Racial differences in systemic sclerosis disease presentation: a European Scleroderma Trials and Research group study

Objectives. Racial factors play a significant role in SSc. We evaluated differences in SSc presentations between white patients (WP), Asian patients (AP) and black patients (BP) and analysed the effects of geographical locations.Methods. SSc characteristics of patients from the EUSTAR cohort were cross-sectionally compared across racial groups using survival and multiple logistic regression analyses.Results. The study included 9162 WP, 341 AP and 181 BP. AP developed the first non-RP feature faster than WP but slower than BP. AP were less frequently anti-centromere (ACA; odds ratio (OR) = 0.4, P < 0.001) and more frequently anti-topoisomerase-I autoantibodies (ATA) positive (OR = 1.2, P = 0.068), while BP were less likely to be ACA and ATA positive than were WP [OR(ACA) = 0.3, P < 0.001; OR(ATA) = 0.5, P = 0.020]. AP had less often (OR = 0.7, P = 0.06) and BP more often (OR = 2.7, P < 0.001) diffuse skin involvement than had WP.AP and BP were more likely to have pulmonary hypertension [OR(AP) = 2.6, P < 0.001; OR(BP) = 2.7, P = 0.03 vs WP] and a reduced forced vital capacity [OR(AP) = 2.5, P < 0.001; OR(BP) = 2.4, P < 0.004] than were WP. AP more often had an impaired diffusing capacity of the lung than had BP and WP [OR(AP vs BP) = 1.9, P = 0.038; OR(AP vs WP) = 2.4, P < 0.001]. After RP onset, AP and BP had a higher hazard to die than had WP [hazard ratio (HR) (AP) = 1.6, P = 0.011; HR(BP) = 2.1, P < 0.001].Conclusion. Compared with WP, and mostly independent of geographical location, AP have a faster and earlier disease onset with high prevalences of ATA, pulmonary hypertension and forced vital capacity impairment and higher mortality. BP had the fastest disease onset, a high prevalence of diffuse skin involvement and nominally the highest mortality

original article © The American Society of Gene & Cell Therapy Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous

Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy

Changes in Adeno-Associated Virus-Mediated Gene Delivery in Retinal Degeneration

Gene therapies for retinal degeneration have relied on subretinal delivery of viral vectors carrying therapeutic DNA. The subretinal injection is clearly not ideal as it limits the viral transduction profile to a focal region at the injection site and negatively affects the neural retina by detaching it from the supportive retinal pigment epithelium (RPE). We assessed changes in adeno-associated virus (AAV) dispersion and transduction in the degenerating rat retina after intravitreal delivery. We observed a significant increase in AAV-mediated gene transfer in the diseased compared with normal retina, the extent of which depends on the AAV serotype injected. We also identified key structural changes that correspond to increased viral infectivity. Particle diffusion and transgene accumulation in normal and diseased retina were monitored via fluorescent labeling of viral capsids and quantitative PCR. Viral particles were observed to accumulate at the vitreoretinal junction in normal retina, whereas particles spread into the outer retina and RPE in degenerated tissue. Immunohistochemistry illustrates remarkable changes in the architecture of the inner limiting membrane, which are likely to underlie the increased viral transduction in diseased retina. These data highlight the importance of characterizing gene delivery vectors in diseased tissue as structural and biochemical changes can alter viral vector transduction patterns. Furthermore, these results indicate that gene delivery to the outer nuclear layer may be achieved by noninvasive intravitreal AAV administration in the diseased state

Intravitreal Injection of AAV2 Transduces Macaque Inner Retina

Intravitreally injected AAV2 transduced inner retinal cells in a restricted region at the macaque fovea. Because macaque and human eyes are similar, the results suggest a need to improve transduction methods in gene therapy for the human inner retina

Brief Report: Whole-Exome Sequencing to Identify Rare Variants and Gene Networks That Increase Susceptibility to Scleroderma in African Americans.

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146461/1/art40541_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146461/2/art40541.pd

Brief Report: Whole-Exome Sequencing to Identify Rare Variants and Gene Networks That Increase Susceptibility to Scleroderma in African Americans

OBJECTIVE: Whole-exome sequencing (WES) studies in systemic sclerosis (SSc) patients of European American (EA) ancestry have identified variants in the ATP8B4 gene and enrichment of variants in genes in the extracellular matrix (ECM)-related pathway that increase SSc susceptibility. This study was undertaken to evaluate the association of the ATP8B4 gene and the ECM-related pathway with SSc in a cohort of African American (AA) patients. METHODS: SSc patients of AA ancestry were enrolled from 23 academic centers across the US under the Genome Research in African American Scleroderma Patients consortium. Unrelated AA individuals without serologic evidence of autoimmunity who were enrolled in the Howard University Family Study were used as unaffected controls. Functional variants in genes reported in the 2 WES studies in EA patients with SSc were selected for gene association testing using the optimized sequence kernel association test (SKAT-O) and pathway analysis by Ingenuity Pathway Analysis in 379 patients and 411 controls. RESULTS: Principal components analysis demonstrated that the patients and controls had similar ancestral backgrounds, with roughly equal proportions of mean European admixture. Using SKAT-O, we examined the association of individual genes that were previously reported in EA patients and none remained significant, including ATP8B4 (P = 0.98). However, we confirmed the previously reported association of the ECM-related pathway with enrichment of variants within the COL13A1, COL18A1, COL22A1, COL4A3, COL4A4, COL5A2, PROK1, and SERPINE1 genes (corrected P = 1.95 × 10-4 ). CONCLUSION: In the largest genetic study in AA patients with SSc to date, our findings corroborate the role of functional variants that aggregate in a fibrotic pathway and increase SSc susceptibility

HLA contributions to risk and protection for anti-centromere autoantibody-positive scleroderma

Background/Purpose: Anti-nuclear autoantibodies are a hallmark of scleroderma with anti-centromere antibody (ACA) recognizing centromeric antigens. ACA-positive patients have longstanding Raynaud\u27s, limited cutaneous disease and increased risk for pulmonary arterial hypertension. We investigated the role of HLA classical genes and alleles on risk for ACA-positive scleroderma in a large collection of patients with scleroderma and genetically matched controls. Methods: SNP genotypes of 723 scleroderma cases and 5,561 controls, all of European ancestry, were obtained from dbGaP. Classical HLA types were imputed with SNP2HLA using the Type I Diabetes Genetic Consortium reference of 5,225 individuals. Association of HLA classical alleles was tested by a dominant model regression analysis coding the HLA types as numeric values (1 for present, 0 for absent). Regression tests were corrected for genetic dissimilarity by including the top 5 principal components as covariates. Results: Of the 723 scleroderma cases, 238 (32.9%) were positive for ACA. The most significantly ACA-positive scleroderma-associated HLA allele was HLA-DRB1∗07:01, which was disease protective (P-value=1.8x10-18, odds ratio (OR)=0.11 (95%CI=0.05 to 0.22)). This allele was found in only 3.4% of the ACA-positive cases versus 23.6% of controls and 20.8% of the ACA-negative cases. Regression analysis conditioning on the disease-associated alleles identified HLADQB1∗ 05:01 as the most significantly associated disease risk allele (P-value= 3.3x10-08, OR=2.18 (1.66-2.86)) with additional independent risk effects of HLA-DQA1∗04:01 and HLA-DQA1∗03:01). A two-locus analysis of the DQB1∗05:01 disease-risk and the DRB1∗07:01 disease-protective alleles suggested the risk effect of DQB1∗05:01 is overridden by the protective effect of DRB1∗07:01(Figure 1). The odds ratio for ACA-positive disease in individuals carrying both alleles was 0.14, similar to that in individuals carrying DRB1∗07:01 without DQB1∗05:01 (0.15). No effect of DRB1∗07:01 or DQB1∗05:01 was found in the anti-topoisomerase I autoantibody subset (P=0.98 and P=0.054, respectively). For validation, 62 African American ACA-positive cases and 946 matched controls from the Genomic Research in African American Scleroderma Patients (GRASP) Collection, were similarly analyzed. DQB1∗05:01 was associated with disease risk (P=3.6x10-4, OR=2.68 (1.57-4.58)) and DRB1∗07:01 was protective (P=8.6x10-3, OR=0.33 (0.13-0.85)) in the African American sample. Conclusion: HLA-DQB1∗05:01 is associated with risk and HLA-DRB1∗07:01 is associated with protection for ACA-positive scleroderma. The mechanisms responsible for these effects could be exploited to prevent or treat scleroderma. (Figure Presented)

Strong HLA and novel non-HLA associations identified by auto-antibody subset analysis of African Americans with scleroderma from the genome research in African American scleroderma patients cohort

Background/Purpose: Anti-fibrillarin (nucleolar, AFA) and anti-topoisomeraseI (ATA) autoantibodies are specific to systemic sclerosis (SSc) and are common in African Americans (AA). These auto antibodies define clinically distinct phenotypes but the genetic risk factors contributing towards them are largely unknown. Methods: Data from the genome-wide association study (GWAS) of AA SSc collected under the Genome Research in African American Scleroderma Patients (GRASP) cohort were utilized.207 AFA and 245 ATA positive patients were compared to 946 controls. After quality control filtering, SNP imputation was performed using Beagle and classicalHLA types were imputed using HLA∗IMP:03web server. Odds ratios for HLAalleles and amino acid residues were calculated using a dominant model. Results: In the GRASP cohort, 22.2% of patients had nucleolar ANA pattern (AFA) after removing other nucleolar staining, SSc specific auto antibodies. In the AFA+ subset, HLA-DRB1∗08:04 demonstrated the strongest association with P=4.2 x 10-27, OR=5.98 (95% CI4.2-8.5) (Figure 1A). HLA-DRB1∗08:04 is a predominantly African ancestry allele and its leucine 74 residue in the peptide binding groove was strongly associated with AFA+ SSc with OR=5.12(95% CI 3.7-7.1). The top SNP in the HLAregion was rs573310147 with P=1.3 x 10-19. ATA positivity was seen in 26.2% of patients in the GRASPcohort. In the ATA+ subset, rs28667353 was the top SNP with P=1.4x 10-19 (Figure 1B). HLA-DPB1∗13:01 was seen in 15.8% ofATA+ SSc as compared to 6.2% in ATA- SSc and 5.1% in controls. HLA-DPB1∗13:01 demonstrated the strongest association with P=1.1 x 10-16, OR=4.1 (95% CI 2.9-5.8) in the ATA+ subset. HLA-DPB1∗13:01has been reported as a risk lociin ATA+ subset in SSc patients from several different ancestries. Meta-analysis with the European ancestry SSc yielded a highly significant P=2.18 x 10-61. On examining the amino acid residues, isoleucine 76 in the peptide binding groove of HLA-DPB1 increased risk for SScwith OR=2.8 (95% CI 2.1-3.8). Six other previously unreported, non-HLA loci were identified in the AFA +subset and two in the ATA + subset at genome-wide significance with the top one being FSD2 (Fibronectin type III andSPRY domain containing 2). Conclusion: HLA-DRB1∗08:04 is a predominantly African ancestryallele that increases SSc risk 6-fold in AFAsubset which is primarily observed in AAs with SSc.This may help explain the increased prevalence and severity of SScin AAs. HLADPB1∗ 13:01 increases risk of SSc in not only AAs but all ancestral populations. Also, HLA-DPB1∗13:01 control frequency in a population correlates with the prevalence of SSc in that ancestral population. Functional roles of these novel non- HLA loci need to be experimentally evaluated. Understanding the mechanism of peptide presentation by these two HLA alleles will lead to a better recognition of the trigger that leads to autoimmunity in SSc