Pathogenic variants in SHROOM3 associated with hemifacial microsomia
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Mechanisms of NLRP3 activation and inhibition elucidated by functional analysis of disease-associated variants
The NLRP3 inflammasome is a multiprotein complex that mediates caspase-1 activation and the release of proinflammatory cytokines, including interleukin (IL)-1β and IL-18. Gain-of-function variants in the gene encoding NLRP3 (also called cryopyrin) lead to constitutive inflammasome activation and excessive IL-1β production in cryopyrin-associated periodic syndromes (CAPS). Here we present functional screening and automated analysis of 534 NLRP3 variants from the international INFEVERS registry and the ClinVar database. This resource captures the effect of NLRP3 variants on ASC speck formation spontaneously, at low temperature, after inflammasome stimulation and with the specific NLRP3 inhibitor MCC950. Most notably, our analysis facilitated the updated classification of NLRP3 variants in INFEVERS. Structural analysis suggested multiple mechanisms by which CAPS variants activate NLRP3, including enhanced ATP binding, stabilizing the active NLRP3 conformation, destabilizing the inactive NLRP3 complex and promoting oligomerization of the pyrin domain. Furthermore, we identified pathogenic variants that can hypersensitize the activation of NLRP3 in response to nigericin and cold temperature exposure. We also found that most CAPS-related NLRP3 variants can be inhibited by MCC950; however, NLRP3 variants with changes to proline affecting helices near the inhibitor binding site are resistant to MCC950, as are variants in the pyrin domain, which likely trigger activation directly with the pyrin domain of ASC. Our findings could help stratify the CAPS population for NLRP3 inhibitor clinical trials and our automated methodologies can be implemented for molecules with a different mechanism of activation and in laboratories worldwide that are interested in adding new functionally validated NLRP3 variants to the resource. Overall, our study provides improved diagnosis for patients with CAPS, mechanistic insight into the activation of NLRP3 and stratification of patients for the future application of targeted therapeutics.
Discordance between a deep learning model and clinical-grade variant pathogenicity classification in a rare disease cohort
Genetic testing is essential for diagnosing and managing clinical conditions, particularly rare Mendelian diseases. Although efforts to identify rare phenotype-associated variants have focused on protein-truncating variants, interpreting missense variants remains challenging. Deep learning algorithms excel in various biomedical tasks1,2, yet distinguishing pathogenic from benign missense variants remains elusive3,4,5. Our investigation of AlphaMissense (AM)5, a deep learning tool for predicting the potential functional impact of missense variants and assessing gene essentiality, reveals limitations in identifying pathogenic missense variants over 45 rare diseases, including very early onset inflammatory bowel disease. For the expert-curated pathogenic variants identified in our cohort, AM’s precision was 32.9%, and recall was 57.6%. Notably, AM struggles to evaluate pathogenicity in intrinsically disordered regions (IDRs), resulting in unreliable gene-level essentiality scores for genes containing IDRs. This observation underscores ongoing challenges in clinical genetics, highlighting the need for continued refinement of computational methods in variant pathogenicity prediction.
The genetic landscape of autism spectrum disorder in an ancestrally diverse cohort
Autism spectrum disorder (ASD) comprises neurodevelopmental disorders with wide variability in genetic causes and phenotypes, making it challenging to pinpoint causal genes. We performed whole exome sequencing on a modest, ancestrally diverse cohort of 195 families, including 754 individuals (222 with ASD), and identified 38,834 novel private variants. In 68 individuals with ASD (~30%), we identified 92 potentially pathogenic variants in 73 known genes, including BCORL1, CDKL5, CHAMP1, KAT6A, MECP2, and SETD1B. Additionally, we identified 158 potentially pathogenic variants in 120 candidate genes, including DLG3, GABRQ, KALRN, KCTD16, and SLC8A3. We also found 34 copy number variants in 31 individuals overlapping known ASD loci. Our work expands the catalog of ASD genetics by identifying hundreds of variants across diverse ancestral backgrounds, highlighting convergence on nervous system development and signal transduction. These findings provide insights into the genetic underpinnings of ASD and inform molecular diagnosis and potential therapeutic targets.
Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency
Somatic variants accumulate in non-malignant tissues with age. Functional variants, leading to clonal advantage of hepatocytes, accumulate in the liver of patients with acquired chronic liver disease (CLD). Whether somatic variants are common to CLD from differing etiologies is unknown. We analyzed liver somatic variants in patients with genetic CLD from alpha-1 antitrypsin (A1AT) deficiency or hemochromatosis. We show that somatic variants in SERPINA1, the gene encoding A1AT, are strongly selected for in A1AT deficiency, with evidence of convergent evolution. Acquired SERPINA1 variants are clustered at the carboxyl terminus of A1AT, leading to truncation. In vitro and in vivo, C-terminal truncation variants reduce disease-associated Z-A1AT polymer accumulation and disruption of the endoplasmic reticulum, supporting the C-terminal domain swap mechanism. Therefore, somatic escape variants from a deleterious germline variant are selected for in A1AT deficiency, suggesting that functional somatic variants are disease-specific in CLD and point to disease-associated mechanisms.
Genotype of PAX2-related disorders correlates with kidney and ocular manifestations
PAX2-related disorders encompass renal coloboma syndrome (RCS) and hereditary focal segmental glomerulosclerosis (FSGS) type 7. We retrospectively analyzed 27 Korean patients with PAX2 pathogenic variants detected between 2004 and 2022 and conducted a literature review of 328 cases, including 301 previously reported. In our cohort, 19 had RCS, 4 had FSGS, and 4 had isolated congenital anomalies of the kidneys and urinary tract. Patients were classified by variant type into predicted loss of function (pLoF) and non-pLoF variant groups, and by variant location into paired domain and other sites group. pLoF variants were predominantly associated with RCS, observed in 82% of patients in both our data (18 of 22, P = 0.017) and the literature (140 of 171, P < 0.001). Kidney failure developed in 52% of Korean patients at a median age of 14.5 years, with no difference in kidney survival between variant types. However, the literature review indicated faster progression to kidney failure in patients with pLoF variants (11.0 vs. 24.0 years; pLoF, n = 138 vs. non-pLoF, n = 71; P = 0.002), with no significant difference by variant location. Ocular manifestations were more common, had earlier onset, and were more severe in the pLoF variants group in our cohort (P = 0.038). The literature confirmed a higher prevalence of ocular involvement in patients with pLoF variants (pLoF, n = 175 vs. non-pLoF, n = 88; P < 0.001) and in those with paired domain variants (P = 0.01). pLoF variants in PAX2 were associated with worse kidney and ocular outcomes. These findings support genotype-phenotype correlations, contributing to tailored management in patients with PAX2-related disorders.
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