% vs 14%; P=0·035) were also higher with arginine than with tyrosine substitutions. Corrected for multiple testing, the associations of arginine with any phaeochromocytoma and bilateral phaeochromocytoma remained statistically significant. Progression of MTC, evidenced by largest primary tumour diameter, nodal status, distant metastasis and biochemical cure, did not differ by amino acid substitution. In codon 634, arginine substitutions for cysteine may cause slightly higher penetrance rates of MEN 2A which, overall, are too small to treat carriers differently. The mode of action by which arginine exerts these subtle effects warrants further research.

Molecular Targeted Therapies of Aggressive Thyroid Cancer.

Differentiated thyroid carcinomas (DTCs) that arise from follicular cells account >90% of thyroid cancer (TC) [papillary thyroid cancer (PTC) 90%, follicular thyroid cancer (FTC) 10%], while medullary thyroid cancer (MTC) accounts <5%. Complete total thyroidectomy is the treatment of choice for PTC, FTC, and MTC. Radioiodine is routinely recommended in high-risk patients and considered in intermediate risk DTC patients. DTC cancer cells, during tumor progression, may lose the iodide uptake ability, becoming resistant to radioiodine, with a significant worsening of the prognosis. The lack of specific and effective drugs for aggressive and metastatic DTC and MTC leads to additional efforts toward the development of new drugs. Several genetic alterations in different molecular pathways in TC have been shown in the past few decades, associated with TC development and progression. Rearranged during transfection (RET)/PTC gene rearrangements, RET mutations, BRAF mutations, RAS mutations, and vascular endothelial growth factor receptor 2 angiogenesis pathways are some of the known pathways determinant in the development of TC. Tyrosine kinase inhibitors (TKIs) are small organic compounds inhibiting tyrosine kinases auto-phosphorylation and activation, most of them are multikinase inhibitors. TKIs act on the aforementioned molecular pathways involved in growth, angiogenesis, local, and distant spread of TC. TKIs are emerging as new therapies of aggressive TC, including DTC, MTC, and anaplastic thyroid cancer, being capable of inducing clinical responses and stabilization of disease. Vandetanib and cabozantinib have been approved for the treatment of MTC, while sorafenib and lenvatinib for DTC refractory to radioiodine. These drugs prolong median progression-free survival, but until now no significant increase has been observed on overall survival; side effects are common. New efforts are made to find new more effective and safe compounds and to personalize the therapy in each TC patient.

Early, Prophylactic Thyroidectomy in Hereditary Medullary Thyroid Carcinoma

Prophylactic thyroidectomy has been encouraged for children with REarranged during Transfection (RET) germline mutations to prevent the onset, persistence, or recurrence of medullary thyroid carcinoma (MTC). The American Thyroid Association (ATA) recently published guidelines on the timing of prophylactic thyroidectomy. Our aim here was to seek information on the optimal timing of surgery for carriers of RET gene mutations with no clinical evidence of disease, bearing in mind the ATA recommendations. From 1986 to 2012, total thyroidectomy was performed at our institute on 31 carriers of RET gene mutations, 28 of them found on family screening in the post-RET era, and the other 3 under 20 years of age and classified as "early cases" in the pre-RET era. The following parameters were studied: age at surgery, MTC risk, basal calcitonin (bCT) and pentagastrin-stimulated calcitonin (sCT), surgery outcomes, and persistence of disease. By family, the most prevalent mutation was codon 634 (30%) RET mutation. The youngest MTC patient was 5 years old. Overall, MTC was found in 68% of cases; 52% of the sample had a normal bCT and 25% had an sCT unresponsive to pentagastrin. The only factor predicting the risk of MTC at final histology was an ATA-RET risk level C. On receiver oparating curves analysis, a cutoff at age over 24 years predicted (P=0.06) a yield of MTC in the resected specimen. Interestingly, none of the patients with MTC had nodal involvement (0/21 patients with MTC). Yet, none of the patients had permanent nerve palsy, and only 1 patient had permanent hypocalcemia. bCT was normal postoperatively and during the follow-up in all but 3 patients. It is noteworthy that the yield of cancer in removed thyroid was 100% for codon 634 (9/9 patients, 5 families) and for codons 891 and 768 (2/2 patients in each of the 2 families with those codon mutations), followed by 67% for codon 609 (4/6 patients, 1 family), and 60% for codon 618 (3/5 patients in 4 families) RET mutation. In cases of ATA-RET levels B and C, waiting for an increase in bCT and/or sCT may not guarantee that prophylactic surgery is performed before MTC develops (which would assure patients a life free of diseases and a less-invasive surgical procedure, without any need for central lymph-node dissection).

Screening of RET gene mutations in Chinese patients with medullary thyroid carcinoma and their relatives.

The rearranged during transfection (RET) gene is a proto-oncogene; active mutations frequently occur in medullary thyroid carcinoma (MTC). This study investigated the spectrum of germline RET mutations and clinical features in Chinese hereditary MTC patients. A total of 53 family members from 11 different hereditary MTC families were recruited for detection of RET exon 8, 10, 11, 13, 14, 15, and 16 mutations, in genomic DNA from peripheral blood leucocytes using polymerase chain reaction (PCR) and direct DNA sequencing. Of the 53 participants, eight different germline RET mutations were detected in 37 individuals. These RET mutations were distributed in exons 10, 11, 13, and 16. The most frequent RET mutation was localized at exon 11 codon 634 (67.6 %; 25/37) and the most prevalent mutation was C634R (37.8 %; 14/37). The most frequent phenotype was multiple endocrine neoplasia type 2A (MEN2A). The incidences of MTC, pheochromocytoma, and hyperparathyroidism in the MEN2A patients were 100, 36.4 and 18.2 %, respectively. The phenotype of families with Y606C or L790F mutation was categorized as familial medullary thyroid carcinoma. Moreover, one proband was identified with multiple endocrine neoplasia type 2B and carried a de novo mutation of M918T. Two families with C618S/Y mutation were categorized as unclassified multiple endocrine neoplasia type 2. Our results further substantiate that most germline mutations of the RET proto-oncogene were localized at codon 634 in Chinese hereditary MTC patients and carriers. RET mutation at codon 634 was always associated to the phenotype of MEN2A. Screening of RET mutations should be probably limited to exons 10, 11, 13 and 16 initially to be cost-effective in China.

Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway.

Constitutive activation of the Rearranged during Transfection (RET) proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC). The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.

Molecular analysis of the rearranged during transfection proto-oncogene in Moroccan patients with medullary thyroid carcinoma

Background: Germline mutations of the proto-oncogene rearranged during transfection ( RET ) are pathognomonic of hereditary medullary thyroid carcinoma (MTC). In this study, genetic analysis and familial testing of the RET proto-oncogene in Moroccan families with MTC were performed. Patients and Methods: Thirty-one index cases with MTC and 115 of their relatives were included in this study. The entire coding region of RET was investigated by direct sequencing of polymerase chain reaction products. Once a mutation was identified, the target exon was sequenced in available relatives. Results: Seven distinct germline mutations of RET were identified in 45.2% (14/31) of probands. The most prevalent mutations were located at codon 634 (p.C634R/Y/F) and restricted to families with multiple endocrine neoplasia type 2A (MEN2A) (50% of the 14 mutation carriers, 7/14), followed by mutation at codon 918 (p.M918T) in all MEN2B cases (21.4%, 3/14), then by mutations at codons 804 (p.V804L/M) (14.3%, 2/14); and 891 (p.S891A) (14.3%, 2/14) detected in all patients with apparently sporadic MTC. Familial screening detected RET mutations in 19.1% (22/115) of the studied relatives; 36.4% (8/22) were found with MEN2A symptoms, and 63.6% (14/22) were asymptomatic. About 55% (12/22) were subjected to total therapeutic or prophylactic thyroidectomy. Conclusion: This is the first comprehensive genetic screening showing the spectrum of mutations in RET in Moroccan patients with MTC, which showed a predominance of mutations at codon 634. These results further support the necessity of genetic testing in MTC patients to provide early diagnosis and adequate initial treatment of these patients. This will moreover, contribute to the definition of a national policy for the control of this cancer in Morocco.

Exome sequencing reveals mutant genes with low penetrance involved in MEN2A-associated tumorigenesis.

Activating rearranged during transfection (RET) mutations function as the initiating causative mutation for multiple endocrine neoplasia type 2A (MEN2A). However, no conclusive findings regarding the non-RET genetic events have been reported. This is the first study, to our knowledge, examining genomic alterations in matched MEN2A-associated tumors. We performed exome sequencing and SNP array analysis of matched MEN2A tumors and germline DNA. Somatic alterations were validated in an independent set of patients using Sanger sequencing. Genes of functional interest were further evaluated. The germline RET mutation was found in all MEN2A-component tumors. Thirty-two somatic mutations were identified in the nine MEN2A-associated tumors, of which 28 (87.5%) were point mutations and 4 (12.5%) were small insertions, duplications, or deletions. We sequenced all the mutations as well as coding sequence regions of the 12 genes in an independent sample set including 35 medullary thyroid cancers (20 MEN2A) and 34 PCCs (22 MEN2A), but found no recurrent mutations. Recurrent alterations were found in 13 genes with either mutations or alterations in copy number, including an EIF4G1 mutation (p. E1147V). Mutation of EIF4G1 led to increased cell proliferation and RET/MAPK phosphorylation, while knockdown of EIF4G1 led to reduced cell proliferation and RET/MAPK phosphorylation in TT, MZ-CRC1, and PC-12 cells. We found fewer somatic mutations in endocrine tumors compared with non-endocrine tumors. RET was the primary driver in MEN2A-associated tumors. However, low-frequency alterations such as EIF4G1 might participate in MEN2A-associated tumorigenesis, possibly by regulating the activity of the RET pathway.

MTOR activation in medullary thyroid carcinoma with RAS mutation.

Rearranged during transfection (RET) mutations are well-known genetic events in sporadic and familial medullary thyroid carcinoma (FMTC). The presence of RAS mutations in sporadic cases, challenging the RET paradigm in these tumors, has been recently reported. We intend to evaluate mTOR pathway activation in RET- and RAS-mutated MTC. In this study, we analysed the presence of RET, H-RAS, and K-RAS mutations in a series of 87 MTCs (82 apparently sporadic and five FMTCs; five apparently sporadic MTCs were eventually found to be familial). We also evaluated mTOR activation--using the expression of its downstream effector phospho-S6 ribosomal protein (p-S6) and the expression of the mTOR inhibitor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN)--by immunohistochemistry. Our results revealed that RET mutations were present in 52.9% of the cases (46/87) and RAS mutations in 12.6% (11/87) of the whole series of MTCs and 14.3% of the 77 sporadic MTCs. The presence of RET and RAS mutations was mutually exclusive. RAS mutations were significantly associated with higher intensity of p-S6 expression (P=0.007), suggesting that the mTOR pathway is activated in such MTCs. We observed also an increased expression of p-S6 in invasive tumors (P=0.042) and in MTCs with lymph node metastases (P=0.046). Cytoplasmic PTEN expression was detected in 58.8% of the cases; cases WT for RAS showed a significantly lower expression of PTEN (P=0.045). We confirmed the presence of RAS mutation in 14.3% of sporadic MTCs and report, for the first time, an association between such mutations and the activation of the mTOR pathway. The evaluation of the mTOR activation by pS6 expression may serve as an indicator of invasive MTC.

Telomerase-dependent and independent telomere maintenance and its clinical implications in medullary thyroid carcinoma.

Context:Telomere maintenance via telomerase activation and the alternative lengthening of telomeres (ALT) mechanism was assessed in medullary thyroid carcinoma.Setting and Design:In total, 42 medullary thyroid carcinomas (MTC) were studied including 24 rearranged during transfection (RET)- mutated cases. Relative telomerase reverse transcriptase (TERT) expression, splice forms, and telomere length were determined by PCR-based methods, and telomerase activity by ELISA. The ALT mechanism was detected by Southern blot analysis and immunofluorescence.Results:TERT expression and telomerase activity were detected in 21/42 tumors (50%), and was independent of the common somatic M918T RET mutation. Mean telomere length was shorter in MTCs compared with thyroids. Telomerase activation was associated with large tumor size (P = .027), advanced clinical stage (P = .0001), and short survival (P = .0001). Full-length TERT and the α− and β−-deletion forms were revealed, and the full-length form was associated with short survival (P = .04). A subset of cases without telomerase activation showed involvement of the ALT mechanism, which was associated with a low MIB-1 proliferation index (P = .024).Conclusions:Stabilization of telomeres by telomerase activation occurs in half of the MTCs and by the ALT mechanism in a subset of cases. Telomerase activation may be used as an additional prognostic marker in medullary thyroid carcinoma.

Hereditary paraganglioma‐pheochromocytoma syndromes associated with SDHD and RET mutations

Hereditary paraganglioma-pheochromocytoma syndromes (PGL/PCC) are rare tumors arising from neuroendocrine cells. The proband, a 59-year-old white man and his 42-year-old elder son had a medical history of bilateral carotid body PGL and both presented for treatment of abdominal PGLs. His 36-year-old daughter had excision of recurrent malignant carotid body PGL and vertebral metastasis. His 33-year-old youngest son presented for excision of a unilateral carotid body PGL. All 4 members had succinate dehydrogenase subunit D (SDHD) mutations, whereas the proband and youngest son also had concurrent rearranged during transfection (RET) mutation. This is the first report of PGL/PCC with SDHD and RET mutations. The role of the RET gene as a modifier remains speculative. Additionally, the family pedigree suggests maternal inheritance of disease from the probands' paternal grandmother. Clinicians should refer PGL/PCC families for mutation analysis as well as being alert to changes in the classification of mutations.

In silico profiling and structural insights of missense mutations in RET protein kinase domain by molecular dynamics and docking approach

A major challenge remaining in drug design efforts towards protein kinase is due to the development of drug resistance initiated by the missense mutations in the kinase catalytic domain. Gain or loss of function mutations in the REarranged during Transfection (RET) tyrosine kinase gene have been associated with the development of a wide range of human associated cancers and Hirschsprung's disease. However, to what extent these mutations might affect bio-molecular functions remains unclear. In this article, the functionally significant mutations in RET were screened with the aid of various sequence and structure based in silico prediction methods. We mapped the deleterious mutants, modelled mutant proteins and deciphered the impact of mutations on drug binding mechanisms in the RET crystal structure of PDB ID: with the potential inhibitor vandetanib by docking analysis. Furthermore, molecular dynamics simulations were undertaken to understand the mechanistic action of cancer associated mutations in altering the protein kinase structure, dynamics, and stability. According to our results, the overall effect of V804M, M918T and S922Y were destabilizing and mostly alter the electrostatic component of the binding energy. Specifically, the mutation of gatekeeper residue valine 804 present in the ATP binding pocket affects the protein stability and confers resistance to the drug vandetanib, which was consistent with previously published experimental results. Overall, our findings may provide useful structural insights for in-depth understanding of the molecular mechanism underlying RET mutation and developing effective drugs.

RET mutations in a large indian family with medullary thyroid carcinoma.

Background:Medullary thyroid carcinoma (MTC) is a tumor arising from the para follicular (C) cells of the thyroid gland and can occur either sporadically or as part of an inherited syndrome. A proportion of these cases carry an autosomal dominant mutation in the RET (REarranged during Transfection) proto-oncogene. Screening for these mutations in the affected patients and the carriers “at risk” which includes the first-degree relatives is of utmost importance for early detection and prompt treatment including prophylactic thyroidectomy in cases that harbor these mutations.Results:This report presents details of screening and subsequent follow-up of a large Indian family, where the index case was found to carry p. Cys634Ser mutation involving exon 11 of the RET gene. These data are of value considering the paucity of information within the region in context of screening large families affected by these mutations.

An extended family with familial medullary thyroid carcinoma and Hirschsprung's disease.

Familial medullary thyroid carcinoma (FMTC) is an autosomal dominant inherited disease that has highly characteristic clinical features, including medullary thyroid carcinoma (MTC). Mutation of the RET proto-oncogene is known to be responsible for development of FMTC and for multiple endocrine neoplasia types 2A and 2B. Hirschsprung's disease is the most common form of structural intestinal obstructive disease in human newborns. Hirschsprung's disease is defined by the absence of neural crest-derived enteric ganglia along a variable length of the bowel that invariably involves the rectoanal junction. Co-segregation of FMTC and Hirschsprung's disease is uncommon; nevertheless, in 3 generations of 1 family, we observed 5 patients with FMTC, 2 patients with Hirschsprung's disease, and 1 patient with characteristics of both FMTC and Hirschsprung's disease. Moreover, a Cys620Ser mutation in RET was identified in 4 of the 8 patients. This mutation had both activating and inactivating effects on the RET (REarranged during Transfection) protein. There were individual differences in the penetrance of Hirschsprung's disease due to the RET mutation, but the penetrance of MTC was uniform and high. Genetic testing is important for making decisions about treatment and follow-up in families of this kind.