Universal Exome Sequencing in Critically Ill Adults: A Diagnostic Yield of 25% and Race-Based Disparities in Access to Genetic Testing.

Numerous studies have underscored the diagnostic and therapeutic potential of exome or genome sequencing in critically ill pediatric populations. However, an equivalent investigation in critically ill adults remains conspicuously absent. We retrospectively analyzed whole exome sequencing (WES) data available through the PennMedicine Biobank (PMBB) from all 365 young adult patients, aged 18-40 years, with intensive care unit (ICU) admissions at the University of Pennsylvania Health System who met inclusion criteria for our study. For each participant, two Medical Genetics and Internal Medicine-trained clinicians reviewed WES reports and patient charts for variant classification, result interpretation, and identification of genetic diagnoses related to their critical illness. Of the 365 individuals in our study, 90 (24.7%) were found to have clearly diagnostic results on WES; an additional 40 (11.0%) had a suspicious variant of uncertain significance (VUS) identified; and an additional 16 (4.4%) had a medically actionable incidental finding. The diagnostic rate of exome sequencing did not decrease with increasing patient age. Affected genes were primarily involved in cardiac function (18.8%), vascular health (16.7%), cancer (16.7%), and pulmonary disease (11.5%). Only half of all diagnostic findings were known and documented in the patient chart at the time of ICU admission. Significant disparities emerged in subgroup analysis by EHR-reported race, with genetic diagnoses known/documented for 63.5% of White patients at the time of ICU admission but only for 28.6% of Black or Hispanic patients. There was a trend towards patients with undocumented genetic diagnoses having a 66% increased mortality rate, making these race-based disparities in genetic diagnosis even more concerning. Altogether, universal exome sequencing in ICU-admitted adult patients was found to yield a new definitive diagnosis in 11.2% of patients. Of these diagnoses, 76.6% conferred specific care-altering medical management recommendations. Our study suggests that the diagnostic utility of exome sequencing in critically ill young adults is similar to that observed in neonatal and pediatric populations and is age-independent. The high diagnostic rate and striking race-based disparities we find in genetic diagnoses argue for broad and universal approaches to genetic testing for critically ill adults. The widespread implementation of comprehensive genetic sequencing in the adult population promises to enhance medical care for all individuals and holds the potential to rectify disparities in genetic testing referrals, ultimately promoting more equitable healthcare delivery.

Sex-Specific Effect of MTSS1 Downregulation on Dilated Cardiomyopathy.

MTSS1 (metastasis suppressor 1) is an I-BAR protein that regulates cytoskeleton dynamics through interactions with actin, Rac, and actin-associated proteins. In a prior study, we identified genetic variants in a cardiac-specific enhancer upstream of MTSS1 that reduce human left ventricular (LV) MTSS1 expression and associate with protection against dilated cardiomyopathy (DCM). We sought to probe these effects further using population genomics and in vivo murine models. We crossed Mtss1-/- mice with a transgenic (Tg) murine model of human DCM caused by the D230N pathogenic mutation in Tpm1 (tropomyosin 1). In females, Tg/Mtss1+/- mice had significantly increased LV ejection fraction and reduced LV volumes relative to their Tg/Mtss1+/+ counterparts, signifying partial rescue of DCM due to Mtss1 haploinsufficiency. No differences were observed in males. To study effects in humans, we fine-mapped the MTSS1 locus with 82 cardiac magnetic resonance (CMR) traits in 22,381 UK Biobank participants. MTSS1 enhancer variants showed interaction with biological sex in their associations with several CMR traits. After stratification by biological sex, associations with CMR traits and colocalization with MTSS1 expression in the Genotype-Tissue Expression (GTEx) Project were observed principally in women and were substantially weaker in men. These findings suggest sex dimorphism in the effects of MTSS1-lowering alleles, and parallel the increased LV ejection fraction and reduced LV volumes observed female Tg/Mtss1+/- mice. Together, our findings at the MTSS1 locus suggest a genetic basis for sex dimorphism in cardiac remodeling and motivate sex-specific study of common variants associated with cardiac traits and disease.

Phenotypes of undiagnosed adults with actionable OTC and GLA variants.

Inherited metabolic disorders (IMDs) are variably expressive, complicating identification of affected individuals. A genotype-first approach can identify individuals at risk for morbidity and mortality from undiagnosed IMDs and can lead to protocols that improve clinical detection, counseling, and management. Using data from 57,340 participants in two hospital biobanks, we assessed the frequency and phenotypes of individuals with pathogenic/likely pathogenic variants (PLPVs) in two IMD genes: GLA, associated with Fabry disease, and OTC, associated with ornithine transcarbamylase deficiency. Approximately 1 in 19,100 participants harbored an undiagnosed PLPV in GLA or OTC. We identified three individuals (2 male, 1 female) with PLPVs in GLA, all of whom were undiagnosed, and three individuals (3 female) with PLPVs in OTC, two of whom were undiagnosed. All three individuals with PLPVs in GLA (100%) had symptoms suggestive of mild Fabry disease, and one individual (14.2%) had an ischemic stroke at age 33, likely indicating the presence of classic disease. No individuals with PLPVs in OTC had documented hyperammonemia despite exposure to catabolic states, but all (100%) had chronic symptoms suggestive of attenuated disease, including mood disorders and migraines. Our findings suggest that GLA and OTC variants identified via a genotype-first approach are of high penetrance and that population screening of these genes can be used to facilitate stepwise phenotyping and appropriate care.

A genotype-first approach identifies high incidence of NF1 pathogenic variants with distinct disease associations.

Loss of function variants in the NF1 gene cause neurofibromatosis type 1 (NF1), a genetic disorder characterized by complete penetrance, prevalence of 1 in 3,000, characteristic physical exam findings, and a substantially increased risk for malignancy. However, our understanding of the disorder is entirely based on patients ascertained through phenotype-first approaches. Leveraging a genotype-first approach in two large patient cohorts, we demonstrate unexpectedly high prevalence (1 in 450-750) of NF1 pathogenic variants. Half were identified in individuals lacking clinical features of NF1, with many appearing to have post-zygotic mosaicism for the identified variant. Incidentally discovered variants were not associated with classic NF1 features but were associated with an increased incidence of malignancy compared to a control population. Our findings suggest that NF1 pathogenic variants are substantially more common than previously thought, often characterized by somatic mosaicism and reduced penetrance, and are important contributors to cancer risk in the general population.

Unmasking the challenges of Kabuki syndrome in adulthood: A case series.

Kabuki syndrome is a recognizable Mendelian disorder characterized by the clinical constellation of childhood hypotonia, developmental delay or intellectual impairment, and characteristic dysmorphism resulting from monoallelic pathogenic variants in KMT2D or KDM6A. In the medical literature, most reported patients are children, and data is lacking on the natural history of the condition across the lifespan, with little known about adult-specific presentations and symptoms. Here, we report the results of a retrospective chart review of eight adult patients with Kabuki syndrome, seven of whom are molecularly confirmed. We use their trajectories to highlight the diagnostic challenges unique to an adult population, expand on neurodevelopmental/psychiatric phenotypes across the lifespan, and describe adult-onset medical complications, including a potential cancer risk and unusual and striking premature/accelerated aging phenotype.

An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications.

The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl ß-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy.

Basal exon skipping and genetic pleiotropy: A predictive model of disease pathogenesis.

Genetic pleiotropy, the phenomenon by which mutations in the same gene result in markedly different disease phenotypes, has proven difficult to explain with traditional models of disease pathogenesis. We have developed a model of pleiotropic disease that explains, through the process of basal exon skipping, how different mutations in the same gene can differentially affect protein production, with the total amount of protein produced correlating with disease severity. Mutations in the centrosomal protein of 290 kDa (CEP290) gene are associated with a spectrum of phenotypically distinct human diseases (the ciliopathies). Molecular biologic examination of CEP290 transcript and protein expression in cells from patients carrying CEP290 mutations, measured by quantitative polymerase chain reaction and Western blotting, correlated with disease severity and corroborated our model. We show that basal exon skipping may be the mechanism underlying the disease pleiotropy caused by CEP290 mutations. Applying our model to a different disease gene, CC2D2A (coiled-coil and C2 domains-containing protein 2A), we found that the same correlations held true. Our model explains the phenotypic diversity of two different inherited ciliopathies and may establish a new model for the pathogenesis of other pleiotropic human diseases.

CEP290 and the primary cilium.

The protein CEP290 has recently emerged as a major player in the biology of the cilium and as a causative protein in a number of human syndromic diseases, most of which are associated with the devastating blinding disease Leber congenital amaurosis. (Coppieters et al., Hum Mutat 31, 2010, 1097-1108) CEP290 is known to be an important component of the primary cilium, localizing to the Y-links of the ciliary transition zone and having a role in the regulation of transport in and out of the ciliary compartment (Craige et al., J Cell Biol 190, 2010, 927-940). While many mutations in CEP290 have been identified in human patients, how these mutations result in the spectrum of human disease attributed to the protein remain unknown. As we begin to learn more about the normal role of CEP290, it is likely that we will begin to shed light on how these mutations result in the various CEP290 disease phenotypes. Here we discuss many of these diverse aspects of CEP290 biology and pathology in an attempt to link what we know about the molecular mechanisms of CEP290 function with what we know about CEP290-associated disease.

Disruption of CEP290 microtubule/membrane-binding domains causes retinal degeneration.

Mutations in the gene centrosomal protein 290 kDa (CEP290) cause an array of debilitating and phenotypically distinct human diseases, ranging from the devastating blinding disease Leber congenital amaurosis (LCA) to Senior-Løken syndrome, Joubert syndrome, and the lethal Meckel-Gruber syndrome. Despite its critical role in biology and disease, very little is known about CEP290's function. Here, we have identified 4 functional domains of the protein. We found that CEP290 directly binds to cellular membranes through an N-terminal domain that includes a highly conserved amphipathic helix motif and to microtubules through a domain located within its myosin-tail homology domain. Furthermore, CEP290 activity was regulated by 2 autoinhibitory domains within its N and C termini, both of which were found to play critical roles in regulating ciliogenesis. Disruption of the microtubule-binding domain in a mouse model of LCA was sufficient to induce significant deficits in cilium formation, which led to retinal degeneration. These data implicate CEP290 as an integral structural and regulatory component of the cilium and provide insight into the pathological mechanisms of LCA and related ciliopathies. Further, these data illustrate that disruption of particular CEP290 functional domains may lead to particular disease phenotypes and suggest innovative strategies for therapeutic intervention.