Molecular genetics of congenital atrial septal defects

Congenital heart defects (CHD) are the most common developmental errors in humans, affecting 8 out of 1,000 newborns. Clinical diagnosis and treatment of CHD has dramatically improved in the last decades. Hence, the majority of CHD patients are now reaching reproductive age. While the risk of familial recurrence has been evaluated in various population studies, little is known about the genetic pathogenesis of CHD. In recent years significant progress has been made in uncovering genetic processes during cardiac development. Data from human genetic studies in CHD patients indicate that the genetic aetiology was presumably underestimated in the past. Inherited mutations in genes encoding cardiac transcription factors and sarcomeric proteins were found as an underlying cause for familial recurrence of non-syndromic CHD in humans, in particular cardiac septal defects. Notably, the cardiac phenotypes most frequently seen in mutation carriers are ostium secundum atrial septal defects (ASDII). This review outlines experimental approaches employed for the detection of CHD-related genes in humans and summarizes recent findings in molecular genetics of congenital cardiac septal defects with an emphasis on ASDII

Regulation of hippocampal synaptic plasticity thresholds and changes in exploratory and learning behavior in dominant negative NPR-B mutant rats

The second messenger cyclic GMP affects synaptic transmission and modulates synaptic plasticity and certain types of learning and memory processes. The impact of the natriuretic peptide receptor B (NPR-B) and its ligand C-type natriuretic peptide (CNP), one of several cGMP producing signaling systems, on hippocampal synaptic plasticity and learning is, however, less well understood. We have previously shown that the NPR-B ligand CNP increases the magnitude of long-term depression (LTD) in hippocampal area CA1, while reducing the induction of long-term potentiation (LTP). We have extended this line of research to show that bidirectional plasticity is affected in the opposite way in rats expressing a dominant-negative mutant of NPR-B (NSE-NPR- BΔKC) lacking the intracellular guanylyl cyclase domain under control of a promoter for neuron-specific enolase. The brain cells of these transgenic rats express functional dimers of the NPR-B receptor containing the dominant- negative NPR-BΔKC mutant, and therefore show decreased CNP-stimulated cGMP- production in brain membranes. The NPR-B transgenic rats display enhanced LTP but reduced LTD in hippocampal slices. When the frequency-dependence of synaptic modification to afferent stimulation in the range of 1–100 Hz was assessed in transgenic rats, the threshold for both, LTP and LTD induction, was shifted to lower frequencies. In parallel, NPR-BΔKC rats exhibited an enhancement in exploratory and learning behavior. These results indicate that bidirectional plasticity and learning and memory mechanism are affected in transgenic rats expressing a dominant-negative mutant of NPR-B. Our data substantiate the hypothesis that NPR-B-dependent cGMP signaling has a modulatory role for synaptic information storage and learning

A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects

BACKGROUND: Ostium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD) and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause for ASDII. However, very little is known about disease penetrance in families and functional consequences of inherited TBX20 mutations. METHODS: The coding region of TBX20 was directly sequenced in 170 ASDII patients. Functional consequences of one novel mutation were investigated by surface plasmon resonance, CD spectropolarymetry, fluorescence spectrophotometry, luciferase assay and chromatin immunoprecipitation. RESULTS: We found a novel mutation in a highly conserved residue in the T-box DNA-binding domain (I121M) segregating with CHD in a three generation kindred. Four mutation carriers revealed cardiac phenotypes in terms of cribriform ASDII, large patent foramen ovale or cardiac valve defects. Interestingly, tertiary hydrophobic interactions within the mutant TBX20 T-box were significantly altered leading to a more dynamic structure of the protein. Moreover, Tbx20-I121M resulted in a significantly enhanced transcriptional activity, which was further increased in the presence of co-transcription factors GATA4/5 and NKX2-5. Occupancy of DNA binding sites on target genes was also increased. CONCLUSIONS: We suggest that TBX20-I121M adopts a more fluid tertiary structure leading to enhanced interactions with cofactors and more stable transcriptional complexes on target DNA sequences. Our data, combined with that of others, suggest that human ASDII may be related to loss- as well as gain-of-function TBX20 mutations

Connective Tissue Growth Factor Overexpression in Cardiomyocytes Promotes Cardiac Hypertrophy and Protection against Pressure Overload

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca2+ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls

Cardiac Alpha-Myosin (MYH6) Is the Predominant Sarcomeric Disease Gene for Familial Atrial Septal Defects

Secundum-type atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD) and are associated with a familial risk. Mutations in transcription factors represent a genetic source for ASDII. Yet, little is known about the role of mutations in sarcomeric genes in ASDII etiology. To assess the role of sarcomeric genes in patients with inherited ASDII, we analyzed 13 sarcomeric genes (MYH7, MYBPC3, TNNT2, TCAP, TNNI3, MYH6, TPM1, MYL2, CSRP3, ACTC1, MYL3, TNNC1, and TTN kinase region) in 31 patients with familial ASDII using array-based resequencing. Genotyping of family relatives and control subjects as well as structural and homology analyses were used to evaluate the pathogenic impact of novel non-synonymous gene variants. Three novel missense mutations were found in the MYH6 gene encoding alpha-myosin heavy chain (R17H, C539R, and K543R). These mutations co-segregated with CHD in the families and were absent in 370 control alleles. Interestingly, all three MYH6 mutations are located in a highly conserved region of the alpha-myosin motor domain, which is involved in myosin-actin interaction. In addition, the cardiomyopathy related MYH6-A1004S and the MYBPC3-A833T mutations were also found in one and two unrelated subjects with ASDII, respectively. No mutations were found in the 11 other sarcomeric genes analyzed. The study indicates that sarcomeric gene mutations may represent a so far underestimated genetic source for familial recurrence of ASDII. In particular, perturbations in the MYH6 head domain seem to play a major role in the genetic origin of familial ASDII

Unequal allelic expression of wild-type and mutated β-myosin in familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, which in about 30% of the patients is caused by missense mutations in one allele of the β-myosin heavy chain (β-MHC) gene (MYH7). To address potential molecular mechanisms underlying the family-specific prognosis, we determined the relative expression of mutant versus wild-type MYH7-mRNA. We found a hitherto unknown mutation-dependent unequal expression of mutant to wild-type MYH7-mRNA, which is paralleled by similar unequal expression of β-MHC at the protein level. Relative abundance of mutated versus wild-type MYH7-mRNA was determined by a specific restriction digest approach and by real-time PCR (RT-qPCR). Fourteen samples from M. soleus and myocardium of 12 genotyped and clinically well-characterized FHC patients were analyzed. The fraction of mutated MYH7-mRNA in five patients with mutation R723G averaged to 66 and 68% of total MYH7-mRNA in soleus and myocardium, respectively. For mutations I736T, R719W and V606M, fractions of mutated MYH7-mRNA in M. soleus were 39, 57 and 29%, respectively. For all mutations, unequal abundance was similar at the protein level. Importantly, fractions of mutated transcripts were comparable among siblings, in younger relatives and unrelated carriers of the same mutation. Hence, the extent of unequal expression of mutated versus wild-type transcript and protein is characteristic for each mutation, implying cis-acting regulatory mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease expression and fraction of mutated mRNA and protein. This strongly suggests that mutation-specific allelic imbalance represents a new pathogenic factor for FHC

Ischemic and nonischemic causes of heart failure

In der vorliegenden Arbeit wurden genetische Prädiktoren einer In-Stent- Restenose bei Hochrisiko-Patienten mit wiederholter In-Stent-Restenose untersucht. Hierbei konnte gezeigt werden, dass Polymorphismen des Renin- Angiotensin-Aldosteron-Systems (RAAS) nicht mit einer höheren Inzidenz der Restenose assoziiert sind. Als weitere wesentliche Komplikation nach koronarem Stenting evaluierten wir die In-Stent-Thrombosehäufigkeit bei KHK-Patienten mit begleitender Tumorerkankung, die sich einem Stenting mit unbeschichten Stents unterzogen. Wir konnten ein mehr als 7-fach höheres Risiko für die In- Stent-Thrombose bei Tumorpatienten nachweisen. In-Stent-Restenosen und In- Stentthrombosen können über chronische Myokardischämie und ungünstigem Remodeling zur systolischen Herzinsuffizienz führen. Eine weitere Ursache der systolischen, primär nicht-ischämischen Herzinsuffizienz ist die Dilatative Kardiomyopathie (DCM). Bei Patienten mit einer Gliedergürteldystrophie Typ 2B, die eine Mutation im Gen Dysferlin tragen, konnten wir erstmals eine kardiale Mitbeteiligung nachweisen. In einem translationalen Ansatz haben wir mit Hilfe von tierexperimentellen Untersuchungen den möglichen Pathomechanismus der kardialen Dysfunktion aufklären können. Durch konditionale Deletion des erbB2-Rezeptors konnte ein weiteres neues DCM-Modell etabliert werden. Der genetische Ansatz, wie auch die klinisch bekannte kardiale Dysfunktion nach pharmakologischer erbB2-Inhibition (Trastuzumab) belegen die essentielle Bedeutung von erbB2 für die kardiale Funktion im adulten Herzen. Im Gegensatz zur DCM zeigen Patienten mit einer Hypertrophen Kardiomyopathie (HCM) meist eine erhaltene systolische Funktion, leiden jedoch unter der eingeschränkten diastolischen Compliance des verdickten Herzmuskels. Bei einer HCM-Familie konnten wir mit Hilfe von Kopplungsanalysen eine Mutation im MLP/CSRP3 als ursächliches Gen der HCM identifizieren. MLP ist im Gegensatz zu den bislang bekannten HCM-Proteinen ein überwiegend im Zytosol lokalisiertes Protein, dass am Stretching-Sensing der Kardiomyozyten beteiligt ist. Darüber hinaus erweitert es die bislang gültige Auffassung, wonach die HCM eine sarkomere Erkrankung ist.The work presented here describes genetic predictors of repetitive in-stent restenosis in high-risk patients. We were able to show that polymorphisms of the Renin-Angiotensin-Aldosterone System (RAAS) are not associated with a higher incidence of coronary in-stent restenosis. Additionally, we evaluated in-stent thrombosis as a further major complication of coronary stenting in patients with coronary artery disease and concomitant malignancy. Our brief retrospective survey supported the clinical impression that cancer patients have a 7-fold higher risk for in-stent thrombosis than patients without known malignancies. In-stent restenosis or in-stent- thrombosis may result in chronic ischemia and adverse myocardial remodeling, thereby contributing to the manifestation of systolic heart failure. Dilated cardiomyopathy (DCM) is another common cause of nonischemic systolic heart failure. In patients with limb-girdle muscular dystrophy 2B, who are carrying a mutation in the dysferlin gene we observed for he first time cardiac involvement. In an translational approach, we identified the underlying pathomechanism of heart dysfunction by establishing new animal models for these disorder. A further essential gene for maintaining cardiac function is erbB2 or HER2. Clinicial trials have shown that pharmacological inhibition (trastuzumab) of erbB2 leads to dilated cardiomyopathy. Our cardiac specific knockout model of erbB2 substantiates the important role of erbB2 in the adult myocardium. In contrast to DCM, patients with hypertrophic cardiomyopathy (HCM) usually display a preserved systolic function. Those patients suffer from heart failure that is related to the altered diastolic compliance of the thick noncompliant heart ventricles. By using linkage analysis we identified a missense mutation in the CSRP3 gene in a German family with HCM. Our data suggest that HCM is not exclusively a sarcomeric disease. An impaired mechano-sensory stress signalling might be involved in the pathogenesis of HCM

Rat Model for Dominant Dystrophic Epidermolysis Bullosa: Glycine Substitution Reduces Collagen VII Stability and Shows Gene-Dosage Effect

<div><p>Dystrophic epidermolysis bullosa, a severely disabling hereditary skin fragility disorder, is caused by mutations in the gene coding for collagen VII, a specialized adhesion component of the dermal-epidermal junction zone. Both recessive and dominant forms are known; the latter account for about 40% of cases. Patients with dominant dystrophic epidermolysis bullosa exhibit a spectrum of symptoms ranging from mild localized to generalized skin manifestations. Individuals with the same mutation can display substantial phenotypic variance, emphasizing the role of modifying genes in this disorder. The etiology of dystrophic epidermolysis bullosa has been known for around two decades; however, important pathogenetic questions such as involvement of modifier genes remain unanswered and a causative therapy has yet to be developed. Much of the failure to make progress in these areas is due to the lack of suitable animal models that capture all aspects of this complex monogenetic disorder. Here, we report the first rat model of dominant dystrophic epidermolysis bullosa. Affected rats carry a spontaneous glycine to aspartic acid substitution, p.G1867D, within the main structural domain of collagen VII. This confers dominant-negative interference of protein folding and decreases the stability of mutant collagen VII molecules and their polymers, the anchoring fibrils. The phenotype comprises fragile and blister-prone skin, scarring and nail dystrophy. The model recapitulates all signs of the human disease with complete penetrance. Homozygous carriers of the mutation are more severely affected than heterozygous ones, demonstrating for the first time a gene-dosage effect of mutated alleles in dystrophic epidermolysis bullosa. This novel viable and workable animal model for dominant dystrophic epidermolysis bullosa will be valuable for addressing molecular disease mechanisms, effects of modifying genes, and development of novel molecular therapies for patients with dominantly transmitted skin disease.</p></div