A multi-cohort genome-wide association study in African ancestry individuals reveals risk loci for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG), the leading cause of irreversible blindness worldwide, disproportionately affects individuals of African ancestry. We conducted a genome-wide association study (GWAS) for POAG in 11,275 individuals of African ancestry (6,003 cases; 5,272 controls). We detected 46 risk loci associated with POAG at genome-wide significance. Replication and post-GWAS analyses, including functionally informed fine-mapping, multiple trait co-localization, and in silico validation, implicated two previously undescribed variants (rs1666698 mapping to DBF4P2; rs34957764 mapping to ROCK1P1) and one previously associated variant (rs11824032 mapping to ARHGEF12) as likely causal. For individuals of African ancestry, a polygenic risk score (PRS) for POAG from our mega-analysis (African ancestry individuals) outperformed a PRS from summary statistics of a much larger GWAS derived from European ancestry individuals. This study quantifies the genetic architecture similarities and differences between African and non-African ancestry populations for this blinding disease.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. In Brief: LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights: LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development.

Recruitment strategies and lessons learned from a large genetic study of African Americans.

Genetic studies must enroll large numbers of participants to obtain adequate statistical power. Data are needed on how researchers can best use limited financial and practical resources to achieve these targets, especially in under-represented populations. This paper provides a retrospective analysis of the recruitment strategies for a large glaucoma genetics study in African Americans. The Primary Open-Angle African American Glaucoma Genetics study enrolled 10,192 African American subjects from the Philadelphia region. Major recruitment approaches included clinic enrollment from University of Pennsylvania (UPenn) sites, clinic enrollment from external sites, sampling of Penn Medicine Biobank (PMBB), and community outreach. We calculated the enrollment yield, cost per subject, and seasonal trends of these approaches. The majority (65%) of subject were enrolled from UPenn sites with an average cost of $133/subject. Over time, monthly case enrollment declined as the pool of eligible subjects was depleted. Expanding to external sites boosted case numbers ($129/subject) and the biobank provided additional controls at low cost ($5/subject), in large part due to the generosity of PMBB providing samples free of cost. Community outreach was costly with low return on enrollment ($978/subject for 220 subjects). Summer months (Jun-Aug) produced the highest recruitment yields (p<0.001). Genetic studies will benefit from a multi-pronged and culturally sensitive recruitment approach. In our experience, the biobank was most cost-effective for control enrollment, while recruitment from clinics (including expansion to new sites) was necessary to recruit fully phenotyped cases.

Fit Factor Change on Quantitative Fit Testing of Duckbill N95 Respirators with the Use of Safety Goggles.

N95 respirators and safety goggles are important components of personal protective equipment to reduce the spread of airborne infections, such as COVID-19, among healthcare workers. Poor N95 respirator seal may reduce its protective effect, thereby increasing transmission. Quantitative fit testing is an established way of assessing the N95 respirator fit, which provides a quantitative measure for seal, called the fit factor. Duckbill N95 respirators frequently fail the fit test. We hypothesized that using safety goggles with a wraparound elastic headband will increase their fit-factor by reinforcing the seal between the face and the upper margin of the respirator. We studied the effect of safety goggles with a wraparound elastic headband (3M™ Chemical Splash Resistant Goggles, ID 70006982741) on the fit factor of two types of Duckbill N95 respirators (Halyard FLUIDSHIELD*3, Model 99SA070M, and ProShield® N95 Model TN01-11) in 63 healthy volunteers in a nonrandomized, before-and-after intervention study design. The mean fit factor increased from 69.4 to 169.1 increased from 17/63 (27%) to 46/63 (73%) after the intervention (p <0.0001, OR 3 [95% CI = 4.9-1223]). This is the first study to explore the impact of safety goggles on N95 respirator fit. We conclude that the use of safety goggles with a wraparound elastic headband increases the fit factor of the tested Duckbill N95 respirators. HOW TO CITE THIS ARTICLE: Johns M, Kyaw S, Lim R, Stewart WC, Thambiraj SR, Shehabi Y, et al. Fit Factor Change on Quantitative Fit Testing of Duckbill N95 Respirators with the Use of Safety Goggles. Indian J Crit Care Med 2021;25(9):981-986.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

Isolation of monoclonal antibodies from anti-synthetase syndrome patients and affinity maturation by recombination of independent somatic variants.

The autoimmune disease known as Jo-1 positive anti-synthetase syndrome (ASS) is characterized by circulating antibody titers to histidyl-tRNA synthetase (HARS), which may play a role in modulating the non-canonical functions of HARS. Monoclonal antibodies to HARS were isolated by single-cell screening and sequencing from three Jo-1 positive ASS patients and shown to be of high affinity, covering diverse epitope space. The immune response was further characterized by repertoire sequencing from the most productive of the donor samples. In line with previous studies of autoimmune repertoires, these antibodies tended to have long complementarity-determining region H3 sequences with more positive-charged residues than average. Clones of interest were clustered into groups with related sequences, allowing us to observe different somatic mutations in related clones. We postulated that these had found alternate structural solutions for high affinity binding, but that mutations might be transferable between clones to further enhance binding affinity. Transfer of somatic mutations between antibodies within the same clonal group was able to enhance binding affinity in a number of cases, including beneficial transfer of a mutation from a lower affinity clone into one of higher affinity. Affinity enhancement was seen with mutation transfer both between related single-cell clones, and directly from related repertoire sequences. To our knowledge, this is the first demonstration of somatic hypermutation transfer from repertoire sequences to further mature in vivo derived antibodies, and represents an additional tool to aid in affinity maturation for the development of antibodies.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

Breath-synchronized electrical stimulation of the expiratory muscles in mechanically ventilated patients: a randomized controlled feasibility study and pooled analysis.

BACKGROUND: Expiratory muscle weakness leads to difficult ventilator weaning. Maintaining their activity with functional electrical stimulation (FES) may improve outcome. We studied feasibility of breath-synchronized expiratory population muscle FES in a mixed ICU population ("Holland study") and pooled data with our previous work ("Australian study") to estimate potential clinical effects in a larger group. METHODS: Holland: Patients with a contractile response to FES received active or sham expiratory muscle FES (30 min, twice daily, 5 days/week until weaned). Main endpoints were feasibility (e.g., patient recruitment, treatment compliance, stimulation intensity) and safety. Pooled: Data on respiratory muscle thickness and ventilation duration from the Holland and Australian studies were combined (N = 40) in order to estimate potential effect size. Plasma cytokines (day 0, 3) were analyzed to study the effects of FES on systemic inflammation. RESULTS: Holland: A total of 272 sessions were performed (active/sham: 169/103) in 20 patients (N = active/sham: 10/10) with a total treatment compliance rate of 91.1%. No FES-related serious adverse events were reported. Pooled: On day 3, there was a between-group difference (N = active/sham: 7/12) in total abdominal expiratory muscle thickness favoring the active group [treatment difference (95% confidence interval); 2.25 (0.34, 4.16) mm, P = 0.02] but not on day 5. Plasma cytokine levels indicated that early FES did not induce systemic inflammation. Using a survival analysis approach for the total study population, median ventilation duration and ICU length of stay were 10 versus 52 (P = 0.07), and 12 versus 54 (P = 0.03) days for the active versus sham group. Median ventilation duration of patients that were successfully extubated was 8.5 [5.6-12.2] versus 10.5 [5.3-25.6] days (P = 0.60) for the active (N = 16) versus sham (N = 10) group, and median ICU length of stay was 10.5 [8.0-14.5] versus 14.0 [9.0-19.5] days (P = 0.36) for those active (N = 16) versus sham (N = 8) patients that were extubated and discharged alive from the ICU. During ICU stay, 3/20 patients died in the active group versus 8/20 in the sham group (P = 0.16). CONCLUSION: Expiratory muscle FES is feasible in selected ICU patients and might be a promising technique within a respiratory muscle-protective ventilation strategy. The next step is to study the effects on weaning and ventilator liberation outcome. TRIAL REGISTRATION: ClinicalTrials.gov, ID NCT03453944. Registered 05 March 2018-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03453944 .

Primary Open-Angle African American Glaucoma Genetics (POAAGG) Study: gender and risk of POAG in African Americans.

The purpose of this study was to investigate the association between gender and primary open-angle glaucoma (POAG) among African Americans and to assess demographic, systemic, and behavioral factors that may contribute to differences between genders. The Primary Open-Angle African American Glaucoma Genetics (POAAGG) study had a case-control design and included African Americans 35 years and older, recruited from the greater Philadelphia, Pennsylvania. Diagnosis of POAG was based on evidence of both glaucomatous optic nerve damage and characteristic visual field loss. Demographic and behavioral information, history of systemic diseases and anthropometric measurements were obtained at study enrollment. Gender differences in risk of POAG were examined using multivariate logistic regression. A total of 2,290 POAG cases and 2,538 controls were included in the study. The percentage of men among cases was higher than among controls (38.6% vs 30.3%, P<0.001). The subjects' mean age at enrollment was significantly higher for cases compared to controls (70.2±11.3 vs. 61.6±11.8 years, P<0.003). Cases had lower rates of diabetes (40% vs. 46%, P<0.001), higher rates of systemic hypertension (80% vs. 72%, P<0.001), and lower body mass index (BMI) (29.7±6.7 vs. 31.9±7.4, P<0.001) than controls. In the final multivariable model, male gender was significantly associated with POAG risk (OR, 1.64; 95% CI, 1.44-1.87; P<0.001), after adjusting for age, systemic hypertension, diabetes, and BMI. Within the POAAGG study, men were at higher risk of having POAG than women. Pending genetic results from this study will be used to better understand the underlying genetic variations that may account for these differences.

Abdominal functional electrical stimulation to assist ventilator weaning in critical illness: a double-blinded, randomised, sham-controlled pilot study.

BACKGROUND: For every day a person is dependent on mechanical ventilation, respiratory and cardiac complications increase, quality of life decreases and costs increase by > $USD 1500. Interventions that improve respiratory muscle function during mechanical ventilation can reduce ventilation duration. The aim of this pilot study was to assess the feasibility of employing an abdominal functional electrical stimulation (abdominal FES) training program with critically ill mechanically ventilated patients. We also investigated the effect of abdominal FES on respiratory muscle atrophy, mechanical ventilation duration and intensive care unit (ICU) length of stay. METHODS: Twenty critically ill mechanically ventilated participants were recruited over a 6-month period from one metropolitan teaching hospital. They were randomly assigned to receive active or sham (control) abdominal FES for 30 min, twice per day, 5 days per week, until ICU discharge. Feasibility was assessed through participant compliance to stimulation sessions. Abdominal and diaphragm muscle thickness were measured using ultrasound 3 times in the first week, and weekly thereafter by a blinded assessor. Respiratory function was recorded when the participant could first breathe independently and at ICU discharge, with ventilation duration and ICU length of stay also recorded at ICU discharge by a blinded assessor. RESULTS: Fourteen of 20 participants survived to ICU discharge (8, intervention; 6, control). One control was transferred before extubation, while one withdrew consent and one was withdrawn for staff safety after extubation. Median compliance to stimulation sessions was 92.1% (IQR 5.77%) in the intervention group, and 97.2% (IQR 7.40%) in the control group (p = 0.384). While this pilot study is not adequately powered to make an accurate statistical conclusion, there appeared to be no between-group thickness changes of the rectus abdominis (p = 0.099 at day 3), diaphragm (p = 0.652 at day 3) or combined lateral abdominal muscles (p = 0.074 at day 3). However, ICU length of stay (p = 0.011) and ventilation duration (p = 0.039) appeared to be shorter in the intervention compared to the control group. CONCLUSIONS: Our compliance rates demonstrate the feasibility of using abdominal FES with critically ill mechanically ventilated patients. While abdominal FES did not lead to differences in abdominal muscle or diaphragm thickness, it may be an effective method to reduce ventilation duration and ICU length of stay in this patient group. A fully powered study into this effect is warranted. TRIAL REGISTRATION: The Australian New Zealand Clinical Trials Registry, ACTRN12617001180303. Registered 9 August 2017.

Mitochondrial haplogroup L1c2 is associated with increased disease severity in African American patients with primary open-angle glaucoma.

OBJECTIVE: The purpose of this study is to evaluate the role mitochondrial inheritance plays in primary open-angle glaucoma (POAG) characteristics in African Americans. METHODS: POAG cases from the L1c2 and L1b mitochondrial haplogroups were compared in a retrospective case-case study. Twenty-six pairs of self-identified African American POAG cases from L1c2 and L1b mitochondrial haplogroups matched on age (mean [SD] = 71.2 [9.6] and 71.3 [9.6] years, respectively; p = 0.97), sex (21 female and 5 male pairs), and family history of glaucoma (positive in 15/26 [58%] pairs) were included. RESULTS: L1c2 subjects displayed higher vertical cup-to-disc ratio (0.75 [0.12] and 0.67 [0.16], respectively; p = 0.01, Bonferroni-corrected p = 0.08), worse pattern standard deviation on visual field (VF) testing (5.5 [3.5] and 3.5 [2.7]; p = 0.005, Bonferroni-corrected p = 0.02), and more severe glaucoma based on American Glaucoma Society staging criteria (p = 0.04, Bonferroni-corrected p = 0.32) compared to L1b subjects. L1c2 also trended towards worse mean deviation on VF compared to L1b (-8.2 [7.6] and -5.8 [6.8], respectively, p = 0.17). Best corrected visual acuity, central corneal thickness, maximum intraocular pressure (IOP), and cataract severity were comparable between L1c2 and L1b haplogroups (p ≥ 0.49), as was retinal nerve fiber layer thickness on optical coherence tomography (75.1 [14.1] and 75.1 [13.0]; p = 0.99). CONCLUSION: Results demonstrated worse glaucomatous cupping and more severe VF loss in the L1c2 compared to the L1b haplogroup despite comparable IOP. Findings implicate mitochondrial inheritance as a factor affecting POAG severity and may ultimately contribute to stratifying POAG patients into phenotypically and genotypically distinct subgroups.

SNP located in an AluJb repeat downstream of TMCO1, rs4657473, is protective for POAG in African Americans.

AIMS: To determine the association of single nucleotide polymorphisms (SNPs) downstream from the TMCO1 gene with primary open-angle glaucoma (POAG) in African Americans (AA). METHODS: AA subjects were recruited for the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study from the Scheie Eye Institute and its satellite sites in Philadelphia. A region containing an AluJb repeat and seven SNPs, including rs4656461 near the TMCO1 gene, were PCR-Sanger sequenced from POAAGG cases (n=1537) and controls (n=1570). Association between POAG and SNPs near TMCO1 was investigated by logistic regression analysis. Phenotypic trait associations with these SNPs were assessed by analysis of variance. Electrophoretic mobility shift assay (EMSA) was performed to assess the affinity of human T-box 5 (TBX5) protein for a predicted binding motif in the TMCO1 region. Dual Luciferase assays were performed by transfecting recombinant plasmids containing the region surrounding the above SNPs in HEK293T and trabecular meshwork cells. RESULTS: The SNP rs4657473 (C>T) was associated with POAG; the TT genotype was protective (OR 0.20, 95% CI 0.09 to 0.42; p<0.001). No significant associations were found between the TMCO1 variants and phenotypic traits. EMSA confirmed the affinity of TBX5 for a predicted binding motif containing TMCO1 SNP rs4657475. Luciferase assays demonstrated a regulatory function for the genomic region around SNP rs4656561, located within AluJb repeat. CONCLUSION: Our results demonstrate that a SNP downstream of TMCO1, rs4657473, is associated with POAG in an AA population. Our studies suggest a regulatory role for the previously POAG-associated locus near the TMCO1 gene that may affect gene expression.

The association of mitochondrial DNA haplogroups with POAG in African Americans.

Mitochondrial dysfunction has been implicated in the pathogenesis of primary open-angle glaucoma (POAG). However, the potential significance of mitochondrial DNA (mtDNA) haplogroups to POAG has not been evaluated in the overaffected African American population. To investigate the association of mtDNA haplogroups with POAG and its phenotypic characteristics, genotyping data from 4081 African American subjects (1919 cases and 2162 controls) was analyzed using 1293 positions on mtDNA. The overall frequency of mtDNA haplogroups in the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study cohort was 37% L3, 29% L2, 21% L1, 4% L0, and 10% non-African haplogroups (non-L). When all haplogroups (L0, L1, L2, and non-L) were compared against theL3 reference group, after adjusting by age and principal component of ancestry, the non-L3 haplogroups showed higher risk of POAG (OR-1.19, p = 0.02), with a particularly strong association among males (OR = 1.41, p = 0.003). More specifically the non-L group was associated with higher POAG risk than the L3 haplogroup (OR = 1.77, p = 0.007, Bonferroni adjusted p = 0.027) and to the L3e (n = 256, OR = 1.92, p = 0.007, Bonferroni adjusted p = 0.029). No significant association was found when genders were analyzed together or in female only analysis. There were no significant differences in various POAG endophenotypes across mtDNA haplogroups. This study expands our knowledge of mitochondrial genetics and mtDNA haplogroup associations in African American POAG. Further work is needed to better understand the functional role of mtDNA polymorphisms and their interactions with nuclear genes that affect POAG.

Functional classification of long non-coding RNAs by k-mer content.

The functions of most long non-coding RNAs (lncRNAs) are unknown. In contrast to proteins, lncRNAs with similar functions often lack linear sequence homology; thus, the identification of function in one lncRNA rarely informs the identification of function in others. We developed a sequence comparison method to deconstruct linear sequence relationships in lncRNAs and evaluate similarity based on the abundance of short motifs called k-mers. We found that lncRNAs of related function often had similar k-mer profiles despite lacking linear homology, and that k-mer profiles correlated with protein binding to lncRNAs and with their subcellular localization. Using a novel assay to quantify Xist-like regulatory potential, we directly demonstrated that evolutionarily unrelated lncRNAs can encode similar function through different spatial arrangements of related sequence motifs. K-mer-based classification is a powerful approach to detect recurrent relationships between sequence and function in lncRNAs.

The MT-CO1 V83I Polymorphism is a Risk Factor for Primary Open-Angle Glaucoma in African American Men.

Purpose: We investigate the function of the V83I polymorphism (m.6150G>A, rs879053914) in the mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) gene and its role in African American (AA) primary open-angle glaucoma (POAG). Methods: This study used Sanger sequencing (1339 cases, 850 controls), phenotypic characterization of Primary Open-Angle African American Glaucoma Genetics study (POAAGG) cases, a masked chart review of CO1 missense cases (V83I plus M117T, n = 29) versus wild type cases (n = 29), a yeast 2-hybrid (Y2H) cDNA library screen, and quantification of protein-protein interactions by Y2H and ELISA. Results: The association of V83I with POAG in AA was highly significant for men (odds ratio [OR] 6.5; 95% confidence interval [CI] 2.0-21.3, P = 0.0001), but not for women (OR 1.1; 95% CI, 0.62-2.00, P = 0.78). POAG cases having CO1 double missense mutation (V83I + M117T, L1c2 haplogroup) had a higher cup-to-disc ratio (0.77 vs. 0.71, P = 0.04) and significantly worse visual function (average pattern standard deviation, 6.5 vs. 4.3, P = 0.009; average mean deviation -10.4 vs. -4.5, P = 0.006) when compared to matched wild type cases (L1b haplogroup). Interaction of the V83I region of CO1 with amyloid beta peptide (Aß) was confirmed by ELISA assay, and this interaction was abrogated by V83I. A Y2H screen of an adult human brain cDNA library with the V83 region of CO1 as bait retrieved the UBQLN1 gene. Conclusions: The V83I polymorphism was associated strongly with POAG in AA men and disrupts Aß-binding to CO1. This region also interacts with a neuroprotective protein, UBQLN1.

Characterizing the "POAGome": A bioinformatics-driven approach to primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is a genetically, physiologically, and phenotypically complex neurodegenerative disorder. This study addressed the expanding collection of genes associated with POAG, referred to as the "POAGome." We used bioinformatics tools to perform an extensive, systematic literature search and compiled 542 genes with confirmed associations with POAG and its related phenotypes (normal tension glaucoma, ocular hypertension, juvenile open-angle glaucoma, and primary congenital glaucoma). The genes were classified according to their associated ocular tissues and phenotypes, and functional annotation and pathway analyses were subsequently performed. Our study reveals that no single molecular pathway can encompass the pathophysiology of POAG. The analyses suggested that inflammation and senescence may play pivotal roles in both the development and perpetuation of the retinal ganglion cell degeneration seen in POAG. The TGF-ß signaling pathway was repeatedly implicated in our analyses, suggesting that it may be an important contributor to the manifestation of POAG in the anterior and posterior segments of the globe. We propose a molecular model of POAG revolving around TGF-ß signaling, which incorporates the roles of inflammation and senescence in this disease. Finally, we highlight emerging molecular therapies that show promise for treating POAG.

Risk Factors Associated with Progression to Blindness from Primary Open-Angle Glaucoma in an African-American Population.

PURPOSE: To determine the risk factors associated with progression to blindness from primary open-angle glaucoma (POAG) in an African-American population. METHODS: This study examined 2119 patients enrolled in the Primary Open-Angle African-American Glaucoma Genetics (POAAGG) study. A total of 59 eyes were identified as legally blind as a result of POAG (cases) and were age-and sex-matched to 59 non-blind eyes with glaucoma (controls). Chart reviews were performed to record known and suspected risk factors. RESULTS: Cases were diagnosed with POAG at an earlier age than controls (p = 0.005). Of the 59 eyes of cases, 16 eyes (27.1%) presented with blindness at diagnosis. Cases had worse visual acuity (VA) at diagnosis (p < 0.0001), with VA worse than 20/40 conferring a 27 times higher risk of progression to blindness (p = 0.0005). Blind eyes also demonstrated more visual field defects (p = 0.01), higher pre-treatment intraocular pressure (IOP; p < 0.0001), and higher cup-to-disc ratio (p = 0.006) at diagnosis. IOP was less controlled in cases, and those with IOP ≥21 mmHg at more than 20% of follow-up visits were 73 times more likely to become blind (p < 0.0001). Cases missed a greater number of appointments per year (p = 0.003) and had non-adherence issues noted in their charts more often than controls (p = 0.03). However, other compliance data did not significantly differ between groups. CONCLUSION: Access to care, initial VA worse than 20/40, and poor control of IOP were the major risk factors associated with blindness from POAG. Future studies should examine earlier, more effective approaches to glaucoma screening as well as the role of genetics in these significantly younger patients who progress to blindness.

Association of primary open-angle glaucoma with mitochondrial variants and haplogroups common in African Americans.

PURPOSE: To estimate the population frequencies of all common mitochondrial variants and ancestral haplogroups among 1,999 subjects recruited for the Primary Open-Angle African American Glaucoma Genetics (POAAGG) Study, including 1,217 primary open-angle glaucoma (POAG) cases and 782 controls, and to identify ancestral subpopulations and mitochondrial mutations as potential risk factors for POAG susceptibility. METHODS: Subject classification by characteristic glaucomatous optic nerve findings and corresponding visual field defects, as defined by enrolling glaucoma specialists, stereo disc photography, phlebotomy, extraction of total DNA from peripheral blood or saliva, DNA quantification and normalization, PCR amplification of whole mitochondrial genomes, Ion Torrent deep semiconductor DNA sequencing on DNA pools ("Pool-seq"), Sanger sequencing of 3,479 individual mitochondrial DNAs, and bioinformatic analysis. RESULTS: The distribution of common African haplogroups within the POAAGG study population was broadly similar to prior surveys of African Americans. However, the POAG case population was found to be enriched in L1c2 haplogroups, which are defined in part by missense mutations m.6150G>A (Val83Ile, odds ratio [OR] 1.8, p=0.01), m.6253C>T (Met117Thr, rs200165736, OR 1.6, p=0.04), and m.6480G>A (Val193Ile, rs199476128, OR 4.6, p=0.04) in the cytochrome c oxidase subunit 1 (MT-CO1) gene and by a variant, m.2220A>G (OR 2.0, p=0.01), in MT-RNR2, which encodes the mitochondrial ribosomal 16s RNA gene. L2 haplogroups were predicted to be overrepresented in the POAG case population by Pool-seq, and the difference was confirmed to be significant with Sanger sequencing, that targeted the L2-associated variants m.2416T>C (rs28358580, OR 1.2, p=0.02) and m.2332C>T (OR 1.2, p=.02) in MT-RNR2. Another variant within MT-RNR2, m.3010G>A (rs3928306), previously implicated in sensitivity to the optic neuropathy-associated antibiotic linezolid, and arising on D4 and J1 lineages, associated with Leber hereditary optic neuropathy (LHON) severity, was confirmed to be common (>5%) but was not significantly enriched in the POAG cases. Two variants linked to the composition of the gut microbiome, m.15784T>C (rs527236194, haplogroup L2a1) and m.16390G>A (rs41378955, L2 haplogroups), were also enriched in the case DNA pools. CONCLUSIONS: These results implicate African mtDNA haplogroups L1c2, L1c2b, and L2 as risk factors for POAG. Approximately one in four African Americans have these mitochondrial ancestries, which may contribute to their elevated glaucoma risk. These haplogroups are defined in part by ancestral variants in the MT-RNR2 and/or MT-CO1 genes, several of which have prior disease associations, such as MT-CO1 missense variants that have been implicated in prostate cancer.

Saliva DNA quality and genotyping efficiency in a predominantly elderly population.

BACKGROUND: The question of whether DNA obtained from saliva is an acceptable alternative to DNA from blood is a topic of considerable interest for large genetics studies. We compared the yields, quality and performance of DNAs from saliva and blood from a mostly elderly study population. METHODS: Two thousand nine hundred ten DNAs from primarily elderly subjects (mean age ± standard deviation (SD): 65 ± 12 years), collected for the Primary Open-Angle African-American Glaucoma Genetics (POAAGG) study, were evaluated by fluorometry and/or spectroscopy. These included 566 DNAs from blood and 2344 from saliva. Subsets of these were evaluated by Sanger sequencing (n = 1555), and by microarray SNP genotyping (n = 94) on an Illumina OmniExpress bead chip platform. RESULTS: The mean age of subjects was 65, and 68 % were female in both the blood and saliva groups. The mean ± SD of DNA yield per ml of requested specimen was significantly higher for saliva (17.6 ± 17.8 µg/ml) than blood (13.2 ± 8.5 µg/ml), but the mean ± SD of total DNA yield obtained per saliva specimen (35 ± 36 µg from 2 ml maximum specimen volume) was approximately three-fold lower than from blood (106 ± 68 µg from 8 ml maximum specimen volume). The average genotyping call rates were >99 % for 43 of 44 saliva DNAs and >99 % for 50 of 50 for blood DNAs. For 22 of 23 paired blood and saliva samples from the same individuals, the average genotyping concordance rate was 99.996 %. High quality PCR Sanger sequencing was obtained from ≥ 98 % of blood (n = 297) and saliva (n = 1258) DNAs. DNA concentrations ≥10 ng/µl, corresponding to total yields ≥ 2 µg, were obtained for 94 % of the saliva specimens (n = 2344). CONCLUSIONS: In spite of inferior purity, the performance of saliva DNAs for microarray genotyping was excellent. Our results agree with other studies concluding that saliva collection is a viable alternative to blood. The potential to boost study enrollments and reduce subject discomfort is not necessarily offset by a reduction in genotyping efficiency. Saliva DNAs performed comparably to blood DNAs for PCR Sanger sequencing.