Eliska Vaclavikova

RET mutation Tyr791Phe the genetic cause of different diseases derived from neural crest

Activating germline RET mutations are presented in patients with familial medullary thyroid carcinoma (FMTC) and multiple endocrine neoplasia (MEN) types 2A and 2B, whereas inactivating germline mutations in patients with Hirschsprung’s disease (HSCR). The aim of this study was to evaluate genotype–phenotype correlations of the frequently discussed Tyr791Phe mutation in exon 13 of the RET proto-oncogene. Screening of three groups of patients was performed (276 families with medullary thyroid carcinoma (MTC), 122 families with HSCR, and 29 patients with pheochromocytoma). We found this mutation in 3 families with apparently sporadic MTC, 3 families with FMTC/MEN2, 1 patient with pheochromocytoma, and 3 families with HSCR. All gene mutation carriers have a silent polymorphism Leu769Leu in exon 13. In three families second germline mutations were detected: Cys620Phe (exon 10) in MEN2A family, Met918Thr (exon 16) in MEN2B family, and Ser649Leu (exon 11) in HSCR patient. Detection of the Tyr791Phe mutation in MEN2/MTC and also in HSCR families leads to the question whether this mutation has a dual character (gain-of-function as well as loss-of-function). A rare case of malignant pheochromocytoma in a patient with the Tyr791Phe mutation is presented. This study shows various clinical characteristics of the frequently discussed Tyr791Phe mutation.

Hirschsprung's disease and medullary thyroid carcinoma: 15-year experience with molecular genetic screening of the RET proto-oncogene.

PurposeInactivating germline mutations in the RET proto-oncogene are the major genetic cause of Hirschsprung’s disease (HD). In some cases, HD can be associated with medullary thyroid carcinoma (MTC) that is commonly caused by activating RET mutations.MethodsThe retrospective and prospective genetic analyses of 157 patients with HD operated on between December 1979 and June 2011 were carried out. DNA was isolated from peripheral leukocytes. HD patients and family members were tested for RET mutations by direct sequencing and single-strand conformation polymorphism methods.ResultsRET mutations were detected in 16 patients (10%). Association with MTC was found in two families, other eight families had a mutation with potentially high risk of MTC development and four novel mutations were detected. Total colonic aganglionosis was noted to have a high mutation detection rate (40%). Three patients underwent total thyroidectomy (two had clinical manifestation of MTC, one C-cell hyperplasia).ConclusionResults show the benefit of systematic RET mutation screening in HD patients in order to identify the risk of MTC in the preclinical stage of the disease. All patients should be tested for RET mutations at least in exon 10, and now additionally in exon 11 and 13, as well.

Polymorphisms in selected DNA repair genes and cell cycle regulating genes involved in the risk of papillary thyroid carcinoma.

BACKGROUND Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. In addition to causal somatic mutations in the BRAF gene and RET/PTC rearrangements, the contribution of single nucleotide polymorphisms (SNPs) in low-penetrance genes in the development of PTC has been proposed. METHODS Four SNPs in the XRCC1 (Arg399Gln, Arg280His, Arg194Trp and T-77C) and one SNP from each of three other genes participating in DNA repair pathways and/or cell cycle regulation (ATM Asp1853Asn, TP53 Arg72Pro, CDKN1B Val109Gly) were selected. The allelic and genotypic distributions of these variants as well as haplotypes of the XRCC1 were examined in 583 individuals comprising well-characterized cohorts of 209 PTC patients and 374 healthy volunteers. Correlations of polymorphism with clinical-pathological data and mutation status were performed. RESULTS XRCC1 T-77C polymorphism affects the genetic susceptibility for PTC development in men, the specific combination of XRCC1 haplotypes correlates with RET/PTC incidence, CDKN1B Val109Gly significantly influences the risk of developing PTC regardless of gender and in PTC cases, selected genotypes of TP53 Arg72Pro and ATM Asp1853Asn were significantly associated with monitored tumour characteristics. CONCLUSION It seems that SNPs in studied regulating genes contribute to the development of PTC and modify the tumour behaviour or characteristics.

RET Variants and Haplotype Analysis in a Cohort of Czech Patients with Hirschsprung Disease

Hirschsprung disease (HSCR) is a congenital aganglionosis of myenteric and submucosal plexuses in variable length of the intestine. This study investigated the influence and a possible modifying function of RET proto-oncogenes single nucleotide polymorphisms (SNPs) and haplotypes in the development and phenotype of the disease in Czech patients. Genotyping of 14 SNPs was performed using TaqMan Genotyping Assays and direct sequencing. The frequencies of SNPs and generated haplotypes were statistically evaluated using chi-square test and the association with the risk of HSCR was estimated by odds ratio. SNP analysis revealed significant differences in frequencies of 11 polymorphic RET variants between 162 HSCR patients and 205 unaffected controls. Particularly variant alleles of rs1864410, rs2435357, rs2506004 (intron 1), rs1800858 (exon 2), rs1800861 (exon 13), and rs2565200 (intron 19) were strongly associated with increased risk of HSCR (p<0.00000) and were over-represented in males vs. females. Conversely, variant alleles of rs1800860, rs1799939 and rs1800863 (exons 7, 11, 15) had a protective role. The haploblock comprising variants in intron 1 and exon 2 was constructed. It represented a high risk of HSCR, however, the influence of other variants was also found after pruning from effect of this haploblock. Clustering patients according to genotype status in haploblock revealed a strong co-segregation with several SNPs and pointed out the differences between long and short form of HSCR. This study involved a large number of SNPs along the entire RET proto-oncogene with demonstration of their risk/protective role also in haplotype and diplotype analysis in the Czech population. The influence of some variant alleles on the aggressiveness of the disease and their role in gender manifestation differences was found. These data contribute to worldwide knowledge of the genetics of HSCR.

A novel RET/PTC variant detected in a pediatric patient with papillary thyroid cancer without ionization history

Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer. Its development is often caused by the formation of RET/PTC fused genes. RET/PTC1 is the most prevalent form, where exon 1 of CCDC6 gene is fused with the intracellular portion of RET protooncogene starting with exon 12. We have discovered a novel RET/PTC1 variant which we have named RET/PTC1ex9 in metastatic PTC of 8-year-old boy. RET/PTC1ex9 detection was performed by real-time polymerase chain reaction with melting curve analysis and subsequent Sanger and next-generation sequencing. A fusion of exon 1 of CCDC6 with exon 9 of extracellular domain of RET followed by exon 12 of RET was revealed. This is the first RET/PTC variant among PTC cases that contain the extracellular part of RET. This observation could be probably explained by incorrect splicing of RET due to the somatic 32-bp deletion in exon-intron 11 boundary of RET.

Establishing high resolution melting analysis: method validation and evaluation for c-RET proto-oncogene mutation screening

Abstract Background: Reliable and effective primary screening of mutation carriers is the key condition for common diagnostic use. The objective of this study is to validate the method high resolution melting (HRM) analysis for routine primary mutation screening and accomplish its optimization, evaluation and validation. Due to their heterozygous nature, germline point mutations of c-RET proto-oncogene, associated to multiple endocrine neoplasia type 2 (MEN2), are suitable for HRM analysis. Early identification of mutation carriers has a major impact on patients’ survival due to early onset of medullary thyroid carcinoma (MTC) and resistance to conventional therapy. Methods: The authors performed a series of validation assays according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines for validation of analytical procedures, along with appropriate design and optimization experiments. After validated evaluation of HRM, the method was utilized for primary screening of 28 pathogenic c-RET mutations distributed among nine exons of c-RET gene. Results: Validation experiments confirm the repeatability, robustness, accuracy and reproducibility of HRM. All c-RET gene pathogenic variants were detected with no occurrence of false-positive/false-negative results. Conclusions: The data provide basic information about design, establishment and validation of HRM for primary screening of genetic variants in order to distinguish heterozygous point mutation carriers among the wild-type sequence carriers. HRM analysis is a powerful and reliable tool for rapid and cost-effective primary screening, e.g., of c-RET gene germline and/or sporadic mutations and can be used as a first line potential diagnostic tool.

The influence of polymorphisms in tumor suppressor genes in thyroid carcinomas

 Statistical evaluation NCSS 2004 programme (Statistical Solutions, Saugus, MA, USA) and Chi-square test Introduction: Thyroid carcinomas are the most often endocrine malignancy and their incidence is still growing. But the genetic predisposition to the development of thyroid cancer is still unknown. Beside mutations in the specific oncogenes, the genetic predisposition to the thyroid cancer could be influenced by risk variants in tumor suppressor genes encoding key proteins in regulation of cell cycle and cell surviving.