Donald L. Atkinson

Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias

Genetic factors contribute to the risk of sudden death from cardiac arrhythmias. Here, positional cloning methods establish KVLQT1 as the chromosome 11-linked LQT1 gene responsible for the most common inherited cardiac arrhythmia. KVLQT1 is strongly expressed in the heart and encodes a protein with structural features of a voltage-gated potassium channel. KVLQT1 mutations are present in affected members of 16 arrhythmia families, including one intragenic deletion and ten different missense mutations. These data define KVLQT1 as a novel cardiac potassium channel gene and show that mutations in this gene cause susceptibility to ventricular tachyarrhythmias and sudden death.

SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome

Long QT syndrome (LQT) is an inherited disorder that causes sudden death from cardiac arrhythmias, specifically torsade de pointes and ventricular fibrillation. We previously mapped three LQT loci: LQT1 on chromosome 11p15.5, LQT2 on 7q35-36, and LQT3 on 3p21-24. Here we report genetic linkage between LQT3 and polymorphisms within SCN5A, the cardiac sodium channel gene. Single strand conformation polymorphism and DNA sequence analyses reveal identical intragenic deletions of SCN5A in affected members of two unrelated LQT families. The deleted sequences reside in a region that is important for channel inactivation. These data suggest that mutations in SCN5A cause chromosome 3-linked LQT and indicate a likely cellular mechanism for this disorder.

LIM-kinase1 hemizygosity implicated in impaired visuospatial constructive cognition.

To identify genes important for human cognitive development, we studied Williams syndrome (WS), a developmental disorder that includes poor visuospatial constructive cognition. Here we describe two families with a partial WS phenotype; affected members have the specific WS cognitive profile and vascular disease, but lack other WS features. Submicroscopic chromosome 7q11.23 deletions cosegregate with this phenotype in both families. DNA sequence analyses of the region affected by the smallest deletion (83.6 kb) revealed two genes, elastin (ELN) and LIM-kinase1 (LIMK1). The latter encodes a novel protein kinase with LIM domains and is strongly expressed in the brain. Because ELN mutations cause vascular disease but not cognitive abnormalities, these data implicate LIMK1 hemizygosity in imparied visuospatial constructive cognition.

The elastin gene is disrupted by a translocation associated with supravalvular aortic stenosis

To identify genes involved in vascular disease, we investigated patients with supravalvular aortic stenosis (SVAS), an inherited vascular disorder that causes hemodynamically significant narrowing of large elastic arteries. Pulsed-field gel and Southern analyses showed that a translocation near the elastin gene cosegregated with SVAS in one family. DNA sequence analyses demonstrated that the translocation disrupted the elastin gene and localized the breakpoint to exon 28. Taken together with our previous study linking SVAS to the elastin gene in two additional families and existing knowledge of vascular biology, these data suggest that mutations in the elastin gene can cause SVAS.

Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase.

Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.

Locus heterogeneity of autosomal dominant long QT syndrome.

Autosomal dominant long QT syndrome (LQT) is an inherited disorder that causes syncope and sudden death from cardiac arrhythmias. In genetic linkage studies of seven unrelated families we mapped a gene for LQT to the short arm of chromosome 11 (11p15.5), near the Harvey ras-1 gene (H ras-1). To determine if the same locus was responsible for LQT in additional families, we performed linkage studies with DNA markers from this region (H ras-1 and MUC2). Pairwise linkage analyses resulted in logarithm of odds scores of -2.64 and -5.54 for kindreds 1977 and 1756, respectively. To exclude the possibility that rare recombination events might account for these results, we performed multipoint linkage analyses using additional markers from chromosome 11p15.5 (tyrosine hydroxylase and D11S860). Multipoint analyses excluded approximately 25.5 centiMorgans of chromosome 11p15.5 in K1756 and approximately 13 centiMorgans in K1977. These data demonstrate that the LQT gene in these kindreds is not linked to H ras-1 and suggest that mutations in at least two genes can cause LQT. While the identification of locus heterogeneity of LQT will complicate genetic diagnosis, characterization of additional LQT loci will enhance our understanding of this disorder.

Gene expression signatures in the newt irises during lens regeneration

Lens regeneration in adult newts is possible by transdifferentiation of the pigment epithelial cells (PECs) of the dorsal iris. The same cells in the ventral iris are not capable of such a process. To understand this difference in regenerative competency, we examined gene expression of 373 genes in the intact dorsal and ventral irises as well as in irises during the process of lens regeneration. We found similar signatures of gene expression in dorsal and ventral with several cases of even higher levels in the ventral iris. Such transcriptional activity in the regeneration‐incompetent ventral iris was unexpected and calls for a revision of our views about mechanisms of lens regeneration induction.

Tissue inhibitor of metalloproteinase 1 regulates matrix metalloproteinase activity during newt limb regeneration

Matrix metalloproteinase (MMP) activity is important for newt limb regeneration. In most biological processes that require MMP function, MMP activity is tightly controlled by a variety of mechanisms, including the coexpression of natural inhibitors. Here, we show that gene expression of one such inhibitor, tissue inhibitor of metalloproteinase 1 (NvTIMP1), is upregulated during the wound healing and dedifferentiation stages of regeneration when several MMPs are at their maximal expression levels. Newt MMPs and NvTIMP1 also exhibit similar spatial expression patterns during the early stages of limb regeneration. NvTIMP1 inhibits the proteolytic activity of regeneration‐related newt MMPs and, like human TIMP1, can induce a weak mitogenic response in certain cell types. These results suggest that NvTIMP1 may be functioning primarily to maintain optimal levels of MMP activity during the early stages of limb regeneration, while possibly serving a secondary role as a mitogen. Developmental Dynamics 235:606–616, 2006.

Multi-tissue microarray analysis identifies a molecular signature of regeneration.

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.

Evaluation of multiple biological therapies for ischemic cardiac disease

The development of cell- and gene-based strategies for regenerative medicine offers a therapeutic option for the repair and potential regeneration of damaged cardiac tissue post-myocardial infarction (MI). Human umbilical cord subepithelial cell-derived stem cells (hUC-SECs), human bone marrow-derived mesenchymal stem cells (hBM-MSCs), and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), all derived from human tissue, have been shown to have in vitro and in vivo therapeutic potential. Additionally, S100a1, VEGF165, and stromal-derived factor-1α (SDF-1α) genes all have the potential to improve cardiac function and/or effect adverse remodeling. In this study, we compared the therapeutic potential of hBM-MSCs, hUC-SECs, and hiPSC-CMs along with plasmid-based genes to evaluate the in vivo potential of intramyocardially injected biologics to enhance cardiac function in a mouse MI model. Human cells derived from various tissue types were expanded under hypoxic conditions and injected intramyocardially into mice that had undergone left anterior descending (LAD) artery ligation. Similarly, plasmids were also injected into three groups of mice after LAD ligation. Seven experimental groups were studied in total: (1) control (saline), (2) hBM-MSCs, (3) hiPSC-CMs, (4) hUC-SECs, (5) S100a1 plasmid, (6) VEGF165 plasmid, and (7) SDF-1α plasmid. We evaluated echocardiography, hemodynamic catheterization measurements, and histology at 4 and 12 weeks post-biologic injection. Significant improvement was observed in cardiac function and contractility in hiPSC-CM and S100a1 groups and a significant reduction in left ventricle scar within the hUC-SEC group and a slight improvement in the SDF-1α and VEGF165 groups compared to the control group. These results demonstrate the potential for new biologic therapies to reduce scar burden and improve contractile function.