Dennis Dooijes

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.

Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box.

CD3‐epsilon expression is controlled by a downstream T lymphocyte‐specific enhancer element. We report the identification of a T cell‐specific transcription factor, TCF‐1, binding to this element. The multimerized recognition motif of TCF‐1 constituted a T cell‐specific enhancer. Subsequent cloning of TCF‐1 identified three splice alternatives. TCF‐1 contained a single DNA‐binding HMG box most closely related to similar boxes in the putative mammalian sex‐determining gene SRY and in the Schizosaccharomyces pombe Mc mating type gene. TCF‐1 mRNA was expressed uniquely in T lymphocytes. Upon cotransfection into non‐T cells, TCF‐1 could transactivate through its cognate motif. These results identify TCF‐1 as a T cell‐specific transcription factor, which might play a role in the establishment of the mature T cell phenotype.

A Mutation in the Fibroblast Growth Factor 14 Gene Is Associated with Autosomal Dominant Cerebral Ataxia

Hereditary spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative disorders for which ⩾14 different genetic loci have been identified. In some SCA types, expanded tri- or pentanucleotide repeats have been identified, and the length of these expansions correlates with the age at onset and with the severity of the clinical phenotype. In several other SCA types, no genetic defect has yet been identified. We describe a large, three-generation family with early-onset tremor, dyskinesia, and slowly progressive cerebellar ataxia, not associated with any of the known SCA loci, and a mutation in the fibroblast growth factor 14 (FGF14) gene on chromosome 13q34. Our observations are in accordance with the occurrence of ataxia and paroxysmal dyskinesia in Fgf14-knockout mice. As indicated by protein modeling, the amino acid change from phenylalanine to serine at position 145 is predicted to reduce the stability of the protein. The present FGF14 mutation represents a novel gene defect involved in the neurodegeneration of cerebellum and basal ganglia.

Cardiac Myosin-Binding Protein C Mutations and Hypertrophic Cardiomyopathy: Haploinsufficiency, Deranged Phosphorylation, and Cardiomyocyte Dysfunction

Background— Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3mut). Methods and Results— Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3mut. Protein expression of cMyBP-C was significantly reduced in MYBPC3mut by 33±5%. Cardiac MyBP-C phosphorylation in MYBPC3mut samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84±5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the &bgr;-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3mut (20.2±2.7 kN/m2) compared with donor (34.5±1.1 kN/m2). Moreover, Ca2+ sensitivity was higher in MYBPC3mut (pCa50=5.62±0.04) than in donor (pCa50=5.54±0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic &bgr;-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca2+ sensitivity between MYBPC3mut (pCa50=5.46±0.03) and donor (pCa50=5.48±0.02). Conclusions— Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca2+ sensitivity in MYBPC3mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

Evidence for an oligogenic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a substantial heritable component. In pedigrees affected by its familial form, incomplete penetrance is often observed. We hypothesized that this could be caused by a complex inheritance of risk variants in multiple genes. Therefore, we screened 111 familial ALS (FALS) patients from 97 families, and large cohorts of sporadic ALS (SALS) patients and control subjects for mutations in TAR DNA-binding protein (TARDBP), fused in sarcoma/translated in liposarcoma (FUS/TLS), superoxide dismutase-1 (SOD1), angiogenin (ANG) and chromosome 9 open reading frame 72 (C9orf72). Mutations were identified in 48% of FALS families, 8% of SALS patients and 0.5% of control subjects. In five of the FALS families, we identified multiple mutations in ALS-associated genes. We detected FUS/TLS and TARDBP mutations in combination with ANG mutations, and C9orf72 repeat expansions with TARDBP, SOD1 and FUS/TLS mutations. Statistical analysis demonstrated that the presence of multiple mutations in FALS is in excess of what is to be expected by chance (P = 1.57 × 10(-7)). The most compelling evidence for an oligogenic basis was found in individuals with a p.N352S mutation in TARDBP, detected in five FALS families and three apparently SALS patients. Genealogical and haplotype analyses revealed that these individuals shared a common ancestor. We obtained DNA of 14 patients with this TARDBP mutation, 50% of whom had an additional mutation (ANG, C9orf72 or homozygous TARDBP). Hereby, we provide evidence for an oligogenic aetiology of ALS. This may have important implications for the interpretation of whole exome/genome experiments designed to identify new ALS-associated genes and for genetic counselling, especially of unaffected family members.

Spinocerebellar ataxias in the Netherlands Prevalence and age at onset variance analysis

BackgroundInternational prevalence estimates of autosomal dominant cerebellar ataxias (ADCA) vary from 0.3 to 2.0 per 100,000. The prevalence of ADCA in the Netherlands is unknown. Fifteen genetic loci have been identified (SCA-1-8, SCA-10-14, SCA-16, and SCA-17) and nine of the corresponding genes have been cloned. In SCA-1, SCA2, SCA3, SCA6, SCA7, SCA-12 and SCA-17 the mutation has been shown to be an expanded CAG repeat. Previously, the length of the CAG repeat was found to account for 50 to 80% of variance in age at onset. Because of heterogeneity in encoded proteins, different pathophysiologic mechanisms leading to neurodegeneration could be involved. The relationship between CAG repeat length and age at onset would then differ accordingly. MethodBased on the results of SCA mutation analysis in the three DNA diagnostic laboratories that serve the entire Dutch population, the authors surveyed the number of families and affected individuals per SCA gene, as well as individual repeat length and age at onset. Regression analysis was applied to study the relationship between CAG repeat length and age at onset per SCA gene. The slopes of the different regression curves were compared. ResultsOn November 1, 2000, mutations were found in 145 ADCA families and 391 affected individuals were identified. The authors extrapolated a minimal prevalence of 3.0 per 100,000 (range 2.8 to 3.8/100,000). SCA3 was the most frequent mutation. CAG repeat length contributed to 52 to 76% of age at onset variance. Regression curve slopes for SCA-1, SCA2, SCA3, and SCA7 did not differ significantly. ConclusionsThe estimated minimal prevalence of ADCA in the Netherlands is 3.0 per 100,000 inhabitants. Except for SCA6, the relationship between age at onset and CAG repeat expansion does not differ significantly between SCA-1, SCA2, SCA3, and SCA7 patient groups in our population, indicating that these SCA subtypes share similar mechanisms of polyglutamine-induced neurotoxicity, despite heterogeneity in gene products.

Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Pathogenic Desmosome Mutations in Index-Patients Predict Outcome of Family Screening: Dutch Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Genotype-Phenotype Follow-Up Study

Background— Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an autosomal dominant inherited disease with incomplete penetrance and variable expression. Causative mutations in genes encoding 5 desmosomal proteins are found in ≈50% of ARVD/C index patients. Previous genotype-phenotype relation studies involved mainly overt ARVD/C index patients, so follow-up data on relatives are scarce. Methods and Results— One hundred forty-nine ARVD/C index patients (111 male patients; age, 49±13 years) according to 2010 Task Force criteria and 302 relatives from 93 families (282 asymptomatic; 135 male patients; age, 44±13 years) were clinically and genetically characterized. DNA analysis comprised sequencing of plakophilin-2 ( PKP2 ), desmocollin-2, desmoglein-2, desmoplakin, and plakoglobin and multiplex ligation-dependent probe amplification to identify large deletions in PKP2. Pathogenic mutations were found in 87 index patients (58%), mainly truncating PKP2 mutations, including 3 cases with multiple mutations. Multiplex ligation-dependent probe amplification revealed 3 PKP2 exon deletions. ARVD/C was diagnosed in 31% of initially asymptomatic mutation-carrying relatives and 5% of initially asymptomatic relatives of index patients without mutation. Prolonged terminal activation duration was observed more than negative T waves in V1 to V3, especially in mutation-carrying relatives <20 years of age. In 45% of screened families, ≥1 affected relatives were identified (90% with mutations). Conclusions— Pathogenic desmosomal gene mutations, mainly truncating PKP2 mutations, underlie ARVD/C in the majority (58%) of Dutch index patients and even 90% of familial cases. Additional multiplex ligation-dependent probe amplification analysis contributed to discovering pathogenic mutations underlying ARVD/C. Discovering pathogenic mutations in index patients enables those relatives who have a 6-fold increased risk of ARVD/C diagnosis to be identified. Prolonged terminal activation duration seems to be a first sign of ARVD/C in young asymptomatic relatives. # Clinical Perspective {#article-title-38}

The Importance of Genetic Counseling, DNA Diagnostics, and Cardiologic Family Screening in Left Ventricular Noncompaction Cardiomyopathy

Background—Left ventricular (LV) noncompaction (LVNC) is a distinct cardiomyopathy featuring a thickened bilayered LV wall consisting of a thick endocardial layer with prominent intertrabecular recesses with a thin, compact epicardial layer. Similar to hypertrophic and dilated cardiomyopathy, LVNC is genetically heterogeneous and was recently associated with mutations in sarcomere genes. To contribute to the genetic classification for LVNC, a systematic cardiological family study was performed in a cohort of 58 consecutively diagnosed and molecularly screened patients with isolated LVNC (49 adults and 9 children). Methods and Results—Combined molecular testing and cardiological family screening revealed that 67% of LVNC is genetic. Cardiological screening with electrocardiography and echocardiography of 194 relatives from 50 unrelated LVNC probands revealed familial cardiomyopathy in 32 families (64%), including LVNC, hypertrophic cardiomyopathy, and dilated cardiomyopathy. Sixty-three percent of the relatives newly diagnosed with cardiomyopathy were asymptomatic. Of 17 asymptomatic relatives with a mutation, 9 had noncompaction cardiomyopathy. In 8 carriers, nonpenetrance was observed. This may explain that 44% (14 of 32) of familial disease remained undetected by ascertainment of family history before cardiological family screening. The molecular screening of 17 genes identified mutations in 11 genes in 41% (23 of 56) tested probands, 35% (17 of 48) adults and 6 of 8 children. In 18 families, single mutations were transmitted in an autosomal dominant mode. Two adults and 2 children were compound or double heterozygous for 2 different mutations. One adult proband had 3 mutations. In 50% (16 of 32) of familial LVNC, the genetic defect remained inconclusive. Conclusion—LVNC is predominantly a genetic cardiomyopathy with variable presentation ranging from asymptomatic to severe. Accordingly, the diagnosis of LVNC requires genetic counseling, DNA diagnostics, and cardiological family screening.

Distinct genetic forms of frontotemporal dementia

Background: Frontotemporal dementia (FTD) is the second most common type of presenile dementia and can be distinguished into various clinical variants. The identification of MAPT and GRN defects and the discovery of the TDP-43 protein in FTD have led to the classification of pathologic and genetic subtypes. In addition to these genetic subtypes, there exist familial forms of FTD with unknown genetic defects. Methods: We investigated the frequency, demographic, and clinical data of patients with FTD with a positive family history in our prospective cohort of 364 patients. Genetic analysis of genes associated with FTD was performed on all patients with a positive family history. Immunohistochemical studies were carried out with a panel of antibodies (tau, ubiquitin, TDP-43) in brains collected at autopsy. Results: In the total cohort of 364 patients, 27% had a positive family history suggestive for an autosomal mode of inheritance, including MAPT (11%) and GRN (6%) mutations. We identified a new Gln300X GRN mutation in a patient with a sporadic FTD. The mean age at onset in GRN patients (61.8 ± 9.9 years) was higher than MAPT patients (52.4 ± 5.9 years). In the remaining 10% of patients with suggestive autosomal dominant inheritance, the genetic defect has yet to be identified. Neuropathologically, this group can be distinguished into familial FTLD+MND and familial FTLD-U with hippocampal sclerosis. Conclusion: Future genetic studies need to identify genetic defects in at least two distinct familial forms of frontotemporal dementia (FTD) with unknown genetic defects: frontotemporal lobe degeneration with ubiquitin-positive inclusions with hippocampal sclerosis and frontotemporal lobe degeneration with motor neuron disease. GLOSSARY: CA1 = cornu ammonis field 1; FTD = frontotemporal dementia; FTD-bv = behavioral variant of FTD; FTD+MND = FTD with motor neuron disease; FTLD = frontotemporal lobe degeneration; FTLD-tau = FTLD with tau-positive pathology; FTLD-U = FTLD with tau-negative, ubiquitin-positive inclusions; HS = hippocampal sclerosis; PNFA = progressive nonfluent aphasia; TDP-43 = TAR-DNA binding protein 43.

Peripartum Cardiomyopathy as a Part of Familial Dilated Cardiomyopathy

Background— Anecdotal cases of familial clustering of peripartum cardiomyopathy (PPCM) and familial occurrences of PPCM and idiopathic dilated cardiomyopathy (DCM) together have been observed, suggesting that genetic factors play a role in the pathogenesis of PPCM. We hypothesized that some cases of PPCM are part of the spectrum of familial DCM, presenting in the peripartum period. Methods and Results— We reviewed our database of 90 DCM families, focusing specifically on the presence of PPCM patients. Then, in a reverse approach, we reviewed 10 PPCM patients seen in our clinic since the early 1990s and performed cardiological screening of the first-degree relatives of 3 PPCM patients who did not show a full recovery. Finally, we analyzed the genes known to be most commonly involved in DCM in the PPCM patients. We identified a substantial number (5 of 90, 6%) of DCM families with PPCM patients. Second, cardiological screening of first-degree relatives of 3 PPCM patients who did not show full recovery revealed undiagnosed DCM in all 3 families. Finally, genetic analyses revealed a mutation (c.149A>G, p.Gln50Arg) in the gene encoding cardiac troponin C (TNNC1) segregating with disease in a DCM family with a member with PPCM, supporting the genetic nature of disease in this case. Conclusions— Our findings strongly suggest that a subset of PPCM is an initial manifestation of familial DCM. This may have important implications for cardiological screening in such families.