The Phenotypic Spectrum of COL4A3 Heterozygotes.

Introduction: The penetrance and phenotypic spectrum of autosomal dominant Alport Syndrome (ADAS), affecting 1 in 106, remains understudied. Methods: Using data from 174,418 participants in the Geisinger MyCode/DiscovEHR study, an unselected health system-based cohort with whole exome sequencing, we identified 403 participants who were heterozygous for likely pathogenic COL4A3 variants. Phenotypic data was evaluated using International Classification of Diseases (ICD) codes, laboratory data, and chart review. To evaluate the phenotypic spectrum of genetically-determined ADAS, we matched COL4A3 heterozygotes 1:5 to nonheterozygotes using propensity scores by demographics, hypertension, diabetes, and nephrolithiasis. Results: COL4A3 heterozygotes were at significantly increased risks of hematuria, decreased estimated glomerular filtration rate (eGFR), albuminuria, and kidney failure (P < 0.05 for all comparisons) but not bilateral sensorineural hearing loss (P = 0.9). Phenotypic severity was more severe for collagenous domain glycine missense variants than protein truncating variants (PTVs). For example, patients with Gly695Arg (n = 161) had markedly increased risk of dipstick hematuria (odds ratio [OR] 9.50; 95% confidence interval [CI]: 6.32, 14.28) and kidney failure (OR 7.02; 95% CI: 3.48, 14.16) whereas those with PTVs (n = 119) had moderately increased risks of dipstick hematuria (OR 1.64; 95% CI: 1.03, 2.59) and kidney failure (OR 3.44; 95% CI: 1.28, 9.22). Less than a third of patients had albuminuria screening completed, and fewer than 1 of 3 were taking inhibitors of the renin-angiotensin-aldosterone system. Conclusion: This study demonstrates a wide spectrum of phenotypic severity in ADAS due to COL4A3 with phenotypic variability by genotype. Future studies are needed to evaluate the impact of earlier diagnosis, appropriate evaluation, and treatment of ADAS.

The Phenotypic Spectrum of COL4A3 Heterozygotes.

Most data on Alport Syndrome (AS) due to COL4A3 are limited to families with autosomal recessive AS or severe manifestations such as focal segmental glomerulosclerosis (FSGS). Using data from 174,418 participants in the Geisinger MyCode/DiscovEHR study, an unselected health system-based cohort with whole exome sequencing, we identified 403 participants (0.2%) who were heterozygous for likely pathogenic COL4A3 variants. Phenotypic data was evaluated using International Classification of Diseases (ICD) codes, laboratory data, and chart review. To evaluate the phenotypic spectrum of genetically-determined autosomal dominant AS, we matched COL4A3 heterozygotes 1:5 to non-heterozygotes using propensity scores by demographics, hypertension, diabetes, and nephrolithiasis. COL4A3 heterozygotes were at significantly increased risks of hematuria, decreased estimated glomerular filtration rate (eGFR), albuminuria, and end-stage kidney disease (ESKD) (p<0.05 for all comparisons) but not bilateral sensorineural hearing loss (p=0.9). Phenotypic severity tended to be more severe among patients with glycine missense variants located within the collagenous domain. For example, patients with Gly695Arg (n=161) had markedly increased risk of dipstick hematuria (OR 9.47, 95% CI: 6.30, 14.22) and ESKD diagnosis (OR 7.01, 95% CI: 3.48, 14.12) whereas those with PTVs (n=119) had moderately increased risks of dipstick hematuria (OR 1.63, 95% CI: 1.03, 2.58) and ESKD diagnosis (OR 3.43, 95% CI: 1.28, 9.19). Less than a third of patients had albuminuria screening completed, and fewer than 1/3 were taking inhibitors of the renin-angiotensin-aldosterone system (RAASi). Future studies are needed to evaluate the impact of earlier diagnosis, appropriate evaluation, and treatment of ADAS.

Exome Sequencing of a Clinical Population for Autosomal Dominant Polycystic Kidney Disease.

Importance: Most studies of autosomal dominant polycystic kidney disease (ADPKD) genetics have used kidney specialty cohorts, focusing on PKD1 and PKD2. These can lead to biased estimates of population prevalence of ADPKD-associated gene variants and their phenotypic expression. Objective: To determine the prevalence of ADPKD and contributions of PKD1, PKD2, and other genes related to cystic kidney disease in a large, unselected cohort. Design, Setting, and Participants: This retrospective observational study used an unselected health system-based cohort in central and northeast Pennsylvania with exome sequencing (enrolled from 2004 to 2020) and electronic health record data (up to October 2021). The genotype-first approach included the entire cohort and the phenotype-first approach focused on patients with ADPKD diagnosis codes, confirmed by chart and imaging review. Exposures: Loss-of-function (LOF) variants in PKD1, PKD2, and other genes associated with cystic kidney disease (ie, ALG8, ALG9, DNAJB11, GANAB, HNF1B, IFT140, SEC61B, PKHD1, PRKCSH, SEC63); likely pathogenic missense variants in PKD1 and PKD2. Main Outcomes and Measures: Genotype-first analysis: ADPKD diagnosis code (Q61.2, Q61.3, 753.13, 753.12); phenotype-first analysis: presence of a rare variant in PKD1, PKD2, or other genes associated with cystic kidney disease. Results: Of 174 172 patients (median age, 60 years; 60.6% female; 93% of European ancestry), 303 patients had ADPKD diagnosis codes, including 235 with sufficient chart review data for confirmation. In addition to PKD1 and PKD2, LOF variants in IFT140, GANAB, and HNF1B were associated with ADPKD diagnosis after correction for multiple comparisons. Among patients with LOF variants in PKD1, 66 of 68 (97%) had ADPKD; 43 of 43 patients (100%) with LOF variants in PKD2 had ADPKD. In contrast, only 24 of 77 patients (31.2%) with a PKD1 missense variant previously classified as "likely pathogenic" had ADPKD, suggesting misclassification or variable penetrance. Among patients with ADPKD diagnosis confirmed by chart review, 180 of 235 (76.6%) had a potential genetic cause, with the majority being rare variants in PKD1 (127 patients) or PKD2 (34 patients); 19 of 235 (8.1%) had variants in other genes associated with cystic kidney disease. Of these 235 patients with confirmed ADPKD, 150 (63.8%) had a family history of ADPKD. The yield for a genetic determinant of ADPKD was higher for those with a family history of ADPKD compared with those without family history (91.3% [137/150] vs 50.6% [43/85]; difference, 40.7% [95% CI, 29.2%-52.3%]; P < .001). Previously unreported PKD1, PKD2, and GANAB variants were identified with pedigree data suggesting pathogenicity, and several PKD1 missense variants previously reported as likely pathogenic appeared to be benign. Conclusions and Relevance: This study demonstrates substantial genetic and phenotypic variability in ADPKD among patients within a regional health system in the US.

Large scale clinical exome sequencing uncovers the scope and severity of skin disorders associated with MC1R genetic variants.

PURPOSE: Genetic variation in MC1R is a main determinant of red hair color (RHC) phenotype and confers susceptibility to skin disorders. METHODS: We assessed the effects and function of MC1R variants identified in our clinical cohort of 135,947 participants with available exome sequencing using phenome-wide association scan (PheWAS). Expression and function of several variants were evaluated. RESULTS: We found 24 nonsense and 215 missense variants in MC1R. Many common missense MC1R variants are strongly associated with skin disorders including skin cancer; however, each variant shows different penetrance and expressivity. Severity of skin phenotype was well correlated with the magnitude of functional defect measured as receptor expression and α-MSH stimulated cAMP production. Remarkably, MC1R deletions and nonsense variants are only weakly associated with milder skin phenotypes. CONCLUSION: Our comprehensive assessment of all MC1R variants in a large cohort clearly establish that individuals with some missense variants are more susceptible to severe skin disorders than those with MC1R deletions or nonsense variants.

Electronic health record analysis identifies kidney disease as the leading risk factor for hospitalization in confirmed COVID-19 patients.

BACKGROUND: Empirical data on conditions that increase risk of coronavirus disease 2019 (COVID-19) progression are needed to identify high risk individuals. We performed a comprehensive quantitative assessment of pre-existing clinical phenotypes associated with COVID-19-related hospitalization. METHODS: Phenome-wide association study (PheWAS) of SARS-CoV-2-positive patients from an integrated health system (Geisinger) with system-level outpatient/inpatient COVID-19 testing capacity and retrospective electronic health record (EHR) data to assess pre-COVID-19 pandemic clinical phenotypes associated with hospital admission (hospitalization). RESULTS: Of 12,971 individuals tested for SARS-CoV-2 with sufficient pre-COVID-19 pandemic EHR data at Geisinger, 1604 were SARS-CoV-2 positive and 354 required hospitalization. We identified 21 clinical phenotypes in 5 disease categories meeting phenome-wide significance (P<1.60x10-4), including: six kidney phenotypes, e.g. end stage renal disease or stage 5 CKD (OR = 11.07, p = 1.96x10-8), six cardiovascular phenotypes, e.g. congestive heart failure (OR = 3.8, p = 3.24x10-5), five respiratory phenotypes, e.g. chronic airway obstruction (OR = 2.54, p = 3.71x10-5), and three metabolic phenotypes, e.g. type 2 diabetes (OR = 1.80, p = 7.51x10-5). Additional analyses defining CKD based on estimated glomerular filtration rate, confirmed high risk of hospitalization associated with pre-existing stage 4 CKD (OR 2.90, 95% CI: 1.47, 5.74), stage 5 CKD/dialysis (OR 8.83, 95% CI: 2.76, 28.27), and kidney transplant (OR 14.98, 95% CI: 2.77, 80.8) but not stage 3 CKD (OR 1.03, 95% CI: 0.71, 1.48). CONCLUSIONS: This study provides quantitative estimates of the contribution of pre-existing clinical phenotypes to COVID-19 hospitalization and highlights kidney disorders as the strongest factors associated with hospitalization in an integrated US healthcare system.

ALG9 Mutation Carriers Develop Kidney and Liver Cysts.

BACKGROUND: Mutations in PKD1 or PKD2 cause typical autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic kidney disease. Dominantly inherited polycystic kidney and liver diseases on the ADPKD spectrum are also caused by mutations in at least six other genes required for protein biogenesis in the endoplasmic reticulum, the loss of which results in defective production of the PKD1 gene product, the membrane protein polycystin-1 (PC1). METHODS: We used whole-exome sequencing in a cohort of 122 patients with genetically unresolved clinical diagnosis of ADPKD or polycystic liver disease to identify a candidate gene, ALG9, and in vitro cell-based assays of PC1 protein maturation to functionally validate it. For further validation, we identified carriers of ALG9 loss-of-function mutations and noncarrier matched controls in a large exome-sequenced population-based cohort and evaluated the occurrence of polycystic phenotypes in both groups. RESULTS: Two patients in the clinically defined cohort had rare loss-of-function variants in ALG9, which encodes a protein required for addition of specific mannose molecules to the assembling N-glycan precursors in the endoplasmic reticulum lumen. In vitro assays showed that inactivation of Alg9 results in impaired maturation and defective glycosylation of PC1. Seven of the eight (88%) cases selected from the population-based cohort based on ALG9 mutation carrier state who had abdominal imaging after age 50; seven (88%) had at least four kidney cysts, compared with none in matched controls without ALG9 mutations. CONCLUSIONS: ALG9 is a novel disease gene in the genetically heterogeneous ADPKD spectrum. This study supports the utility of phenotype characterization in genetically-defined cohorts to validate novel disease genes, and provide much-needed genotype-phenotype correlations.

Genetic variants help define the role of the MC4R C-terminus in signaling and cell surface stability.

Screening 92,445 subjects in the Geisinger-Regeneron DiscovEHR cohort, we identified 5 patients heterozygous for nonsense mutations causing early terminations at Glu307 or Leu328 on the C-terminus of melanocortin 4 receptor (MC4R). Two Q307Ter carriers are severely obese (BMI > 40), while one is overweight (BMI > 25). One L328Ter carrier is overweight and the other is lean. Pedigree analysis for two Q307Ter carriers shows segregation of the variant with higher BMI. Functionally, MC4R(Q307Ter) eliminated receptor surface expression and signaling, while MC4R(L328Ter) functioned like the wild-type receptor. MC4R(Q307Ter) is therefore a loss of function (LOF) variant and the region between the two truncation sites identified in our patients is critical to MC4R function. Truncating MC4R at various C-terminal positions between these two variant sites, we find that cysteine318 sits at a critical junction for receptor trafficking and function. We show that MC4R is lipid modified at cysteine318 and cysteine319. Therefore, truncation early in the MC4R C-terminus results in haploinsufficiency in humans while truncation after the first lipid-modification site is well tolerated. MC4R haploinsufficiency clearly segregates with higher BMI; however, severe obesity is not fully penetrant even in MC4R LOF carriers, suggesting critical roles for environmental and lifestyle factors in MC4R monogenic obesity.

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics.

Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP3), not stimulatory G protein (Gαs), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gαi/o) signaling but rather Ca2+ entry through a Gd3+-sensitive channel. Therefore, PIP3 sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca2+ entry.

Long-term weight-loss in gastric bypass patients carrying melanocortin 4 receptor variants.

BACKGROUND: The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in MC4R are predominantly found in obese individuals. Though some rare variants in MC4R discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects. SUBJECTS/METHODS: In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of MC4R. We matched rare variant carriers to patients with the MC4R reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. In vitro, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy. RESULTS: While carrying a rare MC4R allele is associated with obesity, carriers of rare variants exhibited comparable weight-loss after RYGB to non-carriers. However, subjects carrying three of these variants, V95I, I137T or L250Q, lost less weight after surgery. In vitro, the R305Q mutation caused a defect in cell surface expression while only the I137T and C326R mutations showed impaired cAMP signaling. Despite these apparent differences, there was no correlation between in vitro signaling and pre- or post-surgery clinical phenotype. CONCLUSIONS: These data suggest that subtle differences in receptor signaling conferred by rare MC4R variants combined with additional factors predispose carriers to obesity. In the absence of complete MC4R deficiency, these differences can be overcome by the powerful weight-reducing effects of bariatric surgery. In a complex disorder such as obesity, genetic variants that cause subtle defects that have cumulative effects can be overcome after appropriate clinical intervention.

A conserved mechanism for gating in an ionotropic glutamate receptor.

Ionotropic glutamate receptor (iGluR) channels control synaptic activity. The crystallographic structure of GluA2, the prototypical iGluR, reveals a clamshell-like ligand-binding domain (LBD) that closes in the presence of glutamate to open a gate on the pore lining α-helix. How LBD closure leads to gate opening remains unclear. Here, we show that bending the pore helix at a highly conserved alanine residue (Ala-621) below the gate is responsible for channel opening. Substituting Ala-621 with the smaller more flexible glycine resulted in a basally active, nondesensitizing channel with ∼39-fold increase in glutamate potency without affecting surface expression or binding. On GluA2(A621G), the partial agonist kainate showed efficacy similar to a full agonist, and competitive antagonists CNQX and DNQX acted as a partial agonists. Met-629 in GluA2 sits above the gate and is critical in transmitting LBD closure to the gate. Substituting Met-629 with the flexible glycine resulted in reduced channel activity and glutamate potency. The pore regions in potassium channels are structurally similar to iGluRs. Whereas potassium channels typically use glycines as a hinge for gating, iGluRs use the less flexible alanine as a hinge at a similar position to maintain low basal activity allowing for ligand-mediated gating.

Isolation, characterization, and DNA binding kinetics of three dirhodium(II,II) carboxyamidate complexes: Rh2(µ-L)(HNOCCF3)3 where L= [OOCCH3]-, [OOCCF3]-, or [HNOCCF3]-.

Several transition metal compounds are effective antitumor drugs whose biological activity can be attributed to their ability to bind deoxyribonucleic acid (DNA). In this study, DNA-binding experiments reveal that changing one bridging ligand on compounds with the general formula Rh(2)(µ-L)(HNOCCF(3))(3) alters the rate of DNA-binding by greater than 100-fold with µ-L = trifluoroacetate ≫ acetate > trifluoroacetamidate. These three dirhodium compounds are isolated as the major products of the reaction between Rh(2)(OOCCH(3))(4) and trifluoroacetamide in either refluxing chlorobenzene or molten trifluoroacetamide and have been characterized by NMR and LC/MS. By using (15)N-enriched trifluoroacetamide, NMR spectroscopy was used to assign the cis-(2,1) orientations of Rh(2)(µ-L)(HNOCCF(3))(3) compounds where µ-L = trifluoroacetate or acetate. This is the first report of Rh(2)(OOCCF(3))(HNOCCF(3))(3), a novel compound that may play a significant role in the biological and/or catalytic activity of compound mixtures commonly isolated as "Rh(2)(HNOCCF(3))(4)".