Quantification of Proteus syndrome-associated lung disease.

BACKGROUND: Proteus syndrome is an ultra-rare mosaic overgrowth disorder. Individuals with Proteus syndrome can develop emphysematous and cystic changes of the lung that may lead to progressive respiratory symptoms and require surgical intervention. This retrospective study seeks to quantify the radiographic features of Proteus syndrome-associated lung disease using computed tomography (CT) of the chest. The first method derives a Cystic Lung Score (CLS) by using a computer-aided diagnostic tool to quantify the fraction of cystic involvement of the lung. The second method yields a Clinician Visual Score (CVS), an observer reported scale of severity based on multiple radiographic features. The aim of this study was to determine if these measurements are associated with clinical symptoms, pulmonary function test (PFT) measurements, and if they may be used to assess progression of pulmonary disease. RESULTS: One hundred and thirteen imaging studies from 44 individuals with Proteus syndrome were included. Dyspnea and oxygen use were each associated with higher CLS (p = 0.001 and < 0.001, respectively) and higher CVS (p < 0.001 and < 0.001). Decreases in percent predicted FVC, FEV1, and DLCO each correlated with increased CLS and CVS. The annual increase of CLS in children, 5.6, was significantly greater than in adults, 1.6. (p = 0.03). The annual increase in CVS in children, 0.4, was similar to adults, 0.2 (p = 0.36). CONCLUSIONS: Proteus syndrome-associated lung disease is progressive. The rate of cystic progression is increased in children. Increased scores in CLS and CVS were associated with clinical symptoms and decreased pulmonary function. Both methods were able to detect change over time and were associated with clinically meaningful outcomes which may enable their use in interventional studies.

Phenotypic Features of Cystic Lung Disease in Proteus Syndrome: A Clinical Trial.

Rationale: Limited information is available regarding cystic lung disease in Proteus syndrome, a rare overgrowth disorder caused by a somatic activating variant in AKT1. Objectives: To define the phenotype of cystic lung disease in Proteus syndrome. Methods: Medical records, pulmonary function tests, and chest computed tomography of 39 individuals with Proteus syndrome evaluated at a single center were retrospectively reviewed. Lung histopathology from five affected individuals was examined. Results: Cystic lung disease affected 26 (67%) of 39 individuals. The mean age of affected individuals was 17.1 years. The lung cysts varied in size and location. Focal regions of heterogeneous lung parenchyma resembling emphysema were found in 81% of affected individuals. Mass effect was seen in 12% of affected individuals; pneumothorax occurred in one. Dyspnea and respiratory infections were reported by 38% and 35% of affected individuals, respectively. Abnormal pulmonary function and scoliosis were found in 96% of affected individuals. Lung disease progressed in seven of 10 affected individuals, and all five affected individuals younger than 20 years of age had progressive cystic lung disease. Three affected individuals had symptomatic improvement after lung resection. Histopathology showed cystic air space enlargement of varying severity. Conclusions: Cystic lung disease is common in Proteus syndrome and is likely to progress in affected individuals younger than 20 years of age. Screening asymptomatic individuals with Proteus syndrome for cystic lung disease is indicated. Surgical lung resection is a therapeutic option for affected individuals with severe disease. Clinical trial registered with www.clinicaltrials.gov (NCT00001403).

Development of the Clinical Gestalt Assessment: a visual clinical global impression scale for Proteus syndrome.

BACKGROUND: Clinical outcome assessments are important tools for measuring the natural history of disease and efficacy of an intervention. The heterogenous phenotype and difficult to quantity features of Proteus syndrome present challenges to measuring clinical outcomes. To address these, we designed a global clinical assessment for Proteus syndrome, a rare mosaic overgrowth disorder. The Clinical Gestalt Assessment (CGA) aims to evaluate change over time in this phenotypically diverse disorder. RESULTS: We gathered paired serial photographs and radiographs obtained at 12-to-36-month intervals from our natural history study of Proteus syndrome. The chronologic order of each set was blinded and presented to clinicians familiar with overgrowth disorders. They were asked to determine the chronologic order and, based on that response, rate global clinical change using a seven-point scale (Much Worse, Worse, Minimally Worse, No Change, Minimally Improved, Improved, Much Improved). Following a pilot, we tested the inter-rater reliability of the CGA using eight cases rated by eight clinicians. Raters identified the correct chronologic order in 53 of 64 (83%) of responses. There was low inter-rater variance and poor to moderate reliability with an intraclass correlation coefficient of 0.46 (95% CI 0.24-0.75). The overall estimate of global change was Minimally Worse over time, which is an accurate reflection of the natural history of Proteus syndrome. CONCLUSIONS: The CGA is a tool to evaluate clinical change over time in Proteus syndrome and may be a useful adjunct to measure clinical outcomes in prospective therapeutic trials.

Erratum: Orofacial overgrowth with peripheral nerve enlargement and perineuriomatous pseudo-onion bulb proliferations is part of the PIK3CA-related overgrowth spectrum.

[This corrects the article DOI: 10.1016/j.xhgg.2020.100009.].

Case report: five-year experience of AKT inhibition with miransertib (MK-7075) in an individual with Proteus syndrome.

Proteus syndrome is a rare overgrowth disorder caused by postzygotic activating variants in AKT1 Individuals may develop a range of skin, bone, and soft tissue overgrowth leading to functional impairment and disfigurement. Therapy for this disorder is limited to supportive care and surgical intervention. Inhibitors of AKT, originally designed as cancer therapeutics, are a rational, targeted pharmacologic strategy to mitigate the devastating morbidity of Proteus syndrome. We present the 5-yr follow-up of an 18-yr-old male with Proteus syndrome treated with miransertib (MK-7075), an oral pan-AKT inhibitor. At completion of a planned 48-wk phase 1 pharmacodynamic study, the individual derived sufficient benefit that the study was amended to permit continued use and assess the long-term safety of miransertib. The treatment has been well-tolerated with mild treatment-attributed side effects including headache, transient hyperglycemia, and transient elevations of aspartate aminotransferase, alanine aminotransferase, and bilirubin. The patient has experienced sustained improvement of pain and slowed growth of bilateral plantar cerebriform connective tissue nevi. This case report supplements the data from our prior study extending those findings out to 5 years. It shows that at the doses used, miransertib has a favorable safety profile and durable benefit of improving symptoms of pain and slowing progression of overgrowth in Proteus syndrome in a single individual. Although an uncontrolled single report cannot prove safety or efficacy, these data lend support to the encouraging preliminary data of our prior phase 1 pharmacodynamic study.

A systematic literature review of disclosure practices and reported outcomes for medically actionable genomic secondary findings.

PURPOSE: Secondary findings (SFs) are present in 1-4% of individuals undergoing genome/exome sequencing. A review of how SFs are disclosed and what outcomes result from their receipt is urgent and timely. METHODS: We conducted a systematic literature review of SF disclosure practices and outcomes after receipt including cascade testing, family and provider communication, and health-care actions. Of the 1,184 nonduplicate records screened we summarize findings from 27 included research articles describing SF disclosure practices, outcomes after receipt, or both. RESULTS: The included articles reported 709 unique SF index recipients/families. Referrals and/or recommendations were provided 647 SF recipients and outcome data were available for 236. At least one recommended evaluation was reported for 146 SF recipients; 16 reports of treatment or prophylactic surgery were identified. We found substantial variations in how the constructs of interest were defined and described. CONCLUSION: Variation in how SF disclosure and outcomes were described limited our ability to compare findings. We conclude the literature provided limited insight into how the American College of Medical Genetics and Genomics (ACMG) guidelines have been translated into precision health outcomes for SF recipients. Robust studies of SF recipients are needed and should be prioritized for future research.

Low-level variant calling for non-matched samples using a position-based and nucleotide-specific approach.

BACKGROUND: The widespread use of next-generation sequencing has identified an important role for somatic mosaicism in many diseases. However, detecting low-level mosaic variants from next-generation sequencing data remains challenging. RESULTS: Here, we present a method for Position-Based Variant Identification (PBVI) that uses empirically-derived distributions of alternate nucleotides from a control dataset. We modeled this approach on 11 segmental overgrowth genes. We show that this method improves detection of single nucleotide mosaic variants of 0.01-0.05 variant allele fraction compared to other low-level variant callers. At depths of 600 × and 1200 ×, we observed > 85% and > 95% sensitivity, respectively. In a cohort of 26 individuals with somatic overgrowth disorders PBVI showed improved signal to noise, identifying pathogenic variants in 17 individuals. CONCLUSION: PBVI can facilitate identification of low-level mosaic variants thus increasing the utility of next-generation sequencing data for research and diagnostic purposes.

Cardiothoracic imaging findings of Proteus syndrome.

In this work, we sought to delineate the prevalence of cardiothoracic imaging findings of Proteus syndrome in a large cohort at our institution. Of 53 individuals with a confirmed diagnosis of Proteus syndrome at our institution from 10/2001 to 10/2019, 38 individuals (men, n = 23; average age = 24 years) underwent cardiothoracic imaging (routine chest CT, CT pulmonary angiography and/or cardiac MRI). All studies were retrospectively and independently reviewed by two fellowship-trained cardiothoracic readers. Disagreements were resolved by consensus. Differences between variables were analyzed via parametric and nonparametric tests based on the normality of the distribution. The cardiothoracic findings of Proteus syndrome were diverse, but several were much more common and included: scoliosis from bony overgrowth (94%), pulmonary venous dilation (62%), band-like areas of lung scarring (56%), and hyperlucent lung parenchyma (50%). In addition, of 20 individuals who underwent cardiac MRI, 9/20 (45%) had intramyocardial fat, mostly involving the endocardial surface of the left ventricular septal wall. There was no statistically significant difference among the functional cardiac parameters between individuals with and without intramyocardial fat. Only one individual with intramyocardial fat had mildly decreased function (LVEF = 53%), while all others had normal ejection fraction.

Clinical diagnosis of presumed SOX2 gonadosomatic mosaicism.

Purpose: To describe a family with presumed SOX2 gonadosomatic mosaicism diagnosed upon ophthalmic examination of the proband's mother.Methods: The family underwent comprehensive ophthalmic and physical examination. Variant detection was performed using trio exome analysis on peripheral leukocyte DNA from blood and saliva samples. Variant segregation analysis was performed using a custom panel NGS sequencing. An identified variant in the SOX2 gene was confirmed in the proband by Sanger sequencing.Results: We report an individual with bilateral microphthalmia, developmental delay, hearing loss, and dysmorphic features. Her mother was found to have asymptomatic forme fruste uveal coloboma affecting her anterior segment. Her father, aunt, and sisters were unaffected. Trio exome sequence analysis showed an apparent de novo heterozygous deletion in the proband, NM_003106.3:c.70_89del, NP_003097.1:p.(Asn24Argfs*65), classified as pathogenic. Testing of the other family members' peripheral blood and saliva was negative for this variant. The iris transillumination abnormalities in the proband's mother supports a gonadosomatic mosaicism scenario.Conclusions: The results from this family underscore the importance of performing detailed evaluations of the parents of apparently sporadically affected individuals with heritable ophthalmic disorders. The identification of mildly affected individuals could substantially alter recurrence risks.

Hypertrichotic patches as a mosaic manifestation of Proteus syndrome.

BACKGROUND: Proteus syndrome is an overgrowth disorder caused by a mosaic activating AKT1 variant. Hair abnormalities in Proteus syndrome have rarely been reported, and frequencies of such findings have not been elucidated. OBJECTIVE: To define the types and frequencies of hair findings in individuals with Proteus syndrome. METHODS: A cross-sectional study was conducted of individuals with clinical features of Proteus syndrome and a confirmed pathogenic variant in AKT1 evaluated between November 1996 and June 2019 at the National Institutes of Health Clinical Center. Medical records were reviewed for patterning, density, and color of hair on the body and scalp. RESULTS: Of 45 individuals evaluated, 29 (64%) had asymmetric hypertrichosis on the body. This included unilateral blaschkoid hypertrichotic patches overlying normal skin or epidermal nevi in 16 (36%), unilateral nonblaschkoid hypertrichotic patches in 11 (24%), and unilateral limb hypertrichosis in 10 (22%). Diffuse, scattered, or patchy changes in scalp hair density or color were present in 11 individuals (24%). LIMITATIONS: The retrospective, observational design, and limited longitudinal follow-up. CONCLUSIONS: Asymmetric variations in hair distribution, thickness, length, and color contribute to the overall mosaic appearance of the skin in Proteus syndrome, an observation that provides novel insights into the role of phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling in skin appendage development.

Allelic heterogeneity of Proteus syndrome.

Proteus syndrome is a mosaic disorder that can cause progressive postnatal overgrowth of nearly any organ or tissue. To date, Proteus syndrome has been exclusively associated with the mosaic c.49G > A p.(Glu17Lys) pathogenic variant in AKT1, a variant that is also present in many cancers. Here we describe an individual with severe Proteus syndrome who died at 7.5 yr of age from combined parenchymal and restrictive pulmonary disease. Remarkably, this individual was found to harbor a mosaic c.49_50delinsAG p.(Glu17Arg) variant in AKT1 at a variant allele fraction that ranged from <0.01 to 0.46 in fibroblasts established from an overgrown digit. This variant was demonstrated to be constitutively activating by phosphorylation of AKT(S473). These data document allelic heterogeneity for Proteus syndrome. We recommend that individuals with a potential clinical diagnosis of Proteus syndrome who are negative for the p.(Glu17Lys) variant be tested for other variants in AKT1.

Orofacial overgrowth with peripheral nerve enlargement and perineuriomatous pseudo-onion bulb proliferations is part of the PIK3CA-related overgrowth spectrum.

Individuals with orofacial asymmetry due to mucosal overgrowths, ipsilateral bone and dental aberrations with perineurial hyperplasia and/or perineuriomatous pseudo-onion bulb proliferations, comprise a recognizable clinical entity. In this article, we describe three individuals with this clinical entity and mosaic PIK3CA variants c.3140A>G (p. His1047Arg), c.328_330delGAA (p. Glu110del), and c.1353_1364del (p.Glu453_Leu456del). We conclude that the identification of these mosaic variants in individuals with orofacial asymmetry presenting histopathologically perineurial hyperplasia and/or intraneural pseudo-onion bulb perineurial cell proliferations supports the inclusion of this clinical entity in the PIK3CA-related overgrowth spectrum.

A dyadic genotype-phenotype approach to diagnostic criteria for Proteus syndrome.

Phenotype-based diagnostic criteria were developed for Proteus syndrome in 1999 and updated in 2006. Subsequently, the causative mosaic gene alteration was discovered, the c.49G>A p.E17K variant in AKT1. As well, a number of overlapping overgrowth disorders attributable to mosaic PIK3CA variants have now been characterized, leading to the designation of PIK3CA-related overgrowth spectrum (PROS). Finally, ongoing work to better characterize Proteus syndrome has led to identification of additional features of that disorder that could be useful in diagnostic criteria. We have taken the opportunity of these discoveries to re-evaluate the Proteus syndrome diagnostic criteria. Here we propose a new set of diagnostic criteria that establishes a weighted, point-based system for the phenotypic attributes and then integrates that with the potential molecular test results to result in one of two designations: AKT1-related Proteus syndrome or AKT1-related overgrowth spectrum. A patient whose only manifestation is an AKT1 c.49G>A-positive tumor would receive neither of these designations. Here we review the rational basis of diagnostic criteria and argue that a unitary diagnostic entity is a distinct gene-phenotype dyad and that this should be the model for all mendelian disorders. The gene-alone or phenotype-alone approach is inadequate to rigorously delineate a unitary diagnostic entity.

Thrombosis risk factors in PIK3CA-related overgrowth spectrum and Proteus syndrome.

Increased risk of thromboembolism has been recognized in individuals with mosaic overgrowth disorders, Proteus syndrome (PS) and PIK3CA-related overgrowth spectrum (PROS), including Klippel-Trenaunay syndrome and CLOVES syndrome. PS and PROS have distinct, yet overlapping clinical findings and are caused by somatic pathogenic variants in the PI3K/AKT gene signaling pathway. PS is caused by a single somatic activating AKT1 c.49G > A p.E17K variant while PROS can be caused one of multiple variants in PIK3CA. The role of prothrombotic factors, endothelial cell adhesion molecules, and vascular malformations in both PS and PROS have not been previously investigated. A pilot study of prospective clinical and laboratory evaluations with the purposes of identifying potential risk factors for thrombosis was conducted. Doppler ultrasounds and magnetic resonance angiogram/ venography (MRA/MRV) scans identified vascular malformations in PS and PROS that were not appreciated on physical examination. Abnormal D-dimers (0.60-2.0 mcg/ml) occurred in half of individuals, many having vascular malformations, but no thromboses. Soluble vascular endothelial markers, including thrombomodulin, soluble vascular adhesion molecule (sVCAM), soluble intercellular adhesion molecule (sICAM), E-selectin, and P-selectin were significantly higher in PS and PROS compared to controls. However, no single attribute was identified that explained the risk of thrombosis. Predisposition to thrombosis is likely multifactorial with risk factors including chronic stasis within vascular malformations, stasis from impaired mobility (e.g., following surgery), decreased anticoagulant proteins, and effects of AKT1 and PIK3CA variants on vascular endothelium. Based on our findings, we propose clinical recommendations for surveillance of thrombosis in PS and PROS.

Molecular heterogeneity of the cerebriform connective tissue nevus in mosaic overgrowth syndromes.

The clinical diagnostic criteria for Proteus syndrome were defined before the discovery of the AKT1 c.49G>A; p.(Glu17Lys) causal variant and used a combination of general and specific phenotypic attributes that could be combined to make a clinical diagnosis. The most heavily weighted specific criterion was the cerebriform connective tissue nevus (CCTN). Here, we describe two individuals with connective tissue nevi (CTNs) and some general attributes of Proteus syndrome who were found to have mosaic PIK3CA variants. CTNs on the soles of individuals with PIK3CA-related overgrowth typically exhibit thickening of the soft tissues with at most a wrinkled surface, but these two patients had firm plaques with ridges and furrows characteristic of CCTNs, which was histologically confirmed in one. These data show that CCTNs are not specific to Proteus syndrome and that clinicians should be cautious in diagnosing individuals with Proteus syndrome based on the CCTN alone. Rather, a complete evaluation should include careful assessment of other attributes of the diagnostic criteria and, whenever possible, genetic analysis of affected tissue.

NAA10 polyadenylation signal variants cause syndromic microphthalmia.

BACKGROUND: A single variant in NAA10 (c.471+2T>A), the gene encoding N-acetyltransferase 10, has been associated with Lenz microphthalmia syndrome. In this study, we aimed to identify causative variants in families with syndromic X-linked microphthalmia. METHODS: Three families, including 15 affected individuals with syndromic X-linked microphthalmia, underwent analyses including linkage analysis, exome sequencing and targeted gene sequencing. The consequences of two identified variants in NAA10 were evaluated using quantitative PCR and RNAseq. RESULTS: Genetic linkage analysis in family 1 supported a candidate region on Xq27-q28, which included NAA10. Exome sequencing identified a hemizygous NAA10 polyadenylation signal (PAS) variant, chrX:153,195,397T>C, c.*43A>G, which segregated with the disease. Targeted sequencing of affected males from families 2 and 3 identified distinct NAA10 PAS variants, chrX:g.153,195,401T>C, c.*39A>G and chrX:g.153,195,400T>C, c.*40A>G. All three variants were absent from gnomAD. Quantitative PCR and RNAseq showed reduced NAA10 mRNA levels and abnormal 3' UTRs in affected individuals. Targeted sequencing of NAA10 in 376 additional affected individuals failed to identify variants in the PAS. CONCLUSION: These data show that PAS variants are the most common variant type in NAA10-associated syndromic microphthalmia, suggesting reduced RNA is the molecular mechanism by which these alterations cause microphthalmia/anophthalmia. We reviewed recognised variants in PAS associated with Mendelian disorders and identified only 23 others, indicating that NAA10 harbours more than 10% of all known PAS variants. We hypothesise that PAS in other genes harbour unrecognised pathogenic variants associated with Mendelian disorders. The systematic interrogation of PAS could improve genetic testing yields.

Missense Variants in the Histone Acetyltransferase Complex Component Gene TRRAP Cause Autism and Syndromic Intellectual Disability.

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants.

Pharmacodynamic Study of Miransertib in Individuals with Proteus Syndrome.

Proteus syndrome is a life-threatening segmental overgrowth syndrome caused by a mosaic gain-of-function AKT1 variant. There are no effective treatments for Proteus syndrome. Miransertib is an AKT1 inhibitor that, prior to this study, has been evaluated only in adult oncology trials. We designed a non-randomized, phase 0/1 pilot study of miransertib in adults and children with Proteus syndrome to identify an appropriate dosage starting point for a future efficacy trial using a pharmacodynamic endpoint. The primary endpoint was a 50% reduction in the tissue levels of AKT phosphorylation from biopsies in affected individuals. We also evaluated secondary efficacy endpoints. We found that a dose of 5 mg/m2/day (1/7 the typical dose used in oncology) led to a 50% reduction in phosphorylated AKT (pAKT) in affected tissues from five of six individuals. This dose was well tolerated. Two of the six efficacy endpoints (secondary objectives) suggested that this agent may be efficacious. We observed a decrease in a cerebriform connective tissue nevus and a reduction in pain in children. We conclude that 5 mg/m2/day of miransertib is an appropriate starting point for future efficacy trials and that this agent shows promise of therapeutic efficacy in children with Proteus syndrome.

Safety and efficacy of low-dose sirolimus in the PIK3CA-related overgrowth spectrum.

PURPOSE: PIK3CA-related overgrowth spectrum (PROS) encompasses a range of debilitating conditions defined by asymmetric overgrowth caused by mosaic activating PIK3CA variants. PIK3CA encodes the p110α catalytic subunit of phosphatidylinositol-3-kinase (PI3K), a critical transducer of growth factor signaling. As mTOR mediates the growth-promoting actions of PI3K, we hypothesized that the mTOR inhibitor sirolimus would slow pathological overgrowth. METHODS: Thirty-nine participants with PROS and progressive overgrowth were enrolled into open-label studies across three centers, and results were pooled. For the primary outcome, tissue volumes at affected and unaffected sites were measured by dual energy X-ray absorptiometry during 26 weeks of untreated run-in and 26 weeks of sirolimus therapy. RESULTS: Thirty participants completed the study. Sirolimus led to a change in mean percentage total tissue volume of -7.2% (SD 16.0, p = 0.04) at affected sites, but not at unaffected sites (+1.7%, SD 11.5, p = 0.48) (n = 23 evaluable). Twenty-eight of 39 (72%) participants had ≥1 adverse event related to sirolimus of which 37% were grade 3 or 4 in severity and 7/39 (18%) participants were withdrawn consequently. CONCLUSION: This study suggests that low-dose sirolimus can modestly reduce overgrowth, but cautions that the side-effect profile is significant, mandating individualized risk-benefit evaluations for sirolimus treatment in PROS.