A retrograde approach for liver gene transfer

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Random Graph-Homomorphisms and Logarithmic Degree

A graph homomorphism between two graphs is a map from the vertex set of one graph to the vertex set of the other graph, that maps edges to edges. In this note we study the range of a uniformly chosen homomorphism from a graph G to the infinite line Z. It is shown that if the maximal degree of G is `sub-logarithmic', then the range of such a homomorphism is super-constant. Furthermore, some examples are provided, suggesting that perhaps for graphs with super-logarithmic degree, the range of a typical homomorphism is bounded. In particular, a sharp transition is shown for a specific family of graphs C_{n,k} (which is the tensor product of the n-cycle and a complete graph, with self-loops, of size k). That is, given any function psi(n) tending to infinity, the range of a typical homomorphism of C_{n,k} is super-constant for k = 2 log(n) - psi(n), and is 3 for k = 2 log(n) + psi(n)

DNA methylation epi-signature is associated with two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome

Background: Phelan-McDermid syndrome is characterized by a range of neurodevelopmental phenotypes with incomplete penetrance and variable expressivity. It is caused by a variable size and breakpoint microdeletions in the distal long arm of chromosome 22, referred to as 22q13.3 deletion syndrome, including the SHANK3 gene. Genetic defects in a growing number of neurodevelopmental genes have been shown to cause genome-wide disruptions in epigenomic profiles referred to as epi-signatures in affected individuals. Results: In this study we assessed genome-wide DNA methylation profiles in a cohort of 22 individuals with Phelan-McDermid syndrome, including 11 individuals with large (2 to 5.8 Mb) 22q13.3 deletions, 10 with small deletions (\u3c 1 Mb) or intragenic variants in SHANK3 and one mosaic case. We describe a novel genome-wide DNA methylation epi-signature in a subset of individuals with Phelan-McDermid syndrome. Conclusion: We identified the critical region including the BRD1 gene as responsible for the Phelan-McDermid syndrome epi-signature. Metabolomic profiles of individuals with the DNA methylation epi-signature showed significantly different metabolomic profiles indicating evidence of two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome

Comparison of Replication-Competent, First Generation, and Helper-Dependent Adenoviral Vaccines

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and anti-Ad T cell responses. Also, HD vectors have the benefit of being packaged by all subgroup C serotype helper viruses. We found that HD serotypes 1, 2, 5, and 6 induce anti-HIV responses equivalently. By using these HD serotypes in heterologous succession we showed that HD vectors can be used to significantly boost anti-HIV immune responses in mice and in FG-Ad5-immune macaques. Since HD vectors have been show to have an increased safety profile, do not possess any Ad genes, can be packaged by multiple serotype helper viruses, and elicit strong anti-HIV immune responses, they warrant further investigation as alternatives to FG vectors as gene-based vaccines

Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity

Individuals homozygous for the “Z” mutation in alpha-1 antitrypsin deficiency are known to be at increased risk for liver disease. It has also become clear that some degree of risk is similarly conferred by the heterozygous state. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of a single Z alpha-1 antitrypsin (ZAAT) allele on hepatocyte biology. Here, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-hepatocytes (iHeps). We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations including altered endoplasmic reticulum (ER) and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in cell subpopulations. Our model of MZ heterozygosity thus provides evidence that a single Z allele is sufficient to disrupt hepatocyte homeostatic function.This work was supported by an Alpha-1 Foundation John W. Walsh Translational Research Award (to J.E.K.); a CJ Martin Early Career Fellowship from the Australian National Health and Medical Research Council (to R.B.W.); NIH grant R01HL095993 (to D.N.K.); and NIH grants R01DK101501 (to A.A.W.) and R01DK117940 (to A.N.H. and A.A.W.). iPSC distribution and disease modeling is supported by NIH grants U01TR001810 (to D.N.K. and A.A.W.) and N0175N92020C00005 (to D.N.K.); and by The Alpha-1 Project (TAP), a wholly owned subsidiary of the Alpha-1 Foundation (to D.N.K. and A.A.W.)

Clinical and functional consequences of C-terminal variants in MCT8

CONTEXT: Genetic variants in SLC16A2, encoding the thyroid hormone transporter MCT8, can cause intellectual and motor disability and abnormal serum thyroid function tests, known as MCT8 deficiency. The C-terminal domain of MCT8 is poorly conserved, which complicates prediction of the deleteriousness of variants in this region. We studied the functional consequences of 5 novel variants within this domain and their relation to the clinical phenotypes. METHODS: We enrolled male subjects with intellectual disability in whom genetic variants were identified in exon 6 of SLC16A2. The impact of identified variants was evaluated in transiently transfected cell lines and patient-derived fibroblasts. RESULTS: Seven individuals from 5 families harbored potentially deleterious variants affecting the C-terminal domain of MCT8. Two boys with clinical features considered atypical for MCT8 deficiency had a missense variant [c.1724A>G;p.(His575Arg) or c.1796A>G;p.(Asn599Ser)] that did not affect MCT8 function in transfected cells or patient-derived fibroblasts, challenging a causal relationship. Two brothers with classical MCT8 deficiency had a truncating c.1695delT;p.(Val566*) variant that completely inactivated MCT8 in vitro. The 3 other boys had relatively less-severe clinical features and harbored frameshift variants that elongate the MCT8 protein [c.1805delT;p.(Leu602HisfsTer680) and c.del1826-1835;p.(Pro609GlnfsTer676)] and retained ~50% residual activity. Additional truncating variants within transmembrane domain 12 were fully inactivating, whereas those within the intracellular C-terminal tail were tolerated. CONCLUSIONS: Variants affecting the intracellular C-terminal tail of MCT8 are likely benign unless they cause frameshifts that elongate the MCT8 protein. These findings provide clinical guidance in the assessment of the pathogenicity of variants within the C-terminal domain of MCT8

Bi-allelic KARS1 pathogenic variants affecting functions of cytosolic and mitochondrial isoforms are associated with a progressive and multisystem disease

KARS1 encodes a lysyl-transfer RNA synthetase (LysRS) that links lysine to its cognate transfer RNA. Two different KARS1 isoforms exert functional effects in cytosol and mitochondria. Bi-allelic pathogenic variants in KARS1 have been associated to sensorineural hearing and visual loss, neuropathy, seizures, and leukodystrophy. We report the clinical, biochemical, and neuroradiological features of nine individuals with KARS1-related disorder carrying 12 different variants with nine of them being novel. The consequences of these variants on the cytosol and/or mitochondrial LysRS were functionally validated in yeast mutants. Most cases presented with severe neurological features including congenital and progressive microcephaly, seizures, developmental delay/intellectual disability, and cerebral atrophy. Oculo-motor dysfunction and immuno-hematological problems were present in six and three cases, respectively. A yeast growth defect of variable severity was detected for most variants on both cytosolic and mitochondrial isoforms. The detrimental effects of two variants on yeast growth were partially rescued by lysine supplementation. Congenital progressive microcephaly, oculo-motor dysfunction, and immuno-hematological problems are emerging phenotypes in KARS1-related disorder. The data in yeast emphasize the role of both mitochondrial and cytosolic isoforms in the pathogenesis of KARS1-related disorder and supports the therapeutic potential of lysine supplementation at least in a subset of patients

Population analysis of the GLB1 gene in South Brazil

Infantile GM1 gangliosidosis is caused by the absence or reduction of lysosomal beta-galactosidase activity. Studies conducted in Brazil have indicated that it is one of the most frequent lysosomal storage disorders in the southern part of the country. To assess the incidence of this disorder, 390 blood donors were tested for the presence of two common mutations (1622–1627insG and R59H) in the GLB1 gene. Another group, consisting of 26 GM1 patients, and the blood donors were tested for the presence of two polymorphisms (R521C and S532G), in an attempt to elucidate whether there is a founder effect. The frequencies of the R59H and 1622–1627insG mutations among the GM1 patients studied were 19.2% and 38.5%, respectively. The frequency of polymorphism S532G was 16.7%, whereas R521C was not found in the patients. The overall frequency of either R59H or 1622–1627insG was 57.7% of the disease-causing alleles. This epidemiological study suggested a carrier frequency of 1:58. Seven different haplotypes were found. The 1622–1627insG mutation was not found to be linked to any polymorphism, whereas linkage disequilibrium was found for haplotype 2 (R59H, S532G) (p < 0.001). These data confirm the high incidence of GM1 gangliosidosis and the high frequency of two common mutations in southern Brazil