Development of a Genomic DNA Reference Material Panel for Rett Syndrome (MECP2-Related Disorders) Genetic Testing

Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett syndrome patients have severe neurological and behavioral problems. Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process; however, none are commercially available for MECP2 genetic testing. The Centers for Disease Control and Prevention’s Genetic Testing Reference Material Coordination Program, in collaboration with the genetic testing community and the Coriell Cell Repositories, established 27 new cell lines and characterized the MECP2 mutations in these and in 8 previously available cell lines. DNA samples from the 35 cell lines were tested by eight clinical genetic testing laboratories using DNA sequence analysis and methods to assess copy number (multiplex ligation-dependent probe amplification, semiquantitative PCR, or array-based comparative genomic hybridization). The eight common point mutations known to cause approximately 60% of Rett syndrome cases were identified, as were other MECP2 variants, including deletions, duplications, and frame shift and splice-site mutations. Two of the 35 samples were from males with MECP2 duplications. These MECP2 and other characterized genomic DNA samples are publicly available from the NIGMS Repository at the Coriell Cell Repositories.Rett syndrome is a dominant X-linked disorder usually caused by point mutations (approximately 80% in classical and 40% in atypical cases) and deletions or insertions (approximately 15% to 18% in classical and 3% in atypical cases) in the MECP2 gene, although patients with mutations in two other genes, CDKL5 and FOXG1, may also exhibit a Rett-like phenotype.^1,2 MECP2, located on Xq28 and comprising four exons, encodes methyl-CpG binding protein 2 (MeCP2). In males, Rett syndrome is usually lethal, because of abnormal MeCP2 function from the single X chromosome and severe neonatal encephalopathy, although Rett syndrome in males with an XXY karyotype has been reported.³ Duplications in MECP2 have also been observed, and these can cause severe neurodevelopmental disability in males.⁴ The prevalence of Rett syndrome in females is approximately 1:10,000.⁵Girls with classic Rett syndrome (OMIM #312750) exhibit a rapid decline in language and motor skills at approximately 1 year of age. These patients exhibit a loss of acquired purposeful hand use, loss of communication, gait ataxia and apraxia, and stereotypic hand movements. They may also exhibit additional symptoms, including bruxism, seizures, episodic apnea or hyperpnea, abnormal muscle tone, and often acquired microcephaly.^4,6,7Patients with Rett syndrome can present with various phenotypes. Those diagnosed with atypical Rett syndrome may have either more mild or more severe presentation than patients with the classical form. These patients share some of the same symptoms with classical Rett syndrome cases, but must also have some of the additional symptoms listed above.^4,6 Some patients present with only mild learning disabilities (females) or intellectual disability (males).^4,8Molecular diagnosis of Rett syndrome is performed by examination of the patient’s DNA for MECP2 mutations, using a variety of molecular diagnostic methods. Most MECP2 mutations are sporadic, and testing is performed on the proband, although predictive prenatal and preimplantation genetic testing may also be performed. DNA sequence analysis may detect point mutations and small insertions and deletions (indels). Larger deletions and duplications are detected using multiplex ligation-dependent probe amplification (MLPA), quantitative PCR, and array-based comparative genomic hybridization (CGH). To date, there are no US Food and Drug Administration (FDA)–approved assays for Rett syndrome. All testing is performed using laboratory-developed tests.Reference materials are needed by laboratories to comply with regulatory and accreditation requirements for assay development, assay validation, and quality control, and their use is recommended by professional guidelines for clinical laboratories [eg, http://www.acmg.net/Pages/ACMG_Activities/stds-2002/g.htm, last accessed October 16, 2013; Washington State Legislature, http://www.doh.wa.gov/hsqa/fsl/lqa_home.htm, last accessed January 11, 2013; College of American Pathologists http://www.cap.org/apps/cap.portal, last accessed January 11, 2013 (registration required); New York State Clinical Laboratory Evaluation Program, http://www.wadsworth.org/clep, last accessed January 11, 2013).^9–15 Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process. Ideally, these reference materials should closely resemble patient samples containing variants and types of variants common to the disorder and should be thoroughly characterized using methods different from those used in the user’s laboratory.¹⁵ For Rett syndrome, genomic DNA reference materials derived from females (and, if possible, males) containing common point mutations, indels, and larger deletions and duplications should be used. Careful use of a well-characterized and comprehensive set of reference materials helps to assure the proper design and function of a clinical assay. To date, there are no commercially available reference materials for Rett syndrome genetic testing.To address the need for characterized genomic DNA reference materials for Rett syndrome testing, the Centers for Disease Control and Prevention (CDC) based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the genetic testing community and the National Institute of General Medical Sciences (NIGMS) Repository at the Coriell Cell Repositories, have characterized the MECP2 mutations in 35 publicly available cell lines. Twenty-seven of the 35 cell lines were generated as part of this project, using blood collected with informed consent from Rett syndrome patients with variants not previously represented in cell lines at the Coriell Repository. The availability of a renewable source of characterized reference materials for Rett syndrome helps to assure the accuracy of these genetic tests and facilitate research and test development.     

The morbid anatomy of the dermatologic genome: An update for the third millennium

Background Much progress has been made in recent years in the identification of genes underlying many hereditary skin diseases. Objective To provide an update on the status of the identification of genes involved in hereditary skin disorders and to compare the current standing with that in the last decade. Methods A review of the literature is presented here in a series of lists describing the chromosomal location, specific gene, clinical relevance, and availability of molecular-based genetic tests for each genodermatosis. Results Progress has been made in identifying the genes underlying many disorders of cornification, genodermatoses with malignant potential, bullous disorders, pigmentary disorders, disorders affecting the epidermal appendages and the dermis, and other miscellaneous genodermatoses. Conclusion The great progress made toward the completion of the human gene sequence and the continued efforts of many clinical and molecular scientists to identify disease genes will make diagnosis of hereditary dermatological disorders more precise and allow accurate family counseling as well as possibly leading to more targeted therapies during this millennium.

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Sommaire Antécédents D’normes progrès ont été réalisés au cours des dernières années dans le domaine de l’identification des gènes qui sont à l’origine de nombreuses affections cutanées héréditaires. Objectifs Faire le point sur l’état de l’identification des gènes impliqués dans les affections cutanées héréditaires et comparer la situation actuelle avec celle qui a prévalu au cours de la dernière décennie. Méthodes Nous présentons ici une revue de la littérature sous forme de listes dans lesquelles sont décrits pour chacune des génodermatoses l’emplacement du chromosome, le gène spécifique, la pertinence clinique et la disponibilité des tests génétiques basés sur l’étude des molécules. Résultats Des progrés ont été réalisés relativement à l’identification des gènes responsables de nombreuses affections de kératinisation, de génodermatoses à potentiel malin, d’affections bulleuses, d’affections pigmentaires, d’affections touchant les appendices épidermiques et le derme, et de diverses autres génodermatoses. Conclusion Le parachèvement imminent de la séquence du génome humain ainsi que les efforts soutenus des nombreux chercheurs dans le domaine clinique et moléculaire pour identifier les gènes à l’origine des maladies rendront le diagnostic des affections dermatologiques héréditaires plus précis, permettront de fournir un conseil génétique plus fiable et donneront peut-être également lieu à l’émergence de traitements davantage ciblés au cours du présent millénaire.

     

Understanding the current sexual health service provision for mental health consumers by nurses in mental health settings: Findings from a Survey in Australia and England

Nurses in mental health settings avoid talking to consumers about sexual health concerns. It is unclear whether this avoidance prevents the provision of sexual healthcare. The present study gathered information about how mental health nurses respond to sexual health issues within their routine practice, what issues they address, and their view on their role in promoting sexual health for consumers. A cross‐sectional study using an electronic survey questionnaire, originally generated from a previous study in the united kingdom, was employed. The study occurred in four National Health Service Trusts in England and a national call for participants in Australia. Participants were nurse clinicians (n = 303) who self‐selected by completing surveys available via email and newsletters containing links to the survey. The results demonstrated that mental health nurses do not routinely include sexual health in their practice and are poorly prepared in knowing what to do with a sexual health issue, and what services to assist consumers to use. In conclusion, it has been well established in the literature that mental health consumers experience high sexual health needs that potentially impact on health and recovery. Mental health nurses are ideally placed to promote sexual health and refer consumers to sexual health and family planning services. Training to improve the confidence and responsiveness of mental health nurses to sexual health is an urgent need.     

Identifying early behavioral and molecular markers of future stress sensitivity

Puberty is a plastic period of neurological development when critical maturation of stress pathways occurs. Abnormal maturation may be predictive of future stress sensitivity and affective disorder risk. To identify potential early markers of stress-related disease predisposition, we examined physiological and behavioral stress responses in male pubertal mice compared with adults, using a genetic model of elevated stress sensitivity, CRF receptor-2 (CRFR2)-deficient mice. Juvenile mice of both genotypes exhibited greater basal and stress-induced corticosterone levels than adult mice, indicating that overall hypothalamic-pituitary-adrenal axis sensitivity diminishes in adulthood. However, juvenile CRFR2-deficient mice displayed a delayed stress recovery typical of adults of this genotype, suggesting an early marker of stress sensitivity. The adult phenotype of reduced hippocampal glucocorticoid receptor expression in these sensitive mice was also detected during puberty. This reduction may account for an impaired hypothalamic-pituitary-adrenal axis negative feedback and as such be an early indicator of a stress-sensitive phenotype. Examination of behavioral responses to stress revealed that CRFR2-deficient mice show exaggerated postpubertal maturation. Although wild-type mice did not alter their burying response to stress-provoking marbles after puberty, CRFR2-deficient mice showed a dramatic increase in burying behavior. We conclude that identification of abnormal pubertal stress pathway maturation may be predictive of adult heightened stress sensitivity and future susceptibility to stress-related affective disorders.