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      PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution

      research-article
      1 , 2 , , 3 , 4 , 5 , 6 , 7 , 7 , 2 , 8 , 2 , 2 , 9 , 1 , 2 , 10 , 11 , 12 , 13 , 14 , 14 , 14 , 15 , 16 , 1 , 17 , 18 , 18 , 19 , 20 , 21 , 22 , 23 , 22 , 23 , 24 , 24 , 12 , 25 , 26 , 27 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 7 , 48 , 49 , 4 , 1 , 2 , 35
      JCI Insight
      American Society for Clinical Investigation

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          Abstract

          Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals ( n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing ( n = 6), whole-exome sequencing ( n = 5), or Sanger sequencing ( n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations.

          Abstract

          The clinical and molecular spectrum of PIK3CA-related developmental disorders are correlated with types of mutations, tissue distributions, and levels of mosaicism with the clinical phenotype.

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          Most cited references39

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          Activating mutations of the stimulatory G protein in the McCune-Albright syndrome.

          The McCune-Albright syndrome is a sporadic disease characterized by polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and hyperfunction of multiple endocrine glands. These manifestations may be explained by a somatic mutation in affected tissues that results in activation of the signal-transduction pathway generating cyclic AMP (cAMP). We analyzed DNA from tissues of patients with the McCune-Albright syndrome for the presence of activating mutations of the gene for the alpha subunit of the G protein (Gs alpha) that stimulates cAMP formation. Genomic DNA fragments encompassing regions (exons 8 and 9) previously found to contain activating missense mutations of the Gs alpha gene (gsp mutations) in sporadically occurring pituitary tumors were amplified in tissues from four patients with the McCune-Albright syndrome by the polymerase chain reaction. The amplified DNA was analyzed for mutations by denaturing gradient gel electrophoresis and allele-specific oligonucleotide hybridization. We detected one of two activating mutations within exon 8 of the Gs alpha gene in tissues from all four patients, including affected endocrine organs (gonads, adrenal glands, thyroid, and pituitary) and tissues not classically involved in the McCune-Albright syndrome. In two of the patients, histidine was substituted for arginine at position 201 of Gs alpha, and in the other two patients cysteine was substituted for the same arginine residue. In each patient the proportion of cells affected varied from tissue to tissue. In two endocrine organs, the highest proportion of mutant alleles was found in regions of abnormal cell proliferation. Mutations within exon 8 of the Gs alpha gene that result in increased activity of the Gs protein and increased cAMP formation are present in various tissues of patients with the McCune-Albright syndrome. Somatic mutation of this gene early in embryogenesis could result in the mosaic population of normal and mutant-bearing tissues that may underlie the clinical manifestations of this disease.
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            Brain somatic mutations in MTOR cause focal cortical dysplasia type II leading to intractable epilepsy.

            Focal cortical dysplasia type II (FCDII) is a sporadic developmental malformation of the cerebral cortex characterized by dysmorphic neurons, dyslamination and medically refractory epilepsy. It has been hypothesized that FCD is caused by somatic mutations in affected regions. Here, we used deep whole-exome sequencing (read depth, 412-668×) validated by site-specific amplicon sequencing (100-347,499×) in paired brain-blood DNA from four subjects with FCDII and uncovered a de novo brain somatic mutation, mechanistic target of rapamycin (MTOR) c.7280T>C (p.Leu2427Pro) in two subjects. Deep sequencing of the MTOR gene in an additional 73 subjects with FCDII using hybrid capture and PCR amplicon sequencing identified eight different somatic missense mutations found in multiple brain tissue samples of ten subjects. The identified mutations accounted for 15.6% of all subjects with FCDII studied (12 of 77). The identified mutations induced the hyperactivation of mTOR kinase. Focal cortical expression of mutant MTOR by in utero electroporation in mice was sufficient to disrupt neuronal migration and cause spontaneous seizures and cytomegalic neurons. Inhibition of mTOR with rapamycin suppressed cytomegalic neurons and epileptic seizures. This study provides, to our knowledge, the first evidence that brain somatic activating mutations in MTOR cause FCD and identifies mTOR as a treatment target for intractable epilepsy in FCD.
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              PTEN: one gene, many syndromes.

              Charis Eng (2003)
              PTEN, on 10q23.3, encodes a major lipid phosphatase which signals down the phosphoinositol-3-kinase/Akt pathway and effects G1 cell cycle arrest and apoptosis. Germline PTEN mutations have been found to occur in 80% of classic Cowden syndrome (CS), 60% of Bannayan-Riley-Ruvalcaba syndrome (BRRS), up to 20% of Proteus syndrome (PS), and approximately 50% of a Proteus-like syndrome (PSL). CS is a heritable multiple hamartoma syndrome with a high risk of breast, thyroid, and endometrial carcinomas. BRRS is a congenital autosomal dominant disorder characterized by megencephaly, developmental delay, lipomatosis, and speckled penis. PS and PSL had never been associated with risk of malignancy. Finding germline PTEN mutations in patients with BRRS, PS, and PSL suggests equivalent risks of developing malignancy as in CS with implications for medical management. The mutational spectra of CS and BRRS overlap, with many of the mutations occurring in exons 5, 7, and 8. Genotype-phenotype association analyses have revealed that the presence of germline PTEN mutations is associated with breast tumor development, and that mutations occurring within and 5' of the phosphatase motif were associated with multi-organ involvement. Pooled analysis of PTEN mutation series of CS and BRRS occurring in the last five years reveals that 65% of CS-associated mutations occur in the first five exons encoding the phosphatase domain and the promoter region, while 60% of BRRS-associated mutations occur in the 3' four exons encoding mainly the C2 domain. Somatic PTEN mutations occur with a wide distribution of frequencies in sporadic primary tumors, with the highest frequencies in endometrial carcinomas and glioblastoma multiform. Several mechanisms of PTEN inactivation occur in primary malignancies derived from different tissues, but a favored mechanism appears to occur in a tissue-specific manner. Inappropriate subcellular compartmentalization and increased/decreased proteosome degradation may be two novel mechanisms of PTEN inactivation. Further functional work could reveal more effective means of molecular-directed therapy and prevention. Copyright 2003 Wiley-Liss, Inc.

                Author and article information

                Contributors
                Journal
                JCI Insight
                JCI Insight
                JCI Insight
                JCI Insight
                American Society for Clinical Investigation
                2379-3708
                16 June 2016
                16 June 2016
                16 June 2016
                : 1
                : 9
                : e87623
                Affiliations
                [1 ]Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, Washington, USA.
                [2 ]Center for Integrative Brain Research and
                [3 ]Center for Developmental Biology and Regenerative Medicine, Seattle Children’s Research Institute, Seattle, Washington, USA.
                [4 ]Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Neuroscience Department, A. Meyer Children’s Hospital, University of Florence, Florence, Italy.
                [5 ]Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
                [6 ]Department of Medical Genetics, Kasturba Medical College, Manipal University, Manipal, Karnataka, India.
                [7 ]Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
                [8 ]Whole Exome Sequencing Program, GeneDx, Gaithersburg, Maryland, USA.
                [9 ]Regional Genetics Program, The Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada.
                [10 ]Department of Pediatrics, and Pediatric Neurology and Developmental Medicine, University Children’s Hospital, Tübingen, Germany.
                [11 ]Mount Sinai Hospital, The Prenatal Diagnosis and Medical Genetics Division, Department of Obstetrics and Gynecology, and
                [12 ]Department of Pediatrics, Division of Clinical and Metabolic Genetics, University of Toronto, Toronto, Ontario, Canada.
                [13 ]Center for Human Genetics, University Hospitals Case Medical Center, Cleveland, Ohio, USA.
                [14 ]Department of Pediatrics, Division of Medical Genetics, Saint Louis University, St. Louis, Missouri, USA.
                [15 ]Department of Pediatrics, University of Vermont College of Medicine, Burlington, Vermont, USA.
                [16 ]Department of Clinical Genetics, Royal North Shore Hospital, St Leonards, New South Wales, Australia.
                [17 ]Belfast Health and Social Care Trust, Belfast, United Kingdom.
                [18 ]Genetics, Metabolism and Pediatrics, London, Ontario, Canada.
                [19 ]Clinical Genetics, Center for Personalized Medicine, Children’s Hospital Los Angeles, Keck School of Medicine at University of Southern California, Los Angeles, California, USA.
                [20 ]Neurology Unit, Schneider Children’s Medical Center of Israel, Petach Tikva, and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
                [21 ]School of Medicine, Griffith University, Brisbane, Queensland, Australia.
                [22 ]Division of Genetics, Department of Pediatrics, and
                [23 ]Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA.
                [24 ]Genetics Service, Department of Pediatric Medicine, KK Women’s and Children’s Hospital, Singapore.
                [25 ]British Columbia Medical Genetics Provincial Program, University of British Columbia, Vancouver, British Columbia, Canada.
                [26 ]Service de génétique médicale, Centre Hospitalier Universitaire Vaudois CHUV, Switzerland.
                [27 ]Greenwood Genetic Center, Greenwood, South Carolina, USA.
                [28 ]Department of Dermatology, Children Hospital Prof. Dr. J. P. Garrahan, Buenos Aires, Argentina.
                [29 ]Children’s Hospital of Michigan, Wayne State University, Detroit, Michigan, USA.
                [30 ]Center for Human Genetics, University Hospitals Leuven and KU Leuven, Leuven, Belgium.
                [31 ]South West Thames Regional Genetics Service, St George’s University NHS Foundation Trust, London, and Section of Cancer Genetics, Institute of Cancer Research, Sutton, United Kingdom.
                [32 ]Department of Pediatrics and Ophthalmology, University of Minnesota, Minneapolis, Minnesota, USA.
                [33 ]Department of Child Neurology, University of Florida, Jacksonville, Florida, USA.
                [34 ]Department of Genetics, Liverpool Hospital, Liverpool, New South Wales, Australia.
                [35 ]Department of Neurology, University of Washington, Seattle, Washington, USA.
                [36 ]Providence Child Neurology, Providence Sacred Heart Medical Center and Children’s Hospital, Spokane, Washington, USA.
                [37 ]Department of Pediatrics and Translational Genetics, Department of Pediatrics, Academic Medical Center, University of Amsterdam Medical Center, Amsterdam, The Netherlands.
                [38 ]Department of Laboratories, Seattle Children’s Hospital and
                [39 ]Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.
                [40 ]División de Pediatría, Pontificia Universidad Católica de Chile, Pediatra-Genetista, Unidad de Genética, Hospital Dr. Luis Calvo Mackenna, Santiago, Chile.
                [41 ]Department of Pediatrics, Sidney Kimmel Medical School at T. Jefferson University, Chief of Division of Medical Genetics, A.I. duPont Hospital for Children, Wilmington, Delaware, USA.
                [42 ]Departments of Dermatology and Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
                [43 ]Center for Human Genetics, University Hospitals Leuven, KU Leuven, Leuven, Belgium.
                [44 ]Clinical Genetics Unit, IRCCS Santa Maria Nuova Hospital, Reggio Emilia, Italy.
                [45 ]University of California, San Francisco, San Francisco/Genetic Medicine Central California, San Francisco, California, USA.
                [46 ]Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.
                [47 ]Koç University, School of Medicine, Medical Genetics Department, Koç University Hospital, Istanbul, Turkey.
                [48 ]Howard Hughes Medical Institute, Seattle, Washington, USA.
                [49 ]Department of Pediatrics, Cedars-Sinai Medical Center, Harbor-UCLA Medical Center, David Geffen School of Medicine Los Angeles, California, USA.
                Author notes
                Address correspondence to: Ghayda Mirzaa, Center for Integrative Brain Research, Seattle Children’s Research Institute, 1900 9th Avenue, Mailstop C9S-10, Seattle, Washington 98101, USA. Phone: 206.884.1276; E-mail: gmirzaa@ 123456uw.edu .
                Author information
                http://orcid.org/0000-0003-2648-7657
                http://orcid.org/0000-0003-4494-9771
                http://orcid.org/0000-0001-9278-5471
                http://orcid.org/0000-0002-4476-1309
                http://orcid.org/0000-0001-6762-8407
                http://orcid.org/0000-0002-7780-6542
                http://orcid.org/0000-0003-2017-4270
                http://orcid.org/0000-0003-1406-5000
                Article
                87623
                10.1172/jci.insight.87623
                5019182
                27631024
                f21fb6e0-a105-455c-8a78-56f93a1ab4b7
                Copyright © 2016 Mirzaa et al.

                This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 23 March 2016
                : 17 May 2016
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