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.^1,2MECP2, 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.