Adeno-Associated Viral Transfer of Glyoxalase-1 Blunts Carbonyl and Oxidative Stresses in Hearts of Type 1 Diabetic Rats

Accumulation of methylglyoxal (MG) arising from downregulation of its primary degrading enzyme glyoxalase-1 (Glo1) is an underlying cause of diabetic cardiomyopathy (DC). This study investigated if expressing Glo1 in rat hearts shortly after the onset of Type 1 diabetes mellitus (T1DM) would blunt the development of DC employing the streptozotocin-induced T1DM rat model, an adeno-associated virus containing Glo1 driven by the endothelin-1 promoter (AAV2/9-Endo-Glo1), echocardiography, video edge, confocal imaging, and biochemical/histopathological assays. After eight weeks of T1DM, rats developed DC characterized by decreased E:A ratio, fractional shortening, and ejection fraction, and increased isovolumetric relaxation time, E: e’ ratio, and circumferential and longitudinal strains. Evoked Ca2+ transients and contractile kinetics were also impaired in ventricular myocytes. Hearts from eight weeks T1DM rats had lower Glo1 and GSH levels, elevated carbonyl/oxidative stress, microvascular leakage, inflammation, and fibrosis. A single injection of AAV2/9 Endo-Glo1 (1.7×1012 viron particles/kg) one week after onset of T1DM, potentiated GSH, and blunted MG accumulation, carbonyl/oxidative stress, microvascular leakage, inflammation, fibrosis and impairments in cardiac and myocyte functions that develop after eight weeks of T1DM. These new data indicate that preventing Glo1 downregulation by administering AAV2/9-Endo-Glo1 to rats one week after the onset of T1DM, blunted the DC that develops after eight weeks of diabetes by attenuating carbonyl/oxidative stresses, microvascular leakage, inflammation, and fibrosis

A phased SNP-based classification of sickle cell anemia HBB haplotypes

Background: Sickle cell anemia causes severe complications and premature death. Five common beta-globin gene cluster haplotypes are each associated with characteristic fetal hemoglobin (HbF) levels. As HbF is the major modulator of disease severity, classifying patients according to haplotype is useful. The first method of haplotype classification used restriction fragment length polymorphisms (RFLPs) to detect single nucleotide polymorphisms (SNPs) in the beta-globin gene cluster. This is labor intensive, and error prone. Methods: We used genome-wide SNP data imputed to the 1000 Genomes reference panel to obtain phased data distinguishing parental alleles. Results: We successfully haplotyped 813 sickle cell anemia patients previously classified by RFLPs with a concordance >98%. Four SNPs (rs3834466, rs28440105, rs10128556, and rs968857) marking four different restriction enzyme sites unequivocally defined most haplotypes. We were able to assign a haplotype to 86% of samples that were either partially or misclassified using RFLPs. Conclusion: Phased data using only four SNPs allowed unequivocal assignment of a haplotype that was not always possible using a larger number of RFLPs. Given the availability of genome-wide SNP data, our method is rapid and does not require high computational resources.NIH Bethesda, MDBoston Univ, Sch Med, Dept Med, 72 E Concord St, Boston, MA 02118 USABoston Univ, Bioinformat Program, Boston, MA 02215 USAKing Saud Univ, Coll Med, Sickle Cell Dis Res Ctr, Riyadh, Saudi ArabiaKing Saud Univ, Coll Med, Dept Pediat, Riyadh, Saudi ArabiaKing Faisal Univ, Al Omran Sci Chair, Al Hasa, Saudi ArabiaImam Abdulrahman bin Faisal Univ, Inst Res & Med Consultat, Dammam, Saudi ArabiaEscola Paulista Med, Hematol & Blood Transfus Div, São Paulo, BrazilBoston Univ, Sch Publ Hlth, Dept Biostat, Boston, MA 02118 USAEscola Paulista Med, Hematol & Blood Transfus Div, São Paulo, BrazilNIH: R01 HL 068970NIH: RC2 HL 101212NIH: R01 87681NIH: T32 HL007501Web of Scienc

Concept and design of a genome-wide association genotyping array tailored for transplantation-specific studies

Background: In addition to HLA genetic incompatibility, non-HLA difference between donor and recipients of transplantation leading to allograft rejection are now becoming evident. We aimed to create a unique genome-wide platform to facilitate genomic research studies in transplant-related studies. We designed a genome-wide genotyping tool based on the most recent human genomic reference datasets, and included customization for known and potentially relevant metabolic and pharmacological loci relevant to transplantation. Methods: We describe here the design and implementation of a customized genome-wide genotyping array, the ‘TxArray’, comprising approximately 782,000 markers with tailored content for deeper capture of variants across HLA, KIR, pharmacogenomic, and metabolic loci important in transplantation. To test concordance and genotyping quality, we genotyped 85 HapMap samples on the array, including eight trios. Results: We show low Mendelian error rates and high concordance rates for HapMap samples (average parent-parent-child heritability of 0.997, and concordance of 0.996). We performed genotype imputation across autosomal regions, masking directly genotyped SNPs to assess imputation accuracy and report an accuracy of >0.962 for directly genotyped SNPs. We demonstrate much higher capture of the natural killer cell immunoglobulin-like receptor (KIR) region versus comparable platforms. Overall, we show that the genotyping quality and coverage of the TxArray is very high when compared to reference samples and to other genome-wide genotyping platforms. Conclusions: We have designed a comprehensive genome-wide genotyping tool which enables accurate association testing and imputation of ungenotyped SNPs, facilitating powerful and cost-effective large-scale genotyping of transplant-related studies. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0211-x) contains supplementary material, which is available to authorized users

Perspective: A novel prognostic for sickle cell disease

Sickle hemoglobin (α2βS2) polymerization drives disease pathophysiology in sickle cell anemia. Fetal hemoglobin (α2γ2) restricts disease severity by inhibiting the polymerization of sickle hemoglobin in a concentration-dependent manner. Clinical decision-making relies on diagnostic technologies evaluating fetal hemoglobin as mean percent or mean quantity in blood. Limitation of this approach is exemplified by patients with significant high fetal hemoglobin levels and severe disease, suggesting that fetal hemoglobin is unevenly distributed across F-cells. Therefore, determination of fetal hemoglobin/F-cell would provide a new paradigm for ascertaining prognosis and response to fetal hemoglobin-inducing agents. Measurement of fetal hemoglobin/F-cell, ultimately adapted to widespread standardized analytical use, is a promising fetal hemoglobin-related prognostic approach to monitor the severity of sickle cell disease and the best “phenotype” to follow when developing new candidate fetal hemoglobin inducers or titrating hydroxyurea in treated sickle cell patients

Identification of on-target mutagenesis during correction of a beta-thalassemia splice mutation in iPS cells with optimised CRISPR/Cas9-double nickase reveals potential safety concerns

Precise and accurate gene correction is crucial for enabling iPSC-based therapies, and Cas9-Nickase based approaches are increasingly considered for in vivo correction of diseases such as beta-thalassemia. Here, we generate footprint-free induced pluripotent stem cells from a patient with a beta-thalassemia mutation (IVSII-1 G > A) and employ a double Cas9nickase-mediated correction strategy combined with a piggyBac transposon-modified donor vector for gene correction. Our approach further aimed to minimize the formation of adjacent single-strand breaks at the targeted allele through the destruction of the binding site for one guide and the use of a synonymous protospacer adjacent motif blocking mutation (canonical PAM sequence 5'-NGG-3' is changed to 5'-NCG-3', where N indicates any nucleobase) for the other guide. We show that this strategy indeed not only permits bi-allelic seamless repair of the beta-globin gene splice site mutation and negligible off-target mutagenesis or re-editing of the targeted allele but also results in unexpected on-target mutagenesis with some guide RNAs (gRNAs) in several targeted clones. This study thus not only validates a framework for seamless gene correction with enhanced specificity and accuracy but also highlights potential safety concerns associated with Cas9-nickase based gene correction. (C) 2018 Author(s)

Investigation of KIF6 Trp719Arg gene polymorphism in a case-control study of coronary artery disease and non-fatal myocardial infarction in the Eastern Province of Saudi Arabia

BACKGROUND: Kinesin-like protein 6 (KIF6), a member of the kinesin superfamily, is involved in intracellular transport. A few prospective studies have shown the KIF6 variant Trp719Arg (rs20455) to be associated with coronary artery disease (CAD) in Caucasian populations. However, recent genome-wide association studies on CAD have not proven these associations. OBJECTIVES: Since the role of KIF6 719Arg allele in other ethnic populations is largely unknown, we sought to determine whether the KIF6 719Arg allele is associated with CAD in an ethnic Middle Eastern population. DESIGN: Case-control study. SETTING: CAD patients and control subjects from King Fahd Hospital of the University, Al-Khobar, Saudi Arabia. PATIENTS AND METHODS: The study population included angiographically defined CAD patients (n=1002) and controls (n=984) with a normal electrocardiogram. MAIN OUTCOME MEASURE(S): Association of KIF6 Trp719Arg mutation with CAD. RESULTS: The KIF6 Trp719Arg polymorphism was not associated with CAD (OR 0.976, 95% CI 0.861-1.105; P=.704). In addition, KIF6 Trp719Arg polymorphism showed a lack of association even in stratified myocardial infarction patients (n=802) (OR 1.006, 95% CI 0.881-1.148; P=.929) in comparison to controls. CONCLUSIONS: The absence of Trp719Arg polymorphism association with CAD and CAD in stratified myocardial infarction cases indicates that the polymorphism is not associated with an increased risk among CAD patients from the Eastern Province of Saudi Arabia. LIMITATIONS: Unavailability of data on statin usage among the patient population