Genomic and molecular characterization of preterm birth.

Preterm birth (PTB) complications are the leading cause of long-term morbidity and mortality in children. By using whole blood samples, we integrated whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and DNA methylation data for 270 PTB and 521 control families. We analyzed this combined dataset to identify genomic variants associated with PTB and secondary analyses to identify variants associated with very early PTB (VEPTB) as well as other subcategories of disease that may contribute to PTB. We identified differentially expressed genes (DEGs) and methylated genomic loci and performed expression and methylation quantitative trait loci analyses to link genomic variants to these expression and methylation changes. We performed enrichment tests to identify overlaps between new and known PTB candidate gene systems. We identified 160 significant genomic variants associated with PTB-related phenotypes. The most significant variants, DEGs, and differentially methylated loci were associated with VEPTB. Integration of all data types identified a set of 72 candidate biomarker genes for VEPTB, encompassing genes and those previously associated with PTB. Notably, PTB-associated genes RAB31 and RBPJ were identified by all three data types (WGS, RNA-seq, and methylation). Pathways associated with VEPTB include EGFR and prolactin signaling pathways, inflammation- and immunity-related pathways, chemokine signaling, IFN-γ signaling, and Notch1 signaling. Progress in identifying molecular components of a complex disease is aided by integrated analyses of multiple molecular data types and clinical data. With these data, and by stratifying PTB by subphenotype, we have identified associations between VEPTB and the underlying biology.

Mutations in NOTCH1 cause Adams-Oliver syndrome.

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5' region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743-1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.

Utilization of genomic sequencing for population screening of immunodeficiencies in the newborn.

PurposeImmunodeficiency screening has been added to many state-directed newborn screening programs. The current methodology is limited to screening for severe T-cell lymphopenia disorders. We evaluated the potential of genomic sequencing to augment current newborn screening for immunodeficiency, including identification of non-T cell disorders.MethodsWe analyzed whole-genome sequencing (WGS) and clinical data from a cohort of 1,349 newborn-parent trios by genotype-first and phenotype-first approaches. For the genotype-first approach, we analyzed predicted protein-impacting variants in 329 immunodeficiency-related genes in the WGS data. As a phenotype-first approach, electronic health records were used to identify children with clinical features suggestive of immunodeficiency. Genomes of these children and their parents were analyzed using a separate pipeline for identification of candidate pathogenic variants for rare Mendelian disorders.ResultsWGS provides adequate coverage for most known immunodeficiency-related genes. 13,476 distinct variants and 8,502 distinct predicted protein-impacting variants were identified in this cohort; five individuals carried potentially pathogenic variants requiring expert clinical correlation. One clinically asymptomatic individual was found genomically to have complement component 9 deficiency. Of the symptomatic children, one was molecularly identified as having an immunodeficiency condition and two were found to have other molecular diagnoses.ConclusionNeonatal genomic sequencing can potentially augment newborn screening for immunodeficiency.

Utility of whole-genome sequencing for detection of newborn screening disorders in a population cohort of 1,696 neonates.

PURPOSE: To assess the potential of whole-genome sequencing (WGS) to replicate and augment results from conventional blood-based newborn screening (NBS). METHODS: Research-generated WGS data from an ancestrally diverse cohort of 1,696 infants and both parents of each infant were analyzed for variants in 163 genes involved in disorders included or under discussion for inclusion in US NBS programs. WGS results were compared with results from state NBS and related follow-up testing. RESULTS: NBS genes are generally well covered by WGS. There is a median of one (range: 0-6) database-annotated pathogenic variant in the NBS genes per infant. Results of WGS and NBS in detecting 28 state-screened disorders and four hemoglobin traits were concordant for 88.6% of true positives (n = 35) and 98.9% of true negatives (n = 45,757). Of the five infants affected with a state-screened disorder, WGS identified two whereas NBS detected four. WGS yielded fewer false positives than NBS (0.037 vs. 0.17%) but more results of uncertain significance (0.90 vs. 0.013%). CONCLUSION: WGS may help rule in and rule out NBS disorders, pinpoint molecular diagnoses, and detect conditions not amenable to current NBS assays.

Expanding the phenotypic spectrum in EP300-related Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome (RSTS) can be caused by heterozygous mutations or deletions involving CREBBP or, less commonly, EP300. To date, only 15 patients with EP300 mutations have been clinically described. Frequently reported manifestations in these patients include characteristic facial and limb features, varying degrees of neurocognitive dysfunction, and maternal preeclampsia. Other congenital anomalies are less frequently reported. We describe a child found to have a de novo EP300 mutation (c.4933C>T, predicted to result in p.Arg1645X) through research-based whole-genome sequencing of the family trio. The child's presentation involved dysmorphic features as well as unilateral renal agenesis, a myelomeningocele, and minor genitourinary anomalies. The involvement of congenital anomalies in all 16 clinically described patients with EP300 mutations (25% of which have been identified by "hypothesis free" methods, including microarray, exome, and whole-genome sequencing) is reviewed. In summary, genitourinary anomalies have been identified in 38%, cardiovascular anomalies in 25%, spinal/vertebral anomalies in 19%, other skeletal anomalies in 19%, brain anomalies in 13%, and renal anomalies in 6%. Our patient expands the phenotypic spectrum in EP300-related RSTS; this case demonstrates the evolving practice of clinical genomics related to increasing availability of genomic sequencing methods.

Chromosome 4q deletion syndrome: narrowing the cardiovascular critical region to 4q32.2-q34.3.

The 4q deletion syndrome is a rare chromosome deletion syndrome with a wide range of clinical phenotypes. There is limited clinical phenotype and molecular correlation for congenital heart defects (CHDs) reported so far for this region primarily because many cases are large deletions, often terminal, and because high-resolution array has not been reported in the evaluation of this group of patients. CHDs are reported in about 60% of patients with 4q deletion syndrome, occurring in the presence or absence of dHAND deletion, implying the existence of additional genes in 4q whose dosage influences cardiac development. We report an 8-month-old patient with a large mid-muscular to outlet ventricular septal defect (VSD), moderate-sized secundum-type atrial septal defect (ASD), thickened, dysplastic pulmonary valve with mild stenosis and moderate pulmonic regurgitation, and patent ductus arteriosus (PDA). Illumina CytoSNP array analysis disclosed a de novo, heterozygous, interstitial deletion of 11.6 Mb of genomic material from the long arm of chromosome 4, at 4q32.3-q34.3 (Chr4:167236114-178816031; hg18). The deleted region affects 37 RefSeq genes (hg18), including two provisional microRNA stemloops. Three genes in this region, namely TLL1 (Tolloid-like-1), HPGD (15-hydroxyprostaglandin dehydrogenase), and HAND2 (Heart and neural crest derivatives-expressed protein 2), are known to be involved in cardiac morphogenesis. This report narrows the critical region responsible for CHDs seen in 4q deletion syndrome.

An evidence-based approach to establish the functional and clinical significance of copy number variants in intellectual and developmental disabilities.

PURPOSE: Copy number variants have emerged as a major cause of human disease such as autism and intellectual disabilities. Because copy number variants are common in normal individuals, determining the functional and clinical significance of rare copy number variants in patients remains challenging. The adoption of whole-genome chromosomal microarray analysis as a first-tier diagnostic test for individuals with unexplained developmental disabilities provides a unique opportunity to obtain large copy number variant datasets generated through routine patient care. METHODS: A consortium of diagnostic laboratories was established (the International Standards for Cytogenomic Arrays consortium) to share copy number variant and phenotypic data in a central, public database. We present the largest copy number variant case-control study to date comprising 15,749 International Standards for Cytogenomic Arrays cases and 10,118 published controls, focusing our initial analysis on recurrent deletions and duplications involving 14 copy number variant regions. RESULTS: Compared with controls, 14 deletions and seven duplications were significantly overrepresented in cases, providing a clinical diagnosis as pathogenic. CONCLUSION: Given the rapid expansion of clinical chromosomal microarray analysis testing, very large datasets will be available to determine the functional significance of increasingly rare copy number variants. This data will provide an evidence-based guide to clinicians across many disciplines involved in the diagnosis, management, and care of these patients and their families.

A complex 6p25 rearrangement in a child with multiple epiphyseal dysplasia.

Genomic rearrangements are increasingly recognized as important contributors to human disease. Here we report on an 11½-year-old child with myopia, Duane retraction syndrome, bilateral mixed hearing loss, skeletal anomalies including multiple epiphyseal dysplasia, and global developmental delay, and a complex 6p25 genomic rearrangement. We have employed oligonucleotide-based comparative genomic hybridization arrays (aCGH) of different resolutions (44 and 244K) as well as a 1 M single nucleotide polymorphism (SNP) array to analyze this complex rearrangement. Our analyses reveal a complex rearrangement involving a ∼2.21 Mb interstitial deletion, a ∼240 kb terminal deletion, and a 70-80 kb region in between these two deletions that shows maintenance of genomic copy number. The interstitial deletion contains eight known genes, including three Forkhead box containing (FOX) transcription factors (FOXQ1, FOXF2, and FOXC1). The region maintaining genomic copy number partly overlaps the dual specificity protein phosphatase 22 (DUSP22) gene. Array analyses suggest a homozygous loss of genomic material at the 5' end of DUSP22, which was corroborated using TaqMan® copy number analysis. It is possible that this homozygous genomic loss may render both copies of DUSP22 or its products non-functional. Our analysis suggests a rearrangement mechanism distinct from a previously reported replication-based error-prone mechanism without template switching for a specific 6p25 rearrangement with a 1.22 Mb interstitial deletion. Our study demonstrates the utility and limitations of using oligonucleotide-based aCGH and SNP array technologies of increasing resolutions in order to identify complex DNA rearrangements and gene disruptions.

Deletion at chromosomal band Xp22.12-Xp22.13 involving PDHA1 in a patient with congenital lactic acidosis.

We present a patient with congenital lactic acidosis, agenesis of the corpus callosum, and profound developmental delay. Assays of pyruvate dehydrogenase complex function were normal in lymphocytes, but decreased in fibroblasts. Sequencing of the PDHA1 gene did not reveal deleterious mutations, and BAC based microarray analysis did not reveal any chromosomal abnormality. However, gene dosage analysis with oligonucleotide-based chromosomal microarray revealed a deletion of Xp22.12-Xp22.13 involving complete deletion of PDHA1. This is the first report of a whole gene deletion of PDHA1 detected by oligonucleotide-based microarray.

Duplication 16p11.2 in a child with infantile seizure disorder.

Submicroscopic recurrent 16p11.2 rearrangements are associated with several neurodevelopmental disorders, including autism, mental retardation, and schizophrenia. The common 16p11.2 region includes 24 known genes, of which 22 are expressed in the developing human fetal nervous system. As yet, the mechanisms leading to neurodevelopmental abnormalities and the broader phenotypes associated with deletion or duplication of 16p11.2 have not been clarified. Here we report a child with spastic quadriparesis, refractory infantile seizures, severe global developmental delay, hypotonia, and microcephaly, and a de novo 598 kb 16p11.2 microduplication. Family history is negative for any of these features in parents and immediate family members. Sequencing analyses showed no mutations in DOC2A, QPRT, and SEZ6L2, genes within the duplicated 16p11.2 region that have been implicated in neuronal function and/or seizure related phenotypes. The child's clinical course is consistent with a rare seizure disorder called malignant migrating partial seizure disorder of infancy, raising the possibility that duplication or disruption of genes in the 16p11.2 interval may contribute to this severe disorder.

Increased plasma and tissue guanidino compounds in a mouse model of hyperargininemia.

In humans, arginase I (AI)-deficiency results in hyperargininemia, a metabolic disorder with symptoms of progressive neurological and intellectual impairment, spasticity, persistent growth retardation, and episodic hyperammonemia. A deficiency of arginase II (AII) has never been detected and the clinical disorder, if any, associated with its deficiency has not been defined. Since the spasticity and paucity of hyperammonemic crises seen in human AI-deficient patients are not features of the other urea cycle disorders, the likelihood of ammonia as the main neuropathogenic agent becomes extremely low, and the modest elevations of arginine seen in the brains of our mouse model of hyperargininemia make it an unlikely candidate as well. Specific guanidino compounds, direct or indirect metabolites of arginine, are elevated in the blood of patients with uremia. Other guanidino compounds are also increased in plasma and cerebrospinal fluid of hyperargininemic patients making them plausible as neurotoxins in these disorders. We analyzed several guanidino compounds in our arginase single and double knockout animals and found that alpha-keto-delta-guanidinovaleric acid, alpha-N-acetylarginine, and argininic acid were increased in the brain tissue from the AI knockout and double knockout animals. Several compounds were also increased in the plasma, liver, and kidneys. This is the first time that several of the guanidino compounds have been shown to be elevated in the brain tissue of an arginase-deficient mammal, and it further supports their possible role as the neuropathogenic agents responsible for the complications seen in arginase deficiency.

Mouse model for human arginase deficiency.

Deficiency of liver arginase (AI) causes hyperargininemia (OMIM 207800), a disorder characterized by progressive mental impairment, growth retardation, and spasticity and punctuated by sometimes fatal episodes of hyperammonemia. We constructed a knockout mouse strain carrying a nonfunctional AI gene by homologous recombination. Arginase AI knockout mice completely lacked liver arginase (AI) activity, exhibited severe symptoms of hyperammonemia, and died between postnatal days 10 and 14. During hyperammonemic crisis, plasma ammonia levels of these mice increased >10-fold compared to those for normal animals. Livers of AI-deficient animals showed hepatocyte abnormalities, including cell swelling and inclusions. Plasma amino acid analysis showed the mean arginine level in knockouts to be approximately fourfold greater than that for the wild type and threefold greater than that for heterozygotes; the mean proline level was approximately one-third and the ornithine level was one-half of the proline and ornithine levels, respectively, for wild-type or heterozygote mice--understandable biochemical consequences of arginase deficiency. Glutamic acid, citrulline, and histidine levels were about 1.5-fold higher than those seen in the phenotypically normal animals. Concentrations of the branched-chain amino acids valine, isoleucine, and leucine were 0.4 to 0.5 times the concentrations seen in phenotypically normal animals. In summary, the AI-deficient mouse duplicates several pathobiological aspects of the human condition and should prove to be a useful model for further study of the disease mechanism(s) and to explore treatment options, such as pharmaceutical administration of sodium phenylbutyrate and/or ornithine and development of gene therapy protocols.

New observations on maternal age effect on germline de novo mutations.

Germline mutations are the source of evolution and contribute substantially to many health-related processes. Here we use whole-genome deep sequencing data from 693 parents-offspring trios to examine the de novo point mutations (DNMs) in the offspring. Our estimate for the mutation rate per base pair per generation is 1.05 × 10(-8), well within the range of previous studies. We show that maternal age has a small but significant correlation with the total number of DNMs in the offspring after controlling for paternal age (0.51 additional mutations per year, 95% CI: 0.29, 0.73), which was not detectable in the smaller and younger parental cohorts of earlier studies. Furthermore, while the total number of DNMs increases at a constant rate for paternal age, the contribution from the mother increases at an accelerated rate with age.These observations have implications related to the incidence of de novo mutations relating to maternal age.

A novel method for creating artificial mutant samples for performance evaluation and quality control in clinical molecular genetics.

The lack of readily available, patient-derived materials for molecular genetic testing of many heterozygous or rare disorders creates a major impediment for laboratory proficiency and quality control procedures. The paucity of clinically derived mutation-positive samples could be surmounted if it were possible to construct artificial samples containing mutations of interest that would sufficiently resemble natural human samples. Such samples could then function as acceptable and realistic performance evaluation challenges and quality control reagents for recipient laboratories. Using the cystic fibrosis gene (CFTR) as a prototype, we have devised and executed experiments designed to generate unique DNA samples that could be used for these purposes. We used site-directed mutagenesis to generate mutations of interest in plasmid DNA derived from common bacterial artificial chromosome sources containing the cystic fibrosis transmembrane conductance receptor gene. CFTR mutations G85E and 1078delT were chosen to represent mutations in the original American College of Medical Genetics-recommended population-screening panel of 25 mutations. DNA samples containing predetermined concentrations and ratios of wild-type and mutated plasmids, bacterial artificial chromosomes of interest, and nonhuman genomic carrier DNA were characterized and tested in-house and in a group of nine pilot testing laboratories using a variety of technical platforms. The results indicate that these constructs, containing CFTR mutations in heterozygous and homozygous states, can serve as valid and accessible materials for quality assurance, including performance evaluation, proficiency testing, and assay quality control.

Arginase expression in mouse embryonic development.

We are using the model of the developing mouse embryo to elucidate the pattern of arginase expression in mammalian cells in normal animals and in arginase I (AI) deficiency during development by digoxigenin-labeled RNA in situ hybridization. Our goal is to understand the regulation of these isozymes, with the expectation that this knowledge will help patients suffering from AI deficiency. We found that AI mRNA was widely and strongly expressed in the normal developing mouse embryo; in contrast, a relatively strong AII mRNA signal was found only in the intestine. In the AI knockout mouse embryo, no AII overexpression was found. These results indicated that arginases are needed in mouse embryonic development and AI is the principal form required. The strong AI expression in the peripheral nervous system suggests that the pathogenesis of the neurological retardation in AI deficiency may be conditioned by AI deficiency in the nervous system during embryonic development.

Diagnosis of D-Bifunctional Protein Deficiency through Whole-Genome Sequencing: Implications for Cost-Effective Care.

D-Bifunctional protein deficiency, caused by recessive mutations in HSD17B4, is a severe disorder of peroxisomal fatty acid oxidation. Nonspecific clinical features may contribute to diagnostic challenges. We describe a newborn female with infantile-onset seizures and nonspecific mild dysmorphisms who underwent extensive genetic workup that resulted in the detection of a novel homozygous mutation (c.302+1_4delGTGA) in the HSD17B4 gene, consistent with a diagnosis of D-bifunctional protein deficiency. By comparing the standard clinical workup to diagnostic analysis performed through research-based whole-genome sequencing (WGS), which independently identified the causative mutation, we demonstrated the ability of genomic sequencing to serve as a timely and cost-effective diagnostic tool for the molecular diagnosis of apparent and occult newborn diseases. As genomic sequencing becomes more available and affordable, we anticipate that WGS and related omics technologies will eventually replace the traditional tiered approach to newborn diagnostic workup.

Widespread expression of arginase I in mouse tissues. Biochemical and physiological implications.

Arginase I (AI), the fifth and final enzyme of the urea cycle, detoxifies ammonia as part of the urea cycle. In previous studies from others, AI was not found in extrahepatic tissues except in primate blood cells, and its roles outside the urea cycle have not been well recognized. In this study we undertook an extensive analysis of arginase expression in postnatal mouse tissues by in situ hybridization (ISH) and RT-PCR. We also compared arginase expression patterns with those of ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). We found that, outside of liver, AI was expressed in many tissues and cells such as the salivary gland, esophagus, stomach, pancreas, thymus, leukocytes, skin, preputial gland, uterus and sympathetic ganglia. The expression was much wider than that of arginase II, which was highly expressed only in the intestine and kidney. Several co-localization patterns of AI, ODC, and OAT have been found: (a) AI was co-localized with ODC alone in some tissues; (b) AI was co-localized with both OAT and ODC in a few tissues; (c) AI was not co-localized with OAT alone in any of the tissues examined; and (d) AI was not co-localized with either ODC or OAT in some tissues. In contrast, AII was not co-localized with either ODC or OAT alone in any of the tissues studied, and co-localization of AII with ODC and OAT was found only in the small intestine. The co-localization patterns of arginase, ODC, and OAT suggested that AI plays different roles in different tissues. The main roles of AI are regulation of arginine concentration by degrading arginine and production of ornithine for polyamine biosynthesis, but AI may not be the principal enzyme for regulating glutamate biosynthesis in tissues and cells.

Germline variation in cancer-susceptibility genes in a healthy, ancestrally diverse cohort: implications for individual genome sequencing.

Technological advances coupled with decreasing costs are bringing whole genome and whole exome sequencing closer to routine clinical use. One of the hurdles to clinical implementation is the high number of variants of unknown significance. For cancer-susceptibility genes, the difficulty in interpreting the clinical relevance of the genomic variants is compounded by the fact that most of what is known about these variants comes from the study of highly selected populations, such as cancer patients or individuals with a family history of cancer. The genetic variation in known cancer-susceptibility genes in the general population has not been well characterized to date. To address this gap, we profiled the nonsynonymous genomic variation in 158 genes causally implicated in carcinogenesis using high-quality whole genome sequences from an ancestrally diverse cohort of 681 healthy individuals. We found that all individuals carry multiple variants that may impact cancer susceptibility, with an average of 68 variants per individual. Of the 2,688 allelic variants identified within the cohort, most are very rare, with 75% found in only 1 or 2 individuals in our population. Allele frequencies vary between ancestral groups, and there are 21 variants for which the minor allele in one population is the major allele in another. Detailed analysis of a selected subset of 5 clinically important cancer genes, BRCA1, BRCA2, KRAS, TP53, and PTEN, highlights differences between germline variants and reported somatic mutations. The dataset can serve a resource of genetic variation in cancer-susceptibility genes in 6 ancestry groups, an important foundation for the interpretation of cancer risk from personal genome sequences.

Diagnosis of an imprinted-gene syndrome by a novel bioinformatics analysis of whole-genome sequences from a family trio.

Whole-genome sequencing and whole-exome sequencing are becoming more widely applied in clinical medicine to help diagnose rare genetic diseases. Identification of the underlying causative mutations by genome-wide sequencing is greatly facilitated by concurrent analysis of multiple family members, most often the mother-father-proband trio, using bioinformatics pipelines that filter genetic variants by mode of inheritance. However, current pipelines are limited to Mendelian inheritance patterns and do not specifically address disorders caused by mutations in imprinted genes, such as forms of Angelman syndrome and Beckwith-Wiedemann syndrome. Using publicly available tools, we implemented a genetic inheritance search mode to identify imprinted-gene mutations. Application of this search mode to whole-genome sequences from a family trio led to a diagnosis for a proband for whom extensive clinical testing and Mendelian inheritance-based sequence analysis were nondiagnostic. The condition in this patient, IMAGe syndrome, is likely caused by the heterozygous mutation c.832A>G (p.Lys278Glu) in the imprinted gene CDKN1C. The genotypes and disease status of six members of the family are consistent with maternal expression of the gene, and allele-biased expression was confirmed by RNA-Seq for the heterozygotes. This analysis demonstrates that an imprinted-gene search mode is a valuable addition to genome sequence analysis pipelines for identifying disease-causative variants.

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.