MEF2C regulates NK cell effector functions through control of lipid metabolism.

Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR-Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.

Parental age effects and Rett syndrome.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder, and pathogenic Methyl-CpG-binding Protein 2 (MECP2) variants are identified in >95% of individuals with typical RTT. Most of RTT-causing variants in MECP2 are de novo and usually on the paternally inherited X chromosome. While paternal age has been reported to be associated with increased risk of genetic disorders, it is unknown whether parental age contributes to the risk of the development of RTT. Clinical data including parental age, RTT diagnostic status, and clinical severity are collected from 1226 participants with RTT and confirmed MECP2 variants. Statistical analyses are performed using Student t-test, single factor analysis of variance (ANOVA), and multi-factor regression. No significant difference is observed in parental ages of RTT probands compared to that of the general population. A small increase in parental ages is observed in participants with missense variants compared to those with nonsense variants. When we evaluate the association between clinical severity and parental ages by multiple regression analysis, there is no clear association between clinical severity and parental ages. Advanced parental ages do not appear to be a risk factor for RTT, and do not contribute to the clinical severity in individuals with RTT.

Anthropometric Measures Correspond with Functional Motor Outcomes in Females with Rett Syndrome.

OBJECTIVE: To characterize growth and anthropometric measurements in females with Rett syndrome and compare these measurements with functional outcomes. STUDY DESIGN: We obtained longitudinal growth and anthropometric measurements from 1154 females with classic and atypical Rett syndrome seen between 2006 and 2019 in the US Natural History Study. We calculated the Clinical Severity Score, Motor Behavior Assessment score, and arm and leg muscle areas and recorded the functional assessments of arm and hand use and ambulation. We compared growth and anthropometric variables from females with Rett syndrome in regard to normative data. We analyzed Clinical Severity Score, Motor Behavior Assessment, and anthropometric measurements in regard to functional assessments. RESULTS: Growth and anthropometric measurements were significantly lower in females with classic and severe atypical Rett syndrome compared with those classified as mild atypical Rett syndrome and deviated from normative patterns among all 3 groups. Suprailiac skinfold measurements correlated with body mass index measurements in each group. Lower leg muscle area measurements were significantly greater among females in all 3 Rett syndrome groups who ambulated independently compared with those who did not. In females with classic Rett syndrome, arm, thigh, and lower leg muscle area measurements increased significantly over time and were significantly greater among those who had purposeful arm and hand use and independent ambulation compared with those who did not. CONCLUSIONS: The pattern of growth and anthropometric measures in females with Rett syndrome differs from normative data and demonstrates clear differences between classic and mild or severe atypical Rett syndrome. Anthropometric measures correspond with functional outcomes and could provide markers supporting efficacy outcomes in clinical trials.

Mosaicism of common pathogenic MECP2 variants identified in two males with a clinical diagnosis of Rett syndrome.

Rett (RTT) syndrome, a neurodevelopmental disorder caused by pathogenic variation in the MECP2 gene, is characterized by developmental regression, loss of purposeful hand movements, stereotypic hand movements, abnormal gait, and loss of spoken language. Due to the X-linked inheritance pattern, RTT is typically limited to females. Recent studies revealed somatic mosaicism in MECP2 in male patients with RTT-like phenotypes. While detecting mosaic variation using Sanger sequencing is theoretically possible for mosaicism over ~15%-20%, several variables, including efficiency of PCR, background noise, and/or human error, contribute to a low detection rate using this technology. Mosaic variants in two males were detected by next generation sequencing (NGS; Case 1) and by Sanger re-sequencing (Case 2). Both had targeted digital PCR (dPCR) to confirm the variants. In this report, we present two males with classic RTT syndrome in whom we identified pathogenic variation in the MECP2 gene in the mosaic state (c.730C > T (p.Gln244*) in Patient 1 and c.397C > T (p.Arg133Cys) in Patient 2). In addition, estimates and measures of mosaic variant fraction were surprisingly similar between Sanger sequencing, NGS, and dPCR. The mosaic state of these variants contributed to a lengthy diagnostic odyssey for these patients. While NGS and even Sanger sequencing may be viable methods of detecting mosaic variation in DNA or RNA samples, applying targeted dPCR to supplement these sequencing technologies would provide confirmation of somatic mosaicism and mosaic fraction.

Biliary Tract Disease in Girls and Young Women With Rett Syndrome.

OBJECTIVE: We reviewed medical records and conducted a nationwide survey to characterize the clinical features and determine the prevalence of biliary tract disease in girls and women with Rett syndrome (RTT). METHODS: Sixty-two individuals with RTT and biliary tract disease were identified from the membership of Rett Syndrome Organization and patient files of the principal investigator. Medical records of 46 individuals were reviewed for presenting features, diagnostic tests, and treatment outcomes of biliary tract disease. We designed a questionnaire that probed the frequency of risk factors and treatment outcomes of biliary tract disease in RTT. The questionnaire was completed by 271 parents whose daughters met the clinical criteria for RTT and/or had MECP2 mutations and participated in the Natural History of Rett Syndrome Study. RESULTS: Presenting symptoms identified by record review included abdominal pain (94%), irritability (88%), weight loss (64%), and vomiting (52%). Biliary dyskinesia, cholecystitis, and cholelithiasis were identified in 90%, 77%, and 70%, respectively, by cholescintigraphy, surgical pathology, and abdominal ultrasound. The prevalence of biliary tract disease was 4.4% (n = 12) in the RTT cohort. Risk factors included older age (P < 0.001) and a positive family history (P < 0.01). Diagnoses included cholecystitis (n = 5), biliary dyskinesia (n = 6), and cholelithiasis (n = 7). Ten individuals underwent surgery; 7 had resolution of symptoms after surgical intervention. CONCLUSIONS: Biliary tract disease is not unique to RTT, but may be under-recognized because of the cognitive impairment of affected individuals. Early diagnostic evaluation and intervention may improve the health and quality of life of individuals affected with RTT and biliary tract disease.

Enrichment of mutations in chromatin regulators in people with Rett syndrome lacking mutations in MECP2.

PURPOSE: Rett syndrome (RTT) is a neurodevelopmental disorder caused primarily by de novo mutations in MECP2 and sometimes in CDKL5 and FOXG1. However, some RTT patients lack mutations in these genes. METHODS: Twenty-two RTT patients without apparent MECP2, CDKL5, and FOXG1 mutations were subjected to both whole-exome sequencing and single-nucleotide polymorphism array-based copy-number variant (CNV) analyses. RESULTS: Three patients had MECP2 mutations initially missed by clinical testing. Of the remaining 19, 17 (89.5%) had 29 other likely pathogenic intragenic mutations and/or CNVs (10 patients had 2 or more). Interestingly, 13 patients had mutations in a gene/region previously reported in other neurodevelopmental disorders (NDDs), thereby providing a potential diagnostic yield of 68.4%. These mutations were significantly enriched in chromatin regulators (corrected P = 0.0068) and moderately enriched in postsynaptic cell membrane molecules (corrected P = 0.076), implicating glutamate receptor signaling. CONCLUSION: The genetic etiology of RTT without MECP2, CDKL5, and FOXG1 mutations is heterogeneous, overlaps with other NDDs, and complicated by a high mutation burden. Dysregulation of chromatin structure and abnormal excitatory synaptic signaling may form two common pathological bases of RTT.Genet Med 19 1, 13-19.

Rare Noncoding Mutations Extend the Mutational Spectrum in the PGAP3 Subtype of Hyperphosphatasia with Mental Retardation Syndrome.

HPMRS or Mabry syndrome is a heterogeneous glycosylphosphatidylinositol (GPI) anchor deficiency that is caused by an impairment of synthesis or maturation of the GPI-anchor. The expressivity of the clinical features in HPMRS varies from severe syndromic forms with multiple organ malformations to mild nonsyndromic intellectual disability. In about half of the patients with the clinical diagnosis of HPMRS, pathogenic mutations can be identified in the coding region in one of the six genes, one among them is PGAP3. In this work, we describe a screening approach with sequence specific baits for transcripts of genes of the GPI pathway that allows the detection of functionally relevant mutations also including introns and the 5' and 3' UTR. By this means, we also identified pathogenic noncoding mutations, which increases the diagnostic yield for HPMRS on the basis of intellectual disability and elevated serum alkaline phosphatase. In eight affected individuals from different ethnicities, we found seven novel pathogenic mutations in PGAP3. Besides five missense mutations, we identified an intronic mutation, c.558-10G>A, that causes an aberrant splice product and a mutation in the 3'UTR, c.*559C>T, that is associated with substantially lower mRNA levels. We show that our novel screening approach is a useful rapid detection tool for alterations in genes coding for key components of the GPI pathway.

MECP2 Variants in Males: More Common than Previously Appreciated.

BACKGROUND: To assess the age and MECP2 variants of recently identified males and set the stage for further study of clinical features in males. METHODS: Genetic information on the specific MECP2 variant was acquired from the coordinator (K.F.) of the Parent Group for Males. Data were collected indicating whether these variants were de novo or transmitted from the mother and whether males who appeared to meet the diagnostic criteria for Rett syndrome had mosaicism for the MECP2 variant. RESULTS: Fifty-nine males were identified through the parent group. Their ages ranged from 2 to 28 years, with the median age being 7.0 years and the mean age being 10.8 years. Of these variants, 46 (78.0%) were de novo, nine (15.3%) were maternally inherited, and for four (6.8%) inheritance was not known. Eleven (18.6%) were mosaic, 10 with somatic mosaicism and one with Klinefelter syndrome (47XXY). Together with males reported previously from the US Natural History Study, the total group represents 85 males, of whom 27 are deceased. CONCLUSIONS: These data on males with MECP2 variants are important to caregivers, physicians, and researchers to begin to characterize their historical and clinical features, improve diagnostic recognition and overall care, and accelerate access to therapeutic studies including gene replacement strategies. Equal access to such therapies for males is critical.

Gastrointestinal and nutritional problems occur frequently throughout life in girls and women with Rett syndrome.

OBJECTIVE: We conducted a nationwide survey to determine the prevalence of common gastrointestinal and nutritional disorders in Rett syndrome (RTT) based on parental reporting and related the occurrence of these problems to age and methyl-CpG-binding protein 2 (MECP2) gene status. METHODS: We designed a questionnaire that probed symptoms, diagnoses, diagnostic tests, and treatment interventions related to gastrointestinal and nutritional problems in RTT. The International Rett Syndrome Foundation distributed the questionnaire to 1666 family-based members and forwarded their responses for our review. We interrogated the Rare Disease Clinical Research Network database to supplement findings related to medications used to treat gastrointestinal problems in RTT. RESULTS: Parents of 983 female patients with RTT (59%) responded and identified symptoms and diagnoses associated with gastrointestinal dysmotility (92%), chewing and swallowing difficulties (81%), weight deficits or excess (47%), growth deficits (45%), low bone mineral content or fractures (37%), and biliary tract disorders (3%). Height-for-age, weight-for-age, and body mass index z scores decreased significantly with age; height- and weight-, but not body mass index-for-age z scores were significantly lower in female subjects with MECP2 mutations than in those without. Vomiting, nighttime awakening, gastroesophageal reflux, chewing difficulty, and choking with feeding were significantly less likely to occur with increasing age. Short stature, low bone mineral content, fractures, and gastrostomy placement were significantly more likely to occur with increasing age. Chewing difficulty, choking with feeding, and nighttime awakening were significantly less likely to occur, whereas short stature was significantly more likely to occur, in female subjects with MECP2 mutations than in those without. Diagnostic evaluations and therapeutic interventions were used less frequently than the occurrence of symptoms or diagnoses in the RTT cohort. CONCLUSIONS: Gastrointestinal and nutritional problems perceived by parents are prevalent throughout life in girls and women with RTT and may pose a substantial medical burden for their caregivers. Physician awareness of these features of RTT may improve the health and quality of life of individuals affected with this disorder.

Rett syndrome diagnostic criteria: lessons from the Natural History Study.

Analysis of 819 participants enrolled in the Rett syndrome (RTT) Natural History Study validates recently revised diagnostic criteria. 765 females fulfilled 2002 consensus criteria for classic (653/85.4%) or variant (112/14.6%) RTT. All participants classified as classic RTT fulfilled each revised main criterion; supportive criteria were not uniformly present. All variant RTT participants met at least 3 of 6 main criteria in the 2002, 2 of 4 main criteria in the current format, and 5 of 11 supportive criteria in both. This analysis underscores the critical role of main criteria for classic RTT; variant RTT requires both main and supportive criteria.

Vitamin D deficiency is prevalent in girls and women with Rett syndrome.

OBJECTIVES: The aim of the study was to determine the prevalence of vitamin D deficiency and identify the relation between 25-hydroxyvitamin D (25-(OH)D) levels and the consumption of dietary sources of vitamin D or exposure to anticonvulsants in girls and women with Rett syndrome (RTT). SUBJECTS AND METHODS: Retrospective review of the medical records of 284 girls and women with RTT to determine serum 25-(OH)D and parathyroid hormone levels, nutritional status, dietary sources of vitamin D, exposure to anticonvulsants, degree of mobility, and MECP2 status. RESULTS: Twenty percent of girls and women who were tested (n = 157) had 25-(OH)D levels <50 nmol/L. Multivitamin supplements, vitamin D-fortified milk, and commercial formulas were consumed by 40%, 52%, and 54%, respectively. Anticonvulsants were used by 57%, and 39% ambulated independently. Median 25-(OH)D levels were lower in individuals who did not receive multivitamin supplements (P < 0.05) or commercial formulas (P < 0.001) than in those who did. Median 25-(OH)D levels differed (P < 0.01) among racial and ethnic groups, but the number in some groups was small. Nutritional status, use of anticonvulsants, degree of mobility, and MECP2 status did not influence 25-(OH)D levels. CONCLUSIONS: Vitamin D deficiency is prevalent in girls and women with RTT. The use of multivitamin supplements or commercial formulas is associated with improved vitamin D levels. Attention to vitamin D may enhance bone mineral deposition and reduce the frequency of bone fractures in these individuals.

Longevity in Rett syndrome: analysis of the North American Database.

OBJECTIVE: To determine longevity in Rett syndrome (RTT) from a large cohort. STUDY DESIGN: The North American RTT Database allows the examination of longevity in a large cohort of individuals with RTT from the United States and Canada. This database contains information on 1928 individuals, 85.5% with typical RTT, 13.4% with atypical RTT, and 1.1% with a mutation in the methyl-CpG-binding protein 2 gene (MECP2) but not RTT. Kaplan-Meier analyses were performed to assess longevity. RESULTS: Earlier decennial cohorts exhibited better survival than recent cohorts, with most participants surviving into middle age. Comparing overall survival in persons with typical RTT and atypical RTT revealed greater mortality in typical RTT across the observed lifespan (P < .0001). Comparing survival in persons with RTT and identified MECP2 mutations and persons with unknown MECP2 status demonstrated greater mortality in the latter group (P < .0001, log-rank test). CONCLUSIONS: This analysis provides strong evidence for significant longevity in RTT and indicates the need for careful planning for long-term care of these women. The disproportionately greater survival seen in earlier time periods and in persons with atypical RTT may be attributed to more severely affected individuals dying before diagnosis in the former and to greater numbers with milder variants (ie, preserved speech and delayed onset) in the latter.

Profiling scoliosis in Rett syndrome.

To understand scoliosis, related comorbidities, and phenotype-genotype correlations in individuals with Rett syndrome (RTT), the Rare Disease Clinical Research Network database for RTT was probed. Clinical evaluations included a detailed history and physical examination, comprehensive anthropometric measurements, and two quantitative measures of clinical status, Clinical Severity Scale (CSS) and motor-behavioral analysis (MBA). All data were exported to the Data Technology Coordinating Center (DTCC) at the University of South Florida. Scoliosis assessment was based on direct examination and curvature measurements by radiography (Cobb angle). Statistical analyses included univariate and multiple logistic regression models, adjusting for age at enrollment or mutation type. Scoliosis data were available from 554 classic RTT participants, mean age = 10 y (0-57 y). Scoliosis was noted in 292 (53%); mean age = 15 y with scoliosis and 6 y without. Using multiple regression analysis, MBA severity score, later acquisition, loss or absent walking, and constipation were associated with scoliosis. Two common methyl-CpG-binding protein 2 (MECP2) mutations, R294X and R306C, had reduced risk for scoliosis. These findings corroborated previous reports on scoliosis and extended understanding of comorbidities, clinical severity, and relative risk reduction for specific mutations. Clinical trial design should account for scoliosis and related factors judiciously.

Functional analysis of a novel mutation in the TIMM8A gene that causes deafness-dystonia-optic neuronopathy syndrome.

BACKGROUND: The rare, X-linked neurodegenerative disorder, Mohr-Tranebjaerg syndrome (also called deafness-dystonia-optic neuronopathy [DDON] syndrome), is caused by mutations in the TIMM8A gene. DDON syndrome is characterized by dystonia, early-onset deafness, and various other neurological manifestations. The TIMM8A gene product localizes to the intermembrane space in mitochondria where it functions in the import of nuclear-encoded proteins into the mitochondrial inner membrane. Frameshifts or premature stops represent the majority of mutations in TIMM8A that cause DDON syndrome. However, missense mutations have also been reported that result in loss of the TIMM8A gene product. METHODS: We report a novel TIMM8A variant in a patient with DDON syndrome that alters the initiation codon and employed functional analyses to determine the significance of the variant and its impact on mitochondrial morphology. RESULTS: The novel base change in the TIMM8A gene (c.1A>T, p.Met1Leu) results in no detectable protein and a reduction in TIMM8A transcript abundance. We observed a commensurate decrease in the steady-state level of the Tim13 protein (the binding partner of Tim8a) but no decrease in TIMM13 transcripts. Patient fibroblasts exhibited elongation and/or increased fusion of mitochondria, consistent with prior reports. CONCLUSION: This case expands the spectrum of mutations that cause DDON syndrome and demonstrates effects on mitochondrial morphology that are consistent with prior reports.

Metabolic Signatures Differentiate Rett Syndrome From Unaffected Siblings.

Rett syndrome (RTT, OMIM 312750), a severe neurodevelopmental disorder characterized by regression with loss of spoken language and hand skills, development of characteristic hand stereotypies, and gait dysfunction, is primarily caused by de novo mutations in the X-linked gene Methyl-CpG-binding protein 2 (MECP2). Currently, treatment options are limited to symptomatic management, however, reversal of disease phenotype is possible in mouse models by restoration of normal MECP2 gene expression. A significant challenge is the lack of biomarkers of disease state, disease severity, or treatment response. Using a non-targeted metabolomic approach we evaluated metabolite profiles in plasma from thirty-four people with RTT compared to thirty-seven unaffected age- and gender-matched siblings. We identified sixty-six significantly altered metabolites that cluster broadly into amino acid, nitrogen handling, and exogenous substance pathways. RTT disease metabolite and metabolic pathways abnormalities point to evidence of oxidative stress, mitochondrial dysfunction, and alterations in gut microflora. These observed changes provide insight into underlying pathological mechanisms and the foundation for biomarker discovery of disease severity biomarkers.

When Rett syndrome is due to genes other than MECP2.

Two individuals meeting diagnostic criteria for Rett syndrome (RTT) but lacking a mutation in MECP2, the gene predominantly associated with this disorder, were provided additional genetic testing. This testing revealed pathogenic mutations in a gene not previously associated with RTT, CTNNB1, mutations in which lead to an autosomal dominant neurodevelopmental disorder affecting cell signaling and transcription factors as well as a likely pathogenic mutation in the WDR45 gene, which is associated with developmental delay in early childhood and progressive neurodegeneration in adolescence or adulthood related to iron accumulation in the globus pallidus and substantia nigra. These two individuals are described in relation to previous reports linking multiple other genes with RTT failing to show an MECP2 mutation. These individuals underscore the need to pursue additional molecular testing in RTT when a mutation in MECP2 is not detected.

Rett syndrome: North American database.

The International Rett Syndrome Association (IRSA) North American database is the first comprehensive compilation of information in the United States and Canada on individuals with Rett syndrome or with another diagnosis in association with MECP2 mutations. The database contains specific information by diagnosis, mutation status, and mutation type and frequency on 1928 participants. Among the 1928 participants, 85.5% were typical, 13.4% were atypical, and 1.1% had MECP2 mutations but did not have Rett syndrome. MECP2 mutations were identified in 914 of 1059 participants (86%): 799 of 870 (92%) participants with typical Rett syndrome had an MECP2 mutation, 94 of 162 (58%) with atypical Rett syndrome had a mutation, and all 21 individuals diagnosed as Not Rett syndrome had a mutation. Missense-type mutations (39.0%) were slightly more common than nonsense type (35.1%). Individual mutation frequency for the 8 common mutations varied from 11.9% for T158M to 4.4% for R106W; large deletions accounted for 6.4% and C-terminal truncations occurred in 8.8%. The remaining mutations (14.3%) occurred singly or in small numbers. This database provides a unique resource for expanding our understanding of Rett syndrome, for comparison with other national databases, and for future study including organization of clinical trials based on the expected emergence of fundamental therapies.

Age of diagnosis in Rett syndrome: patterns of recognition among diagnosticians and risk factors for late diagnosis.

PURPOSE: Diagnosis of Rett syndrome (RTT) is often delayed. We sought to determine the type of physician who typically makes the RTT diagnosis and to identify risk factors for delayed diagnosis. METHODS: A total of 1085 participants from the multicenter longitudinal RTT natural history study with classic and atypical RTT were recruited between 2006 and 2014. Age of diagnosis, diagnostician, diagnostic criteria, and clinical and developmental data were collected. RESULTS: Among 919 classic and 166 atypical RTT participants, the median diagnosis age was 2.7 years (interquartile range 2.0-4.1) in classic and 3.8 years (interquartile range 2.3-6.9) in atypical RTT. Pediatricians made the diagnosis of classic RTT rarely (5.2%); however, the proportion diagnosed by pediatricians has increased since 2006. Since the first diagnostic criteria, the age of diagnosis decreased among subspecialists but not pediatricians. Odds of a pediatrician making the diagnosis of classic RTT were higher if a child stopped responding to parental interaction, and lower if they possessed gastroesophageal reflux, specific stereotypies, lost babbling, or the ability to follow commands. Delayed acquisition of basic gross motor skills or finger feeding was associated with younger diagnosis; delayed acquisition of higher level fine motor skills, later onset of supportive features, and normal head circumference were associated with late diagnosis. Thirty-three percent with microcephaly before 2.5 years were diagnosed after the median age of 2.7 years. CONCLUSIONS: Age of RTT diagnosis has improved among subspecialists, and pediatricians have made the diagnosis of classic RTT more frequently since 2006. Strategies for educating diagnosticians should incorporate specific risk factors for delayed diagnosis.

Developmental delay in Rett syndrome: data from the natural history study.

BACKGROUND: Early development appears normal in Rett syndrome (OMIM #312750) and may be more apparent than real. A major purpose of the Rett Syndrome (RTT) Natural History Study (NHS) was to examine achievement of developmental skills or abilities in classic and atypical RTT and assess phenotype-genotype relations in classic RTT. METHODS: Developmental skills in four realms, gross and fine motor, and receptive and expressive communication from initial enrollment and longitudinal assessments for up to 7 years, were assessed from 542 females meeting criteria for classic RTT and 96 females with atypical RTT divided into two groups: 50 with better and 46 with poorer functional scores. Data were analyzed for age at acquisition and loss of developmental features and for phenotype-genotype effects. Acquired, lost, and retained skills were compared between classic RTT and atypical RTT with better or poorer functional scores using Fisher's Exact test. To examine if the mean total score from the Motor Behavioral Assessment during follow-up differed for acquiring a skill, we used a generalized estimating equation assuming compound symmetry correlation structure within a subject. A general linear model was used to examine whether the mean age of acquisition or loss of a developmental skill differed by mutation type. P values <0.05 were considered significant and were two-sided without adjustment for multiple testing. Statistical analyses utilized SAS 9.3 (SAS Institute, Cary, NC, USA). RESULTS: Early developmental skills or abilities were often acquired albeit later than normal. More complex motor and communication acquisitions were delayed or absent. Clinical severity was less in those achieving the respective skill. Individuals with R133C, R294X, and R306C point mutations and 3' truncations tended to have better developmental outcomes. CONCLUSIONS: Early developmental skills were acquired by many, but clear differences from normal emerged, particularly in skills expected after age 6 months. When comparing clinical severity, greater acquisition of specific skills was associated with specific mutations, confirming the impression that these mutations confer milder developmental abnormalities. These data may serve for planning and interpretation of early intervention studies in RTT. TRIAL REGISTRATION: This NHS study, clinicaltrials.gov (NCT00296764), represents the largest group of RTT participants assessed repeatedly by direct examination.