Arleta Lebioda

Assessment of three epigenotypes in colorectal cancer by combined bisulfite restriction analysis

Recent investigations have demonstrated the clear heterogeneity of sporadic colorectal cancer (CRC) with regard to CpG island methylation. Two unsupervised cluster analyses revealed that CRCs form three distinct DNA methylation subsets, which are referred to as the high‐, intermediate‐, and low‐methylation epigenotypes (HME, IME, and LME, respectively). A recent study by Yagi et al. found a fairly sensitive and specific identification of HME, IME, and LME using two marker panels analyzed by MALDI‐TOF mass spectrometry (MassARRAY). However, the expensive equipment required for this method substantially increases the cost and complexity of the assay. In this article, we demonstrate the assessment of HME, IME, and LME in a group of 233 sporadic CRCs using seven markers proposed by Yagi et al. The DNA methylation of each marker was quantified using combined bisulfite restriction analysis (COBRA) and analyzed along with various genetic factors associated with CRC [the BRAF and KRAS mutations, MLH1 methylation and microsatellite instability (MSI)]. The baseline methylation of each marker was generated from pooled DNA isolated from 50 normal colon tissues. We demonstrate that the correlation of HME, IME, and LME epigenotyped by COBRA using different molecular classifiers is similar to that achieved by MassARRAY. Therefore, epigenotyping CRCs using COBRA is a simple, specific, and cost‐effective method that has the potential to be widely used in CRC research.

Mutation of theMYH7 gene in a child with hypertrophic cardiomyopathy and Wolff-Parkinson-White syndrome

Familial hypertrophic cardiomyopathy (HCM) displays autosomal dominant inheritance with incomplete penetration of defective genes. Data concerning the familial occurrence of ventricular preexcitation, i.e. Wolff-Parkinson-White (WPW) syndrome, also indicate autosomal dominant inheritance. In the literature, only a gene mutation on chromosome 7q3 has been described in familial HCM coexisting with WPW syndrome to date. The present paper describes the case of a 7-year-old boy with HCM and coexisting WPW syndrome. On his chromosome 14, molecular diagnostics revealed a C 9123 mutation (arginine changed into cysteine in position 453) in exon 14 in a copy of the gene for beta-myosin heavy chain (MYH7). It is the first known case of mutation of theMYH7 gene in a child with both HCM and WPW. Since no linkage betweenMYH7 mutation and HCM with WPW syndrome has been reported to date, we cannot conclude whether the observed mutation is a common cause for both diseases, or this patient presents an incidental co-occurrence of HCM (caused byMYH7 mutation) and WPW syndrome.

Alternations in genes expression of pathway signaling in esophageal tissue with atresia: results of expression microarray profiling.

Esophageal atresia (EA) is a congenital defect of the esophagus involving the interruption of the esophagus with or without connection to the trachea (tracheoesophageal fistula [TEF]). EA/TEF may occur as an isolated anomaly, may be part of a complex of congenital defects (syndromic), or may develop within the context of a known syndrome or association. The molecular mechanisms underlying the development of EA are poorly understood. It is supposed that a combination of multigenic factors and epigenetic modification of genes play a role in its etiology. The aim of our work was to assess the human gene expression microarray study in esophageal tissue samples. Total RNA was extracted from 26 lower pouches of esophageal tissue collected during thoracoscopic EA repair in neonates with the isolated (IEA) and the syndromic form (SEA). We identified 787 downregulated and 841 upregulated transcripts between SEA and controls, and about 817 downregulated and 765 upregulated probes between IEA and controls. Fifty percent of these genes showed differential expression specific for either IEA or SEA. Functional pathway analysis revealed substantial enrichment for Wnt and Sonic hedgehog, as well as cytokine and chemokine signaling pathways. Moreover, we performed reverse transcription polymerase chain reaction study in a group of SHH and Wnt pathways genes with differential expression in microarray profiling to confirm the microarray expression results. We verified the altered expression in SFRP2 gene from the Wnt pathway as well as SHH, GLI1, GLI2, and GLI3 from the Sonic hedgehog pathway. The results suggest an important role of these pathways and genes for EA/TEF etiology.

A Novel p.E311K Mutation of Thyroid Receptor Beta Gene in Resistance to Thyroid Hormone Syndrome, Inherited in Autosomal Recessive Trait

Resistance to thyroid hormone (RTH) syndrome is caused by mutations in THRB gene and is inherited mainly as an autosomal dominant trait with dominant negative effect. Most of up-to-now described RTH cases were heterozygous. We studied a 19-year-old woman presenting severe mental impairment, hyperkinetic behavior, learning disability, hearing loss, tachycardia, goiter, strabismus, nystagmus, and normal stature. The laboratory findings revealed elevated TSH, T3, and T4 serum levels. Her parents were healthy with normal serum level of TSH, fT3, and fT4. Sequence based prediction of a substitution was analyzed by SDM, PolPhen, and SNAP software whereas structural visualizations were performed in UCSF Chimera. We found a novel mutation in THRB gene in position 1216 (G to A transition, codon 311) resulting in novel Glu-311-Lys (p.E311K) substitution, homozygous in proband presenting with severe symptoms of RTH and heterozygous in both of her healthy parents, thus suggesting autosomal recessive mode of inheritance. p.E311K substitution was not found in 50 healthy, unrelated individuals. p.E311K was shown to be deleterious by SDM, PolPhen, and SNAP software. Structural visualizations of mutated protein performed by UCSF Chimera software disclosed a loss of hydrogen bonds between E311, R383, and R429 along with abnormal residue-residue contact between K311 and L377. This is a very rare case of a homozygous mutation in a patient with severe symptoms of RTH and lack of symptoms in both heterozygous parents. Although, computational analyses have provided the evidence that p.E311K substitution may affect THRB function, lack of dominant negative effect typical for THRB mutations could not be explained by structure-based modeling. Further in vitro analysis is required to assess the functional consequences of this substitution.

KRAS mutation in relation to HER2 overexpression/amplification in colorectal cancer

The development of targeted therapies for KRAS, EGFR and HER2 may increase the range of response in patients with colorectal cancer. Mutation at codons 12 and 13 of the KRAS gene has been shown to be predictive of cetuximab response in colorectal cancer. However, due to the combined effects of multiple oncogenes involved in disease progression of patients with colorectal cancer, it seems to be important to identify the molecular factors that characterize therapy-resistance phenotype of tumors. n nForty six paraffin-embedded colorectal cancer specimens were analyzed for KRAS mutation and HER2 overexpression/amplification. A high–resolution melting (HRM) assay and single-nucleotide polymorphisms (SNPs) were used to detect somatic mutation in exon 2 notably condons 12 and 13 of the KRAS gene. HER2 overexpression was detected using monoclonal antibody and confirmed by fluorescence in situ hybridization (FISH) analysis. n nKRAS mutations for codons 12 and 13 were identified in 16/46 (34.7%) of patients by SNP. The alterations in KRAS gene were observed in similar percentage of both codons. Colorectal cancer showed mainly heterozygous 35G>A and 38G>A KRAS gene mutations. n nHRM analysis showed presence of KRAS exon 2 mutation in 13/46 (28.2%) colorectal cancers. Despite positive SPN results in three cases , HRM technique did not reveal KRAS gene alteration. The concordance rate between the two methodologies was high at 87.5%. KRAS mutation was more frequently observed in poorly differentiated tumors and adenocarcinomas than in other histological types. n nHER2 overexpression was found in 37/46 (80.3%) of all colorectal cancers and in 62.1%, 61.5% of KRAS mutation-positive cases detected by SNP and HRM techniques respectively. HER2 overexpression was accompanied by amplification of HER2 gene. The subgroup of colorectal cancers with KRAS mutation and HER2 overexpression/amplification was poorly differentiated. n nIn summary, the presence of HER2 overexpression/amplification in KRAS mutation–positive colorectal cancers suggests a possible role for the use of specific tyrosine kinase inhibitors in the treatment of disease.

Esophageal atresia and anal atresia in a newborn with heterotaxia combined with other congenital defects

Heterotaxia (HTX) is a heterogeneous group of laterality defects characterized by abnormal discordance of asymmetric thoracic and abdominal organs. Esophageal anomalies occur rarely in HTX cases although additional defects associated with esophageal atresia are common. We report on a rare case of a neonate with HTX and multiple congenital malformations as well as specific facial dysmorphism, corresponding only to a few cases described in literature. Clinical examination of the proband revealed esophageal atresia with distal tracheoesophageal fistula, anal atresia, abdominal situs inversus, dextrocardia with complex congenital heart defect and left lung agenesis. A complex genetic analysis revealed no genetic abnormalities. Despite extensive diagnostic procedures, the cause of the laterality sequence disruption remains unclear, indicating its multifactorial etiology.

Validation of the minisequencing method for detection of G1691A (Leiden) factor V mutation.

Abstract Introduction: The factor V (FV) plays an important role in the coagulation process and belongs to the group of factors that significantly increases the risk of thrombophilia. Our study presents a novel, rapid method for the detection of FV (R506Q) mutation using minisequencing approach. Material and methods: Samples of peripheral blood were obtained from 300 females of the Lower Silesian population. Minisequencing, as one of the polymerase chain reaction (PCR)-based methods, was used for detection the of FV (R506Q) point mutations. The allele restriction mutation system PCR (ARMS-PCR) verifying method was applied. Results: By using minisequencing reaction we examined the FV genotypes in the female group who experienced at least one unexplained spontaneous miscarriage. The results of the ARMS-PCR, as a verifying test, were fully consistent with the results of the minisequencing technique. Discussion: One of the many factors which may cause thrombophilia is the FV gene mutation R506Q. A full validation of the minisequencing method was carried out in order to apply this method to clinical tests. The validation shows that the minisequencing technique is highly precise and may be used in routine diagnostics of the FV R506Q mutation

Normal exon copy number of the GLI2 and GLI3 genes in patients with esophageal atresia

Esophageal atresia (EA) is a congenital developmental defect of the alimentary tract concerning the interruption of the esophagus with or without connection to the trachea. The incidence of EA is 1 in 3000-3500 of live-born infants, and occurs in both isolated and syndromic (in combination with abnormalities in other organ systems) forms. The molecular mechanisms underlying the development of EA are poorly understood. Knockout studies in mice indicate that genes like Sonic hedgehog, Gli2, and Gli3 play a role in the etiology of EA. These facts led us to hypothesize that Sonic hedgehog-GLI gene rearrangements are associated with EA in humans. To test this hypothesis, we screened patients with isolated and syndromic EA for GLI2 and/or GLI3 microrearrangements using methods to estimate the copy number (Multiplex Ligation-dependent Probe Amplification, real-time polymerase chain reaction). To our best knowledge this is the first study assessing copy number of GLI2 and GLI3 genes in patients with EA.