Heterozygous MAP3K20 variants cause ectodermal dysplasia, craniosynostosis, sensorineural hearing loss, and limb anomalies.

Biallelic pathogenic variants in MAP3K20, which encodes a mitogen-activated protein kinase, are a rare cause of split-hand foot malformation (SHFM), hearing loss, and nail abnormalities or congenital myopathy. However, heterozygous variants in this gene have not been definitively associated with a phenotype. Here, we describe the phenotypic spectrum associated with heterozygous de novo variants in the linker region between the kinase domain and leucine zipper domain of MAP3K20. We report five individuals with diverse clinical features, including craniosynostosis, limb anomalies, sensorineural hearing loss, and ectodermal dysplasia-like phenotypes who have heterozygous de novo variants in this specific region of the gene. These individuals exhibit both shared and unique clinical manifestations, highlighting the complexity and variability of the disorder. We propose that the involvement of MAP3K20 in endothelial-mesenchymal transition provides a plausible etiology of these features. Together, these findings characterize a disorder that both expands the phenotypic spectrum associated with MAP3K20 and highlights the need for further studies on its role in early human development.

Genomic newborn screening: Are we entering a new era of screening?

Population newborn screening (NBS) for phenylketonuria began in the United States in 1963. In the 1990s electrospray ionization mass spectrometry permitted an array of pathognomonic metabolites to be identified simultaneously, enabling up to 60 disorders to be recognized with a single test. In response, differing approaches to the assessment of the harms and benefits of screening have resulted in variable screening panels worldwide. Thirty years on and another screening revolution has emerged with the potential for first line genomic testing extending the range of screening conditions recognized after birth to many hundreds. At the annual SSIEM conference in 2022 in Freiburg, Germany, an interactive plenary discussion on genomic screening strategies and their challenges and opportunities was conducted. The Genomics England Research project proposes the use of Whole Genome Sequencing to offer extended NBS to 100 000 babies for defined conditions with a clear benefit for the child. The European Organization for Rare Diseases seeks to include "actionable" conditions considering also other types of benefits. Hopkins Van Mil, a private UK research institute, determined the views of citizens and revealed as a precondition that families are provided with adequate information, qualified support, and that autonomy and data are protected. From an ethical standpoint, the benefits ascribed to screening and early treatment need to be considered in relation to asymptomatic, phenotypically mild or late-onset presentations, where presymptomatic treatment may not be required. The different perspectives and arguments demonstrate the unique burden of responsibility on those proposing new and far-reaching developments in NBS programs and the need to carefully consider both harms and benefits.

Elective genomic testing: Practice resource of the National Society of Genetic Counselors.

Genetic counseling for patients who are pursuing genetic testing in the absence of a medical indication, referred to as elective genomic testing (EGT), is becoming more common. This type of testing has the potential to detect genetic conditions before there is a significant health impact permitting earlier management and/or treatment. Pre- and post-test counseling for EGT is similar to indication-based genetic testing. Both require a complete family and medical history when ordering a test or interpreting a result. However, EGT counseling has some special considerations including greater uncertainties around penetrance and clinical utility and a lack of published guidelines. While certain considerations in the selection of a high-quality genetic testing laboratory are universal, there are some considerations that are unique to the selection of a laboratory performing EGT. This practice resource intends to provide guidance for genetic counselors and other healthcare providers caring for adults seeking pre- or post-test counseling for EGT. Genetic counselors and other genetics trained healthcare providers are the ideal medical professionals to supply accurate information to individuals seeking counseling about EGT enabling them to make informed decisions about testing and follow-up.

Functional and structural analysis of cytokine-selective IL6ST defects that cause recessive hyper-IgE syndrome.

BACKGROUND: Biallelic variants in IL6ST, encoding GP130, cause a recessive form of hyper-IgE syndrome (HIES) characterized by high IgE level, eosinophilia, defective acute phase response, susceptibility to bacterial infections, and skeletal abnormalities due to cytokine-selective loss of function in GP130, with defective IL-6 and IL-11 and variable oncostatin M (OSM) and IL-27 levels but sparing leukemia inhibitory factor (LIF) signaling. OBJECTIVE: Our aim was to understand the functional and structural impact of recessive HIES-associated IL6ST variants. METHODS: We investigated a patient with HIES by using exome, genome, and RNA sequencing. Functional assays assessed IL-6, IL-11, IL-27, OSM, LIF, CT-1, CLC, and CNTF signaling. Molecular dynamics simulations and structural modeling of GP130 cytokine receptor complexes were performed. RESULTS: We identified a patient with compound heterozygous novel missense variants in IL6ST (p.Ala517Pro and the exon-skipping null variant p.Gly484_Pro518delinsArg). The p.Ala517Pro variant resulted in a more profound IL-6- and IL-11-dominated signaling defect than did the previously identified recessive HIES IL6ST variants p.Asn404Tyr and p.Pro498Leu. Molecular dynamics simulations suggested that the p.Ala517Pro and p.Asn404Tyr variants result in increased flexibility of the extracellular membrane-proximal domains of GP130. We propose a structural model that explains the cytokine selectivity of pathogenic IL6ST variants that result in recessive HIES. The variants destabilized the conformation of the hexameric cytokine receptor complexes, whereas the trimeric LIF-GP130-LIFR complex remained stable through an additional membrane-proximal interaction. Deletion of this membrane-proximal interaction site in GP130 consequently caused additional defective LIF signaling and Stüve-Wiedemann syndrome. CONCLUSION: Our data provide a structural basis to understand clinical phenotypes in patients with IL6ST variants.

An online compendium of treatable genetic disorders.

More than 4,000 genes have been associated with recognizable Mendelian/monogenic diseases. When faced with a new diagnosis of a rare genetic disorder, health care providers increasingly turn to internet resources for information to understand the disease and direct care. Unfortunately, it can be challenging to find information concerning treatment for rare diseases as key details are scattered across a number of authoritative websites and numerous journal articles. The website and associated mobile device application described in this article begin to address this challenge by providing a convenient, readily available starting point to find treatment information. The site, Rx-genes.com (https://www.rx-genes.com/), is focused on those conditions where the treatment is directed against the mechanism of the disease and thereby alters the natural history of the disease. The website currently contains 633 disease entries that include references to disease information and treatment guidance, a brief summary of treatments, the inheritance pattern, a disease frequency (if known), nonmolecular confirmatory testing (if available), and a link to experimental treatments. Existing entries are continuously updated, and new entries are added as novel treatments appear in the literature.

De novo loss-of-function KCNMA1 variants are associated with a new multiple malformation syndrome and a broad spectrum of developmental and neurological phenotypes.

KCNMA1 encodes the large-conductance Ca2+- and voltage-activated K+ (BK) potassium channel α-subunit, and pathogenic gain-of-function variants in this gene have been associated with a dominant form of generalized epilepsy and paroxysmal dyskinesia. Here, we genetically and functionally characterize eight novel loss-of-function (LoF) variants of KCNMA1. Genome or exome sequencing and the participation in the international Matchmaker Exchange effort allowed for the identification of novel KCNMA1 variants. Patch clamping was used to assess functionality of mutant BK channels. The KCNMA1 variants p.(Ser351Tyr), p.(Gly356Arg), p.(Gly375Arg), p.(Asn449fs) and p.(Ile663Val) abolished the BK current, whereas p.(Cys413Tyr) and p.(Pro805Leu) reduced the BK current amplitude and shifted the activation curves toward positive potentials. The p.(Asp984Asn) variant reduced the current amplitude without affecting kinetics. A phenotypic analysis of the patients carrying the recurrent p.(Gly375Arg) de novo missense LoF variant revealed a novel syndromic neurodevelopmental disorder associated with severe developmental delay, visceral and cardiac malformations, connective tissue presentations with arterial involvement, bone dysplasia and characteristic dysmorphic features. Patients with other LoF variants presented with neurological and developmental symptoms including developmental delay, intellectual disability, ataxia, axial hypotonia, cerebral atrophy and speech delay/apraxia/dysarthria. Therefore, LoF KCNMA1 variants are associated with a new syndrome characterized by a broad spectrum of neurological phenotypes and developmental disorders. LoF variants of KCNMA1 cause a new syndrome distinctly different from gain-of-function variants in the same gene.

Reducing Sanger confirmation testing through false positive prediction algorithms.

PURPOSE: Clinical genome sequencing (cGS) followed by orthogonal confirmatory testing is standard practice. While orthogonal testing significantly improves specificity, it also results in increased turnaround time and cost of testing. The purpose of this study is to evaluate machine learning models trained to identify false positive variants in cGS data to reduce the need for orthogonal testing. METHODS: We sequenced five reference human genome samples characterized by the Genome in a Bottle Consortium (GIAB) and compared the results with an established set of variants for each genome referred to as a truth set. We then trained machine learning models to identify variants that were labeled as false positives. RESULTS: After training, the models identified 99.5% of the false positive heterozygous single-nucleotide variants (SNVs) and heterozygous insertions/deletions variants (indels) while reducing confirmatory testing of nonactionable, nonprimary SNVs by 85% and indels by 75%. Employing the algorithm in clinical practice reduced overall orthogonal testing using dideoxynucleotide (Sanger) sequencing by 71%. CONCLUSION: Our results indicate that a low false positive call rate can be maintained while significantly reducing the need for confirmatory testing. The framework that generated our models and results is publicly available at https://github.com/HudsonAlpha/STEVE .

Next-generation sequencing for constitutional variants in the clinical laboratory, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Next-generation sequencing (NGS) technologies are now established in clinical laboratories as a primary testing modality in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude. It is now cost-effective to analyze an individual with disease-targeted gene panels, exome sequencing, or genome sequencing to assist in the diagnosis of a wide array of clinical scenarios. While clinical validation and use of NGS in many settings is established, there are continuing challenges as technologies and the associated informatics evolve. To assist clinical laboratories with the validation of NGS methods and platforms, the ongoing monitoring of NGS testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics (ACMG) has developed the following technical standards.

Functional variants in TBX2 are associated with a syndromic cardiovascular and skeletal developmental disorder.

The 17 genes of the T-box family are transcriptional regulators that are involved in all stages of embryonic development, including craniofacial, brain, heart, skeleton and immune system. Malformation syndromes have been linked to many of the T-box genes. For example, haploinsufficiency of TBX1 is responsible for many structural malformations in DiGeorge syndrome caused by a chromosome 22q11.2 deletion. We report four individuals with an overlapping spectrum of craniofacial dysmorphisms, cardiac anomalies, skeletal malformations, immune deficiency, endocrine abnormalities and developmental impairments, reminiscent of DiGeorge syndrome, who are heterozygotes for TBX2 variants. The p.R20Q variant is shared by three affected family members in an autosomal dominant manner; the fourth unrelated individual has a de novo p.R305H mutation. Bioinformatics analyses indicate that these variants are rare and predict them to be damaging. In vitro transcriptional assays in cultured cells show that both variants result in reduced transcriptional repressor activity of TBX2. We also show that the variants result in reduced protein levels of TBX2. Heterologous over-expression studies in Drosophila demonstrate that both p.R20Q and p.R305H function as partial loss-of-function alleles. Hence, these and other data suggest that TBX2 is a novel candidate gene for a new multisystem malformation disorder.

Expanding the phenotype of the CDKL5 deficiency disorder: Are seizures mandatory?

Pathogenic variants in the cyclin-dependent kinase-like 5 (CDKL5) gene cause the neurodevelopmental disorder, the CDKL5 deficiency disorder. Reports of individuals with pathogenic variants in CDKL5 without seizures are exceedingly rare, and in-depth analyses of their variants have been lacking. Whole-genome sequencing was performed on a 29-year-old female with mild intellectual disability who, in the absence of overt seizures, presented with multiple episodes of altered mental status over a 24-year period. Clinical history was supplemented by a parent completed questionnaire from the International CDKL5 Disorder Database. We identified a de novo heterozygous variant in CDKL5 (NM_003159.2:c.645T>A;p.Ser215Arg). In-depth computational analysis performed to predict the impact of the variant on protein structure and function demonstrated that the variant was likely pathogenic. In this light, cell-based studies showed that the S215R substitution causes a marked reduction in CDKL5 kinase activity. Similarities between our case and one previously reported case are striking. These cases, both without seizures but with apparent behavioral symptomatology, together question whether seizures are mandatory in this neurodevelopmental disorder.

Case for genome sequencing in infants and children with rare, undiagnosed or genetic diseases.

Up to 350 million people worldwide suffer from a rare disease, and while the individual diseases are rare, in aggregate they represent a substantial challenge to global health systems. The majority of rare disorders are genetic in origin, with children under the age of five disproportionately affected. As these conditions are difficult to identify clinically, genetic and genomic testing have become the backbone of diagnostic testing in this population. In the last 10 years, next-generation sequencing technologies have enabled testing of multiple disease genes simultaneously, ranging from targeted gene panels to exome sequencing (ES) and genome sequencing (GS). GS is quickly becoming a practical first-tier test, as cost decreases and performance improves. A growing number of studies demonstrate that GS can detect an unparalleled range of pathogenic abnormalities in a single laboratory workflow. GS has the potential to deliver unbiased, rapid and accurate molecular diagnoses to patients across diverse clinical indications and complex presentations. In this paper, we discuss clinical indications for testing and historical testing paradigms. Evidence supporting GS as a diagnostic tool is supported by superior genomic coverage, types of pathogenic variants detected, simpler laboratory workflow enabling shorter turnaround times, diagnostic and reanalysis yield, and impact on healthcare.

Understanding the present and preparing for the future: Exploring the needs of diagnostic and elective genomic medicine patients.

Advances in genomic knowledge and technology have increased the use of comprehensive clinical sequencing tests. Genome sequencing has established utility for diagnosing patients with rare, undiagnosed diseases as well as interest in an elective context, without a clinical indication for testing. The Smith Family Clinic for Genomic Medicine, LLC in Huntsville, AL is a private practice genomic medicine clinic caring for both diagnostic (79%) and elective (21%) patients. Diagnostic and elective patients are seen on a clinical basis and receive standard care. Genome sequencing is provided on a self-pay basis, with assistance available for diagnostic patients who have financial need. Here, we describe demographics and motivations of the distinct patient populations and our experiences engaging patients in online education. Diagnostic patients were motivated by the possibility of receiving an explanation for symptoms (96%) while elective patients were motivated by the chance to learn about future disease risk (57%). Elective patients were less likely to engage with online education, with only 28% reading all assigned topics compared to 54% of diagnostic patients. Understanding the needs, interests, and barriers unique to diagnostic and elective patients is critical to inform individualized and scalable best practices in patient education and engagement.

A toolkit for genetics providers in follow-up of patients with non-diagnostic exome sequencing.

There are approximately 7,000 rare diseases affecting 25-30 million Americans, with 80% estimated to have a genetic basis. This presents a challenge for genetics practitioners to determine appropriate testing, make accurate diagnoses, and conduct up-to-date patient management. Exome sequencing (ES) is a comprehensive diagnostic approach, but only 25%-41% of the patients receive a molecular diagnosis. The remaining three-fifths to three-quarters of patients undergoing ES remain undiagnosed. The Stanford Center for Undiagnosed Diseases (CUD), a clinical site of the Undiagnosed Diseases Network, evaluates patients with undiagnosed and rare diseases using a combination of methods including ES. Frequently these patients have non-diagnostic ES results, but strategic follow-up techniques identify diagnoses in a subset. We present techniques used at the CUD that can be adopted by genetics providers in clinical follow-up of cases where ES is non-diagnostic. Solved case examples illustrate different types of non-diagnostic results and the additional techniques that led to a diagnosis. Frequent approaches include segregation analysis, data reanalysis, genome sequencing, additional variant identification, careful phenotype-disease correlation, confirmatory testing, and case matching. We also discuss prioritization of cases for additional analyses.

A survey of current practices for genomic sequencing test interpretation and reporting processes in US laboratories.

PURPOSE: While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization. METHODS: Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free-text responses that were later clarified with phone interviews. RESULTS: Twenty-one laboratories participated. Practices highly concordant across all groups included consent documentation, multiperson case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing, or patient access to data. CONCLUSION: This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas of development in best practice guidelines that may be helpful.Genet Med advance online publication 03 Novemeber 2016.

High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.

Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.

The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants.(1) In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next-generation sequencing. By adopting and leveraging next-generation sequencing, clinical laboratories are now performing an ever-increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes, and epigenetic assays for genetic disorders. By virtue of increased complexity, this shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context the ACMG convened a workgroup in 2013 comprising representatives from the ACMG, the Association for Molecular Pathology (AMP), and the College of American Pathologists to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP, and College of American Pathologists stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories, including genotyping, single genes, panels, exomes, and genomes. This report recommends the use of specific standard terminology-"pathogenic," "likely pathogenic," "uncertain significance," "likely benign," and "benign"-to describe variants identified in genes that cause Mendelian disorders. Moreover, this recommendation describes a process for classifying variants into these five categories based on criteria using typical types of variant evidence (e.g., population data, computational data, functional data, segregation data). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a Clinical Laboratory Improvement Amendments-approved laboratory, with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or the equivalent.

Recurrent ACADVL molecular findings in individuals with a positive newborn screen for very long chain acyl-coA dehydrogenase (VLCAD) deficiency in the United States.

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.

EFTUD2 deficiency in vertebrates: Identification of a novel human mutation and generation of a zebrafish model.

BACKGROUND: Congenital microphthalmia and coloboma are severe developmental defects that are frequently associated with additional systemic anomalies and display a high level of genetic heterogeneity. METHODS: To identify the pathogenic variant in a patient with microphthalmia, coloboma, retinal dystrophy, microcephaly, and other features, whole exome sequencing analysis of the patient and parental samples was undertaken. To further explore the identified variant/gene, expression and functional studies in zebrafish were performed. RESULTS: Whole exome sequencing revealed a de novo variant, c.473_474delGA, p.(Arg158Lysfs*4), in EFTUD2 which encodes a component of the spliceosome complex. Dominant mutations in EFTUD2 cause Mandibulofacial Dysostosis, Guion-Almeida type, which does not involve microphthalmia, coloboma, or retinal dystrophy; analysis of genes known to cause these ocular phenotypes identified several variants of unknown significance but no causal alleles in the affected patient. Zebrafish eftud2 demonstrated high sequence conservation with the human gene and broad embryonic expression. TALEN-mediated disruption was employed to generate a c.378_385 del, p.(Ser127Aspfs*23) truncation mutation in eftud2. Homozygous mutants displayed a reduced head size, small eye, curved body, and early embryonic lethality. Apoptosis assays demonstrated a striking increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells in the developing brain, eye, spinal cord, and other tissues starting at 30 hours postfertilization. CONCLUSION: This study reports a novel mutation in EFTUD2 in a Mandibulofacial Dysostosis, Guion-Almeida type patient with unusual ocular features and the generation of a first animal model of eftud2 deficiency. The severe embryonic phenotype observed in eftud2 mutants indicates an important conserved role during development of diverse tissues in vertebrates.

A mosaic activating mutation in AKT1 associated with the Proteus syndrome.

BACKGROUND: The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS: We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots. RESULTS: Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation. CONCLUSIONS: The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).