Malignant hyperthermia, a pharmacogenetic disorder of skeletal muscle, is a potentially fatal reaction triggered by exposure to volatile anesthetics or succinylcholine. Genetic testing of the three known involved genes (RYR1, CACNA1S, and STAC3) has become the first line of testing for malignant hyperthermia susceptibility. However, genetic testing has sensitivity of only up to 70%. Contracture testing may be used to rule out the disorder for some individuals. This study outlines a stepwise approach, using clinical history and assessment as well as interpretation of genetic variants to balance risks and benefits for individuals at risk of this disorder. This work has received an Affirmation of Value designation from the American Society of Anesthesiologists (Schaumburg, Illinois) and endorsement from the Canadian Anesthesiologists' Society (Toronto, Canada).

Understanding the yield, predictive power, and utility of a secondary finding is critical for policy development and can help inform discussions for population screening. Because American College of Medical Genetics and Genomics (ACMG) Secondary Findings guidelines are applied in diverse testing contexts, we recruited participants from multiple sources to address these questions. We assessed our first 1,500 inquiries to review the disorders/genes that were returned to these individuals. After eligibility screening, we enrolled 227 recipients and completed genotyping, cascade testing, and phenotyping efforts for 163 probands. From evaluating these families, it became clear that there were highly variable outcomes for the diagnostic yield of secondary findings. To objectively and quantitatively assess this, we developed a method to measure the likelihood that the family was, in fact, affected with the disorder associated with the secondary finding variant. We assessed this in detail for 59 families who had a secondary finding of BRCA1- or BRCA2-related cancer predisposition. Our estimates of the likelihood of a valid clinicomolecular diagnosis ranged from 26.2% to 100%. Over half (51%) of the families met criteria for diagnostic testing, indicating that diagnostic testing for these disorders is underused and that secondary findings testing is being applied inappropriately to these families. These results will be useful for policy refinement for secondary findings and are also relevant to considerations of population genomic screening.

While pharmacogenetic testing has traditionally relied on array-based genotyping platforms, these methods are limited by incomplete variant coverage and inability to detect novel alleles. We hypothesize that the performance of genome sequencing (GS) is superior to that of an array-based genotyping method for gene-drug pairs that are clinically implemented at the NIH Clinical Center. DNA was collected from a cohort of 293 patients from a single-center prospective cohort study at the National Institutes of Health, Bethesda, MD. Post hoc analysis was conducted in probands with genetically inferred ancestry: European, African, African American, and Asian. The primary endpoint was to determine concordance between GS, exome sequencing (ES), and a commercial genotyping array while assessing accuracy of non-concordant alleles using orthogonal sequencing. In a cohort of 293 individuals genotyped with a commonly used array, GS (n = 120) and/or ES (n = 185) was conducted. GS demonstrated superior accuracy, resolving unknown or ambiguous array-based allele calls in 5% of cases, ultimately achieving 99% baseline accuracy. Moreover, we confirmed discordances between array-based calls and GS in favor of GS through validation by orthogonal sequencing methods such as long-read sequencing. We highlight clinically significant inaccuracies that include critical variants missed by arrays and ES. From a cost perspective, GS proved comparable or superior to array-based methods, with significantly greater clinical applicability and flexibility. Our findings underscore the feasibility and superiority of integrating GS-based pharmacogenetics testing into clinical settings, demonstrating robust analytical performance, improved patient management potential, and a pathway toward broader, lifetime utility of genomic data.

Genome-wide association studies (GWAS) have identified >1,200 signals associated with type 2 diabetes (T2D), yet identifying functional variants remains challenging because the majority of them lie in noncoding regions of the genome and are in areas of high linkage disequilibrium (LD). While chromatin accessibility QTL (caQTL) and expression QTL (eQTL) analyses are useful for nominating regulatory mechanisms underlying GWAS signals, limitations still exist in pinpointing functional variants within regions of high LD. A complementary approach that has been less frequently applied is to focus on the allele-specific effect on chromatin accessibility at heterozygous single-nucleotide polymorphisms (SNPs), hereafter referred to as "allelic imbalance". We analyzed the allelic imbalance of reads generated from an assay for transposase-accessible chromatin with sequencing (ATAC-seq) across genotyped samples from 490 donors in T2D-relevant tissues: skeletal muscle, liver, pancreatic islets, adipose tissue, and relevant cell types. We identified 119,949 allelically imbalanced SNPs (FDR<0.05) across the genome. The allelic imbalance was often most prominent in one tissue and showed an enrichment overlapping with tissue-specific transcription factor (TF) binding footprints. Focusing on the 8,581 SNPs in previously published 99% credible sets from 338 T2D GWAS signals, we identified 256 imbalanced SNPs across 123 (36.4% of) signals, each showing allelic imbalance in at least one tissue or cell type. Of these, 71 signals contained only a single imbalanced SNP, representing excellent candidate causative variants. As a proof-of-concept, we showed that 23 of the 256 imbalanced SNPs were supported by allelic assays from previous studies. Further, we experimentally validated two imbalanced SNPs as likely functional variants: rs34584161 among a seven-SNP T2D credible set at the RNF6 signal in islets and rs849134 among a 13-SNP credible set at the JAZF1 signal in liver. This study demonstrates the power of integrating ATAC-seq allelic imbalance (ASAI) with GWAS statistical fine-mapping to identify candidate functional regulatory variants from among tightly linked GWAS variants in disease-relevant tissues. While applied here in T2D, this approach represents a widely applicable high-throughput framework for refining the genetic architecture of complex traits.

Induced pluripotent stem cells (iPSCs) enabled the generation of diverse cell types; however, certain fundamental biological properties, such as the genetic and epigenetic determinants of proliferation, remain poorly characterized. We quantified proliferation across 602 unique donors with a time-lapse imaging-based growth area under the curve (gAUC) phenotype and correlated gAUC with cell line gene expression and genotype. We identified 3,091 differentially expressed genes and found that rare deleterious variants in WDR54, TMEM250, and C2orf81 were associated with reduced iPSC growth. Notably, WDR54 was differentially expressed with respect to gAUC. Although no common variants were associated, common genetic variation explained 71%-75% of the variance. These results indicate a complex genetic architecture of iPSC growth rates, where rare, large-effect variants in important growth regulators are layered onto a highly polygenic background. These findings can impact the design of pooled iPSC-based studies and disease models, which may be confounded by intrinsic growth differences.

BACKGROUND:Proteus syndrome is a progressive asymmetric overgrowth disorder caused by mosaic activating variants in AKT1. It most frequently affects the skin, soft tissues, bones, and central nervous system and increases the risk for certain tumors and venous thromboembolism. A hallmark feature is the progressive plantar cerebriform connective tissue nevus (CCTN). The natural history of skin manifestations of the hand in Proteus syndrome is less well characterized. METHODS:A retrospective review of photography collected from 2000 through 2024 was conducted as part of an ongoing natural history of Proteus syndrome study (NCT00001403). RESULTS:Clinical photography of the hands from 74 individuals (40 male, 34 female) with Proteus syndrome was reviewed. The average age at the earliest photograph was 13.2 (SD 8.8) years. Thirty-five individuals had serial photographs with an average total interval of 5.8 (SD 4.2) years. Connective tissue nevi (CTN) were present in 30 (41%) individuals. These were cerebriform in 16 individuals. Palmar CTN progression through the gradual expansion of nodules was universal in children. Epidermal nevi (EN) were present on the hands of 28 (39%) individuals and were generally stable. Enlargement of digits with nodules or plaques was seen in 45 (61%) individuals. Less frequent features included changes to the nails, dorsum of the hand, and vascular anomalies. CONCLUSION:There are a variety of skin manifestations on the hand in Proteus syndrome. Palmar CTN are often progressive whereas other lesions progress slowly or remain stable over time.

Proteus syndrome is a rare genetic disorder characterized by progressive, abnormal overgrowth that can affect any organ or tissue in the body. This abnormality is caused by a mosaic activating variant in AKT1 that encodes a key serine/threonine kinase of the phosphoinositide-3-kinase (P13K)/AKT signaling pathway and is involved in cell growth, survival, and metabolism. Miransertib (MK-7075, formerly ARQ 092) is a novel, orally bioavailable allosteric pan-AKT inhibitor that selectively targets AKT1, AKT2, and AKT3, demonstrating potent suppression of AKT signaling and tumor growth in murine xenograft models with dysregulated signaling pathways. It has also been evaluated as a potential therapeutic option for individuals with Proteus syndrome, both in pre-clinical studies and in clinical trials. In this study, we have developed and validated a sensitive, robust, and specific LC-MS/MS method for quantifying miransertib in human plasma. The calibration curve ranged from 0.5 to 500 ng/mL in human plasma with a linearity of r2 = 0.9945 ± 0.0019 across multiple days. Accuracy of assay ranged from -3.38 to 3.53% and precision was between 2.90 and 8.42%. Miransertib also showed excellent stability following multiple freeze-thaw cycles, during bench-top storage, and while on the autosampler overnight. This method enabled us to assess the pharmacokinetic parameters of participants enrolled in a phase II clinical trial (NCT04316546).

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder affecting tissues of mesenchymal origin. Most patients harbor a c.1824C>T/p.G608= variant, commonly described as G608G, in exon 11 of LMNA that leads to aberrant splicing and production of the toxic progerin protein. In addition to cardiovascular, dermal, and adipose tissue deterioration, HGPS mouse models also develop progressive bone dysplasia that occurs in patients. Here we characterize the efficacy of in vivo mutation correction with an adenine base editor (ABE) to rescue structural and functional defects in HGPS transgenic murine bone tissue. Treatment of double-copy transgenic osteoblast cultures with a lentiviral-delivered CRISPR-Cas9 ABE achieved nearly 40% gene correction in vitro, resulting in significant reduction of progerin transcripts and protein, in the absence of selective agents. Furthermore, gene correction improved progeroid osteoblasts' capacity to deposit and mineralize extracellular matrix compared to untreated cultures. In vivo, a single intravenous dose of AAV9-delivered ABE corrected the mutation, achieving ~14%, ~22%, ~10% and < 1% correction in bone by six months of age when administered at P3, P14, 1 and 4 months of age, respectively. Partially rescued bone structural and physical parameters were observed in P14-treated mice with concomitant normalization of gene transcriptional programs and intracellular signaling pathways involved in bone remodeling. This work demonstrates in vivo delivery of a locus-specific DNA base editor to bone tissue, delineates the timing of treatment required for maximum efficacy, and suggests that this system might be tailored for application to other monogenic bone disorders.

PURPOSE:The return of secondary findings is well established in clinical testing and is increasingly used in clinical research testing. Variant classification can be challenging because of the lack of monogenic disease entity (MDE, defined as a gene-phenotype pair)-specific variant classification recommendations. A key criterion for variant classification is disease allele frequency thresholds (DAFTs), which are not established for all MDEs recommended for the return of secondary findings. METHODS:We calculated DAFTs for secondary finding MDEs considering prevalence, gene contribution, and penetrance. American College of Medical Genetics and Genomics criterion BS1 was set at the calculated DAFT value, with BA1 set at 10 times the calculated DAFT. For genes associated with multiple secondary finding MDEs without clear genotype-phenotype correlation, DAFT values were combined. GnomAD Grpmax filtering allele frequencies for pathogenic/likely pathogenic classified variants were compared with calculated thresholds. RESULTS:We determined BS1 and BA1 values for 58 secondary findings MDEs (47 genes). No pathogenic/likely pathogenic variant Grpmax filtering allele frequency was greater than the relevant MDE-specific BA1. CONCLUSION:Setting BA1 and BS1 thresholds should improve variant classification consistency and reduce misclassifications. For secondary finding MDEs without current ClinGen Variant Curation Expert Panel specifications, these frequency specifications can be used until full criteria are available.

RNA modifications are critical regulators of gene expression and cellular processes; however, the epitranscriptome is less well studied than the epigenome. Here, we studied transcriptome-wide changes in RNA modifications and expression levels in two human pancreatic beta-cell lines, EndoC-BH1 and EndoC-BH3, after one hour of glucose stimulation. Using direct RNA nanopore sequencing (dRNA-seq), we measured N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, and pseudouridine concurrently across the transcriptome. We developed a differential RNA modification method and identified 1,697 differentially modified sites (DMSs) across all modifications. These DMSs were largely independent of changes in gene expression levels and enriched in transcripts for type 2 diabetes (T2D) genes. Our study demonstrates how dRNA-seq can be used to detect and quantify RNA modification changes in response to cellular stimuli at the single-nucleotide level and provides new insights into RNA-mediated mechanisms that may contribute to normal beta-cell response and potential dysfunction in T2D.

PURPOSE:We previously developed an approach to calibrate computational tools for clinical variant classification, updating recommendations for the reliable use of variant impact predictors to provide evidence strength up to Strong. A new generation of tools using distinctive approaches has since been released, and these methods must be independently calibrated for clinical application. METHODS:Using our local posterior probability-based calibration and our established data set of ClinVar pathogenic and benign variants, we determined the strength of evidence provided by 3 new tools (AlphaMissense, ESM1b, and VARITY) and calibrated scores meeting each evidence strength. RESULTS:All 3 tools reached the Strong level of evidence for variant pathogenicity and Moderate for benignity, although sometimes for few variants. Compared with previously recommended tools, these yielded at best only modest improvements in the trade-offs between evidence strength and false-positive predictions. CONCLUSION:At calibrated thresholds, 3 new computational predictors provided evidence for variant pathogenicity at similar strength to the 4 previously recommended predictors (and comparable with functional assays for some variants). This calibration broadens the scope of computational tools for application in clinical variant classification. Their new approaches offer promise for future advancement of the field.

PURPOSE:To provide diagnostic guidance for individuals with lateralized overgrowth (LO) and implement appropriate screening protocols. LO without a syndromic presentation is considered idiopathic isolated lateralized overgrowth (ILO). METHODS:We performed a literature search of LO syndromes and malignancy risk and reviewed existing guidelines and expert input. RESULTS:We integrated 940 unique articles to form recommendations. We defined LO as significantly larger length and/or girth of aspect(s) of one side of the body compared with its contralateral side. It can be associated with somatic overgrowth syndromes. ILO was previously defined based on clinical features and deemed idiopathic by absence of molecular findings. Much of the tumor risk is likely because of specific LO syndromic causes now identified through improved diagnostic technologies; therefore, the tumor risk in idiopathic ILO is likely lower than previously accepted. CONCLUSION:Mosaicism complicates molecular diagnosis for children with LO. However, conditions such as Beckwith-Wiedemann spectrum and PTEN-related hamartoma tumor syndrome necessitate routine tumor screening. Establishing a specific diagnosis via comprehensive molecular testing on affected tissue will guide screening and management. In cases of idiopathic ILO, location of the overgrowth, estimation of tumor risk, regional practice approaches and family concerns all play roles in determining tumor screening.

BACKGROUND:Hematopoietic cell transplantation (HCT) provides effective long-term management for some inborn errors of immunity. Genetic findings can inform donor selection, considerations in conditioning intensity and agents, and graft-versus-host disease prophylaxis. Exome/genome sequencing is increasingly accessible but of uncertain clinical utility. We aimed to evaluate the clinical utility of comprehensive genomic evaluations through review of HCT at our center. METHODS:We performed exome/genome sequencing on pre-HCT samples from participants between 2017 and 2023. We reported primary findings (PF) and secondary findings (SF). Post hoc, we analyzed medication and pharmacogenetic (PGx) data. RESULTS:We analyzed pre-HCT exome/genome sequencing (n = 84 exome, n = 63 genome, n = 32 with both) for 179 probands. Most (143/179; 79.9%) had a PF underlying the HCT indication, with GATA2 being most common (n = 59). Three percent of participants had an SF predisposing to cancer or cardiovascular disease. Most (n = 108/179; 60.3%) received ≥1 medication(s) that may have been further optimized with PGx. Using Kaplan-Meier survival analysis, we compared the survival rates of participants with 0, 1, and ≥2 genomic risk factors (GRF: absence of PF; presence of SF or PGx). Survival at 3 y was 94.8%, 84.8%, and 58.5% for those with 0, 1, and ≥2 GRF, respectively (log-rank: 16.10, df = 2, P  = 0.0003), indicating statistically significant survival differences by GRF. CONCLUSIONS:Comprehensive genomic evaluation is an emerging avenue for tailoring HCT approaches, and identification of HCT-relevant findings may be common. On multivariate analysis, GRF was associated with survival in this retrospective cohort. Prospective research is warranted to further integrate genomic data into precision treatment.