Truncating Titin (TTN) Variants in Chemotherapy-Induced Cardiomyopathy.

Chemotherapy-induced cardiomyopathy (CCMP) is a complication of chemotherapy treatment occurring in 9% of patients treated with the use of anthracyclines. Currently, risk stratification is based on clinical risk factors that do not adequately account for variable individual susceptibility. This suggests the presence of other determinants. In this case series, we describe 2 women with breast cancer who developed severe heart failure within months after chemotherapy. Genetic screening revealed truncating frameshift mutations in TTN, encoding the myofilament titin, in both women. To our knowledge, this is the 1st report of an association between truncating TTN variants and CCMP. Because truncations in TTN are the most common cause of familial and sporadic dilated cardiomyopathy, further research is needed to establish their prevalence in patients presenting with CCMP.

Arrhythmogenic cardiomyopathy: diagnosis, genetic background, and risk management.

Arrhythmogenic cardiomyopathy (AC), also known as arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), is a hereditary disease characterised by ventricular arrhythmias, right ventricular and/or left ventricular dysfunction, and fibrofatty replacement of cardiomyocytes. Patients with AC typically present between the second and the fourth decade of life with ventricular tachycardias. However, sudden cardiac death (SCD) may be the first manifestation, often at young age in the concealed stage of disease. AC is diagnosed by a set of clinically applicable criteria defined by an international Task Force. The current Task Force Criteria are the essential standard for a correct diagnosis in individuals suspected of AC. The genetic substrate for AC is predominantly identified in genes encoding desmosomal proteins. In a minority of patients a non-desmosomal mutation predisposes to the phenotype. Risk stratification in AC is imperfect at present. Genotype-phenotype correlation analysis may provide more insight into risk profiles of index patients and family members. In addition to symptomatic treatment, prevention of SCD is the most important therapeutic goal in AC. Therapeutic options in symptomatic patients include antiarrhythmic drugs, catheter ablation, and ICD implantation. Furthermore, patients with AC and also all pathogenic mutation carriers should be advised against practising competitive and endurance sports.

Detection of genomic deletions of PKP2 in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease that predominantly affects the right ventricle and is associated with ventricular arrhythmias that may lead to sudden cardiac death. Mutations within at least seven separate genes have been identified to cause ARVC, however a genetic culprit remains elusive in approximately 50% of cases. Although negative genetic testing may be secondary to pathogenic mutations within undiscovered genes, an alternative explanation may be the presence of large deletions or duplications involving known genes. These large copy number variants may not be detected with standard clinical genetic testing which is presently limited to direct DNA sequencing. We describe two cases of ARVC possessing large deletions involving plakophilin-2 (PKP2) identified with microarray analysis and/or multiplex ligation-dependent probe amplification (MLPA) that would have been classified as genotype negative with standard clinical genetic testing. A deletion of the entire coding region of PKP2 excluding exon 1 was identified in patient 1 and his son. In patient 2, MLPA analysis of PKP2 revealed deletion of the entire gene with subsequent microarray analysis demonstrating a de novo 7.9 Mb deletion of chromosome 12p12.1p11.1. These findings support screening for large copy number variants in clinically suspected ARVC cases without clear disease causing mutations following initial sequencing analysis.

Recurrent and founder mutations in the Netherlands : Plakophilin-2 p.Arg79X mutation causing arrhythmogenic right ventricular cardiomyopathy/dysplasia.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited cardiac disease with reduced penetrance and a highly variable expression. Mutations in the gene encoding the plakophilin-2 gene (PKP2) are detected in about 50% of ARVC/D patients. The p.Arg79X mutation in PKP2 has been identified in Europe and North America and has been functionally characterised. We evaluated the prevalence of the p.Arg79X mutation in PKP2 in the Dutch population. METHODS: Twelve index patients and 41 family members were evaluated in three university hospitals in the Netherlands. The diagnosis of ARVC/D was established according to the recently revised Task Force Criteria. Segregation of the p.Arg79X mutation was studied and haplotypes were reconstructed to determine whether the p.Arg79X mutation was a recurrent or a founder mutation. RESULTS: The p.Arg79X mutation in PKP2 was identified in 12 index patients. Haplotype analysis revealed a shared haplotype among Dutch p.Arg79X mutation carriers, indicating a common founder. Six index patients (50%) had a first- or second-degree relative who had died of sudden cardiac death below 40 years of age. At age 60, only 60% of the mutation carriers had experienced any symptoms. There was no significant difference in symptom-free survival and event-free survival between men and women. CONCLUSION: We have identified the largest series of patients with the same desmosome gene mutation in ARVC/D reported to date. This p.Arg79X mutation in PKP2 is a founder mutation in the Dutch population. The phenotypes of PKP2 p.Arg79X mutation carriers illustrate the clinical variability and reduced penetrance often seen in ARVC/D. (Neth Heart J 2010;18:583-91.).

A high incidence of MSH6 mutations in Amsterdam criteria II-negative families tested in a diagnostic setting.

BACKGROUND AND AIMS: In Lynch syndrome, the clinical phenotype in MSH6 mutation families differs from that in MLH1 and MSH2 families. Therefore, MSH6 mutation families are less likely to fulfil diagnostic criteria such as the Amsterdam II criteria (AC II) and the revised Bethesda guidelines (rBG), and will be underdiagnosed. The aim of the present study was to evaluate the contribution of MSH6 gene mutations in families that were analysed for Lynch syndrome in a diagnostic setting. METHODS: Families that had molecular analysis for Lynch syndrome were included in this study. Complete molecular screening of the MLH1, MSH2 and MSH6 genes was performed in all families. Microsatellite instability (MSI) and immunohistochemical (IHC) analysis was performed in almost all families. Clinical data were collected from medical records and family pedigrees. RESULTS: A total of 108 families were included. MSI and IHC analysis was performed in 97 families, and in 40 an MSI-high phenotype with absent protein expression was found. Germline mutation analysis detected mutations in 23 families (7 MLH1, 4 MSH2 and 12 MSH6). The majority of MSH6 families were AC II negative, but fulfilled the rBG. CONCLUSIONS: There is a high incidence of MSH6 mutations in families tested for Lynch syndrome in a diagnostic setting. Many of these families remain underdiagnosed using the AC II. The rBG are more useful to select these families for further analysis. However, to optimise the detection of MSH6 families, MSI and IHC analysis should also be performed in families with clustering of late-onset endometrial carcinoma.

The use of genetic testing in hereditary colorectal cancer syndromes: genetic testing in HNPCC, (A)FAP and MAP.

This study evaluated the use of genetic testing and time trends in hereditary non-polyposis colorectal cancer (HNPCC), (attenuated) familial adenomatous polyposis [(A)FAP] and human MutY homolog (MUTYH) associated polyposis (MAP) families. Eighty-seven families, who were diagnosed with disease-causing mutations between 1995 and 2006, were included in this study. The families consisted of 1547 individuals at risk. Data of these individuals were collected from medical records and family pedigrees. There was considerable interest in genetic testing with test rates of 41% in HNPCC families, 42% in (A)FAP families and 53% in MAP families. The use of genetic testing was associated with age and parenthood. Despite the interest in genetic testing, many risk carriers do not apply for testing. Moreover, time trend analysis showed a decline in test rate in HNPCC families. Studies evaluating the reasons for not testing are needed. Furthermore, a better implementation of genetic testing in clinical practice is desirable.

Hyperechogenic fetal bowel: counseling difficulties.

The detection of echodense fetal bowel on ultrasound examination in the second trimester of pregnancy justifies invasive procedures such as amniocentesis to detect an underlying cause. We present a case in which initial tests identified only one mutation in the cystic fibrosis transmembrane regulator (CFTR)-gene of the fetus, the family history being negative for CF. Strongly reduced intestinal enzyme activities suggested intestinal obstruction and further increased the estimated risk for CF. After the 24th gestational week, a second mutation was found, confirming cystic fibrosis in this child. Problems in counseling in this particular case are discussed.

[From gene to disease: arteriohepatic dysplasia or Alagille syndrome]. / Van gen naar ziekte; arteriohepatische dysplasie--het syndroom van Alagille.

Alagille syndrome (AGS), also known as arteriohepatic dysplasia, is an autosomal dominant disorder with a prevalence of approximately one in 70,000 live births. AGS is characterised by intrahepatic bile duct paucity and other developmental abnormalities affecting the heart, liver, eyes, vertebrae and the craniofacial region. Mutations in the JAG1 gene have been demonstrated to cause Alagille syndrome. JAG1 encodes a cellular membrane-bound ligand for the Notch receptor and is expressed during the normal development of tissues affected in Alagille syndrome. JAG1 mutations are detected in approximately 70% of AGS patients and are mostly protein truncating mutations. JAG1 mutations have also been described in patients that do not demonstrate the complete AGS phenotype, suggesting that the phenotypic spectrum of JAG1 mutations is broader than thus far assumed.

[From gene to disease; neurofibromatosis type 1]. / Van gen naar ziekte; neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease characterised by café-au-lait spots, freckling in the axillary or inguinal region, dermal and plexiform neurofibromas and Lisch nodules. Complications are severe in one third of patients, and the clinical variability is pronounced, even within families. The NF1 gene has been localised to chromosome 17q11.2 and encodes the protein neurofibromin. The gene is proposed to be a tumour suppressor gene. Inactivation of neurofibromin leads to a disruption in cell growth regulation. Mutation analysis is possible but laborious, and therefore NF1 is generally a clinical diagnosis based on diagnostic criteria.

Recombination hotspot in NF1 microdeletion patients.

Neurofibromatosis type 1 (NF1) patients that are heterozygous for an NF1 microdeletion are remarkable for an early age at onset and an excessive burden of dermal neurofibromas. Microdeletions are predominantly maternal in origin and arise by unequal crossover between misaligned NF1REP paralogous sequence blocks which flank the NF1 gene. We mapped and sequenced the breakpoints in several patients and designed primers within each paralog to specifically amplify a 3.4 kb deletion junction fragment. This assay amplified a deletion junction fragment from 25 of the 54 unrelated NF1 microdeletion patients screened. Sequence analysis demonstrated that each of the 25 recombination events occurred in a discrete 2 kb recombination hotspot within each of the flanking NF1REPs. Two recombination events were accompanied by apparent gene conversion. A search for recombination-prone motifs revealed a chi-like sequence; however, it is unknown whether this element stimulates recombination to occur at the hotspot. The deletion-junction assay will facilitate the prospective identification of patients with NF1 microdeletion at this hotspot for genotype-phenotype correlation studies and diagnostic evaluation.

Cell differentiation by interaction of two HMG-box proteins: Mat1-Mc activates M cell-specific genes in S.pombe by recruiting the ubiquitous transcription factor Ste11 to weak binding sites.

The Schizosaccharomyces pombe mfm1 gene is expressed in an M cell-specific fashion. This regulation requires two HMG-box proteins: the ubiquitous Ste11 transcription factor and the M cell-controlling protein Mat1-Mc. Here we report that the mfm1 promoter contains a single, weak Stell-binding site (a so-called TR-box) that can confer M-specificity on a heterologous promoter when present in eight copies. In vitro, both Mat1-Mc and Ste11 can bind this box with approximately the same affinity. The Mat1-Mc protein caused a dramatic increase in the DNA-binding of Ste11 to this box, under conditions where we could not detect Mat1-Mc in the resulting protein-DNA complex. When we changed a single base in the mfm1 TR-box, such that it resembled those boxes found in ubiquitously expressed genes, Ste11 binding was enhanced, and in vivo the mfm1 gene also became expressed in P cells where Mat1-Mc is absent. These findings suggest that M-specificity results from Mat1-Mc-mediated Ste11 binding to weak TR-boxes. We have also defined a novel motif (termed M-box), adjacent to the mfm1 TR-box, to which Mat1-Mc binds strongly. A DNA fragment containing both the TR- and the M-box allowed the formation of a complex containing both Ste11 and Mat1-Mc. A single copy of this fragment was sufficient to activate a heterologous promoter in an M-specific fashion, suggesting that these two boxes act in a synergistic manner.

Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF.

The vertebrate transcription factors TCF (T cell factor) and LEF (lymphocyte enhancer binding factor) interact with beta-catenin and are hypothesized to mediate Wingless/Wnt signaling. We have cloned a maternally expressed Drosophila TCF family member, dTCF. dTCF binds a canonical TCF DNA motif and interacts with the beta-catenin homolog Armadillo. Previous studies have identified two regions in Armadillo required for Wingless signaling. One of these interacts with dTCF, while the other constitutes a transactivation domain. Mutations in dTCF and expression of a dominant-negative dTCF transgene cause a segment polarity phenotype and affect expression of the Wingless target genes engrailed and Ultrabithorax. Epistasis analysis positions dTCF downstream of armadillo. The Armadillo-dTCF complex mediates Wingless signaling as a bipartite transcription factor.

Characterization of adenovirus type 5 insertion and deletion mutants encoding altered DNA binding proteins.

We have introduced insertion and deletion mutations in the cloned DNA binding protein (DBP) gene of adenovirus type 5. The mutated DBP genes were subsequently introduced in the viral genome by a combination of in vitro and in vivo methods. The resulting mutant viruses were tested for their viability in human 293 cells and an initial characterization of these viruses was performed. Viable mutants with insertions in the carboxyl-terminal portion of the gene could not be obtained. In contrast, a number of viable mutants were constructed that contained insertions or deletions in the amino-terminal half of DBP. Several of these, which covered the region between amino acid (aa) residues 39 and 81, were phenotypically wild type, implying that this segment is completely dispensable for DBP function. However, mutations altering the region encompassed by aa 2-38 were, at least, partially defective suggesting that this region is important for full activity of the protein.