Qais Zawaydeh

Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations.

Inherited retinal degenerations (IRDs) are a group of genetically heterogeneous conditions with a broad phenotypic heterogeneity. Here, we report detection and validation of the underlying cause of progressive retinal degeneration in a nuclear family of European descent with a single affected individual. Whole genome sequencing of the proband and her unaffected sibling identified a novel intron 8 donor splice site variant (c.1296 + 1G>A) and a novel 731 base pair deletion encompassing exon 9 (Chr2:g.112751488_112752218 del) resulting in c.1297_1451del; p.K433_G484fsTer3 in the Mer tyrosine kinase protooncogene (MERTK), which is highly expressed in the retinal pigment epithelium (RPE). The proband carried both variants in the heterozygous state, which segregated with disease in the pedigree. These MERTK variants are predicted to result in the defective splicing of exon 8 and loss of exon 9 respectively. To evaluate the impact of these novel variants, peripheral blood mononuclear cells of the proband and her parents were reprogrammed to humaninduced pluripotent stem cell (hiPSC) lines, which were subsequently differentiated to hiPSC-RPE. Analysis of the proband's hiPSC-RPE revealed the absence of both MERTK transcript and its respective protein as well as abnormal phagocytosis when compared with the parental hiPSC-RPE. In summary, whole genome sequencing identified novel compound heterozygous variants in MERTK as the underlying cause of progressive retinal degeneration in a simplex case. Further, analysis using an hiPSC-RPE model established the functional impact of novel MERTK mutations and revealed the potential mechanism underlying pathology in the proband.

Introduction: The COVID pandemic has led to devastating consequences for those afflicted. The use of dexamethasone has proven benefit, although the risks and benefits are not fully characterized nor is the optimal dose of steroid. The benefits of steroids may include reduced inflammation in patients with acute respiratory distress syndrome (ARDS), but the risk might include increased viral replication and/or risk of secondary infection. Using a retrospective review of our ICU experience (prior to RECOVERY NEJM publication) we sought to characterize the relationship between steroid use and secondary infections in COVID-19 patients in order to test the hypothesis that steroid dose may be associated with worse ICU outcomes in some patients. Methods: We conducted a retrospective review of our ICU experience from a large academic medical center. We defined secondary infection as positive bacterial or fungal culture from blood stream, urine or sputum deemed by the clinical team to require intervention. Among 130 consecutive patients who received critical care for COVID-19, we identified 41 who had received steroids in the ICU. Among these, 13 had received steroids prior to secondary infection (GP1) whereas 13 had secondary infection documented prior to receiving steroid (GP2) and 15 received steroids without secondary infection (GP3). Results: We observed mortality in all patients involved in this study (n=130) of 27.7%. Among patients who received steroids (n=41) of 29.3% vs. patients who never received steroids (n=89) of 27%. Mortality specific to the steroids groups was 15.4% for GP1 (n=13) vs. 53.8% for GP2 (n=13) vs. 20% for GP3 (n=15). Conclusions: We did not observe an impact of steroid dosage on the risk of secondary infection in a critically ill cohort of COVID-19 patients. We did observe a high mortality among patients who received steroids following documented secondary infection, but further work will be required to determine if this finding reflects confounding by indication i.e. whether steroids are a marker of sicker patients. Optimal steroid dosage in COVID remains unclear, but higher doses (as were given in the DExa-ARDS study) should be rigorously studied.

Rationale: Prone positioning has proven benefit but is generally underutilized in moderate to severe Acute Respiratory Disease Syndrome (ARDS). Common practice includes administering neuromuscular blockade [paralytic medication] when proning this population, despite equivocal data regarding benefits of routine paralysis in ARDS. During the COVID19 pandemic, drug shortages and other factors have driven clinicians to prone patients without therapeutic paralysis. Recent publications also suggest prone positioning may be beneficial for non-intubated (non-paralyzed patients). We hypothesized that intubated patients with COVID19-related ARDS can be safely proned without therapeutic paralysis and would experience similar improvement in gas exchange (as evidenced by improved PF [Pa02/Fi02] and SF [Sp02/Fi02] ratios). Methods: Observational data on ICU patients were collected at a large tertiary university hospital from March to September 2020. Clinical teams directed use of sedatives, paralytics, mechanical ventilation settings, and timing of prone vs. supine positioning and blood draws. Comparison of demographics, gas exchange in supine and prone positions (within the same subjects), as well as oxygen requirements, ventilator settings, adverse events and hemodynamic parameters were analyzed using descriptive statistics. Results: Among our cohort of 156 patients, a total of 15 patients were identified that changed from supine to prone position with and without the use of paralytics while in the ICU. All 15 patients had moderate to severe ARDS per the Berlin Criteria, secondary to COVID19. PF and SF ratios were compared in the same patient before and after proning with and without paralytics (within subject comparisons). All groups had an improvement in both average PF ratio (34.7% with and 24.0% without paralysis, p=0.52) and average SF ratio (5.2% with and 6.1% without paralysis, p=0.92) after proning (Figure 1). There were no serious adverse events associated with prone positioning with or without paralytics. Conclusions: When proning patients with COVID19 ARDS, we found no statistically significant difference to support routine use of paralytics when proning. Safety and efficacy were similar when COVID19 patients were being proned with or without neuromuscular blockade. Use of paralytic medication should be an individualized decision rather than given routinely. Larger randomized controlled trials comparing proning with or without paralysis are needed to identify the optimal approach in this population.