Bi-allelic Variants in RALGAPA1 Cause Profound Neurodevelopmental Disability, Muscular Hypotonia, Infantile Spasms, and Feeding Abnormalities.

Ral (Ras-like) GTPases play an important role in the control of cell migration and have been implicated in Ras-mediated tumorigenicity. Recently, variants in RALA were also described as a cause of intellectual disability and developmental delay, indicating the relevance of this pathway to neuropediatric diseases. Here, we report the identification of bi-allelic variants in RALGAPA1 (encoding Ral GTPase activating protein catalytic alpha subunit 1) in four unrelated individuals with profound neurodevelopmental disability, muscular hypotonia, feeding abnormalities, recurrent fever episodes, and infantile spasms . Dysplasia of corpus callosum with focal thinning of the posterior part and characteristic facial features appeared to be unifying findings. RalGAPA1 was absent in the fibroblasts derived from two affected individuals suggesting a loss-of-function effect of the RALGAPA1 variants. Consequently, RalA activity was increased in these cell lines, which is in keeping with the idea that RalGAPA1 deficiency causes a constitutive activation of RalA. Additionally, levels of RalGAPB, a scaffolding subunit of the RalGAP complex, were dramatically reduced, indicating a dysfunctional RalGAP complex. Moreover, RalGAPA1 deficiency clearly increased cell-surface levels of lipid raft components in detached fibroblasts, which might indicate that anchorage-dependence of cell growth signaling is disturbed. Our findings indicate that the dysregulation of the RalA pathway has an important impact on neuronal function and brain development. In light of the partially overlapping phenotype between RALA- and RALGAPA1-associated diseases, it appears likely that dysregulation of the RalA signaling pathway leads to a distinct group of genetic syndromes that we suggest could be named RALopathies.

De Novo Variants in MAPK8IP3 Cause Intellectual Disability with Variable Brain Anomalies.

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.

Exome and genome sequencing for pediatric patients with congenital anomalies or intellectual disability: an evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: To develop an evidence-based clinical practice guideline for the use of exome and genome sequencing (ES/GS) in the care of pediatric patients with one or more congenital anomalies (CA) with onset prior to age 1 year or developmental delay (DD) or intellectual disability (ID) with onset prior to age 18 years. METHODS: The Pediatric Exome/Genome Sequencing Evidence-Based Guideline Work Group (n = 10) used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) evidence to decision (EtD) framework based on the recent American College of Medical Genetics and Genomics (ACMG) systematic review, and an Ontario Health Technology Assessment to develop and present evidence summaries and health-care recommendations. The document underwent extensive internal and external peer review, and public comment, before approval by the ACMG Board of Directors. RESULTS: The literature supports the clinical utility and desirable effects of ES/GS on active and long-term clinical management of patients with CA/DD/ID, and on family-focused and reproductive outcomes with relatively few harms. Compared with standard genetic testing, ES/GS has a higher diagnostic yield and may be more cost-effective when ordered early in the diagnostic evaluation. CONCLUSION: We strongly recommend that ES/GS be considered as a first- or second-tier test for patients with CA/DD/ID.

Professional medical education and genomics.

Genomic medicine is a relatively new concept that involves using individual patients' genomic results in their clinical care. Genetic technology has advanced swiftly over the past decade, and most providers have been left behind without an understanding of this complex field. To realize its full potential, genomic medicine must be both understood and accepted by the greater medical community. The current state of professional medical education in genomics and genomic medicine is reviewed, including ongoing plans to expand educational efforts for medical students, clinical geneticists, and nongeneticist physicians.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a ß1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1-CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Mouse model implicates GNB3 duplication in a childhood obesity syndrome.

Obesity is a highly heritable condition and a risk factor for other diseases, including type 2 diabetes, cardiovascular disease, hypertension, and cancer. Recently, genomic copy number variation (CNV) has been implicated in cases of early onset obesity that may be comorbid with intellectual disability. Here, we describe a recurrent CNV that causes a syndrome associated with intellectual disability, seizures, macrocephaly, and obesity. This unbalanced chromosome translocation leads to duplication of over 100 genes on chromosome 12, including the obesity candidate gene G protein ß3 (GNB3). We generated a transgenic mouse model that carries an extra copy of GNB3, weighs significantly more than its wild-type littermates, and has excess intraabdominal fat accumulation. GNB3 is highly expressed in the brain, consistent with G-protein signaling involved in satiety and/or metabolism. These functional data connect GNB3 duplication and overexpression to elevated body mass index and provide evidence for a genetic syndrome caused by a recurrent CNV.

Surveying the current landscape of clinical genetics residency training.

PURPOSE: Further knowledge about medical genetics residency training structure and function could help advance this educational process. METHODS: Medical genetics residency program directors were surveyed about their trainees' backgrounds and skills as well as the recruitment and matching process. RESULTS: Previous resident training was predominantly in pediatrics (49%). Average ratings of residents' beginning clinical knowledge (scale of 1-10, minimal to superior) were: dysmorphology - 3.5, inborn errors of metabolism - 2.5, prenatal genetics - 2.6, and cancer genetics - 2.8. On average, four months of research were required for categorical residency and fifteen months for combined residency. For the 2011 transition to ERAS/NRMP, 69% of program directors were extremely or somewhat prepared; however, 21% felt unprepared. The number of trainees at most institutions remained unchanged. 36% of respondents reported that ERAS/NRMP has had no impact on recruitment of trainees, and 26% felt it has had a slightly positive impact. Continued utilization was recommended by 71% while 5% disagreed. CONCLUSION: Genetics residents come from diverse training backgrounds. Their education can be directed toward specific areas of perceived initial weakness. ERAS/NRMP has not drastically increased entrance into the field. Further discussions are merited regarding enhancement of medical genetics residency recruitment and training.

A description of spina bifida cases and co-occurring malformations, 1976-2011.

Mandatory folic acid fortification in the United States corresponded with a decline in the prevalence of spina bifida (SB). The aim of this study was to describe the epidemiologic characteristics of isolated versus non-isolated SB cases in both pre- and post-fortification periods. SB cases in the Slone Epidemiology Center Birth Defects Study from 1976 to 2011 without chromosomal anomalies and syndromes were included. A maternal interview, conducted within 6 months of delivery, collected information on demographics, reproductive history, diet, and supplement use. Daily folic acid intake in the periconceptional period was calculated using both dietary and supplement information and categorized as low intake (<400 µg/day) or high intake (≥400 µg/day). SB cases (n = 1170) were classified as isolated (80.4%) or non-isolated (19.1%). Non-isolated cases were further divided into subgroups based on accompanying major malformations (midline, renal, genital, heart, laterality). Compared to non-isolated cases, isolated cases were more likely to be white, non-Hispanic and have more than 12 years of education. Cases in the renal, genital, and heart subgroups had the lowest proportions of mothers with a high folic acid intake. The change from pre- to post-fortification was associated with a decrease in the proportion of isolated cases from 83% to 72%, though in both periods isolated cases were more likely to be female and their mothers were more likely to have high folic acid intake. These findings highlight the importance of separating isolated and non-isolated cases in etiologic research of SB.

Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia.

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻5). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.

Keratoconus in Costello syndrome.

Keratoconus is a corneal dystrophy with progressive corneal thinning resulting in abnormal corneal shape and astigmatism. Corneal hydrops and rupture can occur and corneal transplant may become necessary. While keratoconus is rare in the general population occurring in about 1/2,000 individuals, it is more common in individuals with intellectual disability and syndromic conditions. Connective tissue abnormalities, most typically brittle cornea syndrome, have frequently been reported in association with keratoconus. Here, we report on bilateral keratoconus with acute hydrops in the left eye of a 24-year-old male with Costello syndrome. The patient was treated medically. After resolution of the hydrops, he had significant visual impairment from the resulting irregular astigmatism and scarring. This is the second report of keratoconus in Costello syndrome, suggesting an increased risk for this corneal dystrophy in individuals with Costello syndrome. Ongoing ophthalmological surveillance may be necessary for adult individuals with Costello syndrome, and apparent vision changes should be evaluated expediently.

Maternal tea consumption during early pregnancy and the risk of spina bifida.

Studies have demonstrated that catechin, an antioxidant found in tea, can reduce the bioavailability of folate. Because periconceptional folic acid intake has been demonstrated to reduce the risk of spina bifida, tea consumption may put pregnant women at risk because of its possible antifolate properties. Using data collected in the Slone Epidemiology Center Birth Defects Study, we examined whether tea consumption during early pregnancy was associated with an increased risk of spina bifida. Mothers of 518 spina bifida cases and 6424 controls were interviewed within 6 months after delivery about pregnancy events and exposures. Data on tea intake were collected during three periods (1976-1988, 1998-2005 and 2009-2010). Logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), adjusted for study center. Intake of both periconceptional food folate and diet and supplemental folic acid were examined as a potential effect modifier. For 1976 to 1988, ORs were not elevated for daily tea intake. For 1998 and onward, ORs were also close to 1.0, but there was a modest increase for those who drank more than 3 cups/day (OR, 1.92; 95% CI, 0.84-4.38). Among women with total folic acid intake greater than 400 µg, consumption of 3 cups or more of tea per day was associated with an increased risk of spina bifida in 1976 to 1988 (OR, 2.04; 95% CI, 0.69-7.66) and in the later periods (OR, 3.13; 95% CI, 0.87-11.33). Our data do not support an overall association between tea consumption and spina bifida, but there is a suggestion of a possible interaction between higher levels of folic acid intake and tea consumption.

Lessons learned from the introduction of personalized genotyping into a medical school curriculum.

PURPOSE: There is an expanding gap between the availability of direct-to-consumer whole genome testing and physician knowledge regarding interpretation of test results. Advances in the genomic literacy of health care providers will be necessary for genomics to exert its potential to affect clinical practice. However, implementation of a major shift in medical education to include genomics is not easily done. The purpose of this educational report is to describe efforts to incorporate knowledge of personalized medicine into a medical school curriculum. METHODS: In this report, we describe the experiences, both good and bad, of a multidisciplinary faculty group that examined ways to improve genomic education at Tufts University School of Medicine during a 16-month period. RESULTS: The results of the faculty's deliberation process resulted in the use of anonymous, rather than student genomes, to teach material on genomic medicine. CONCLUSION: Increased medical school education regarding genomic analysis and personalized medicine is a necessity, both to be able to translate the advances made by the Human Genome Project into improvements in human health and to begin to think of diseases as disruptions in specific pathways. Our experiences illustrate that adding this material to a medical school curriculum is a complex process that deserves careful thought and broad discussion within the academic community.

Male-to-male transmission of Costello syndrome: G12S HRAS germline mutation inherited from a father with somatic mosaicism.

Costello syndrome is a rare congenital anomaly syndrome associated with mental retardation and predisposition to benign and malignant tumors, caused by heterozygous missense mutations in the HRAS oncogene. Previously, all molecularly analyzed mutations appeared de novo, and most arose in the paternal germline. A single patient with somatic mosaicism for a Costello syndrome causing HRAS mutation has been reported. Here we describe the first documented transmission of an HRAS mutation from a parent with somatic mosaicism to a child with typical Costello syndrome. Prior to the identification of the underlying gene mutation in Costello syndrome, this family had been identified clinically. The proband was subsequently found to carry a G12S HRAS germline mutation. Testing of the parents for parental origin identified his father as mosaic for the same HRAS mutation. The mother was found not to carry an HRAS mutation. The causative familial mutation is identified as a c.34G > A, which is the most common mutation in the HRAS gene in patients with Costello syndrome. The father carries the mutation in 7-8% of his alleles. This is the second case of mosaicism observed in Costello syndrome and the first direct molecular evidence of father-to-son transmission of the disease-causing mutation. Our observation underlines the importance of parental evaluation, and may have implications for genetic counseling and clinical practice.

Prenatal features of Costello syndrome: ultrasonographic findings and atrial tachycardia.

OBJECTIVE: Delineate prenatal features of Costello syndrome (caused by HRAS mutations), which consists of mental retardation, facial, cardiovascular, skin, and musculoskeletal anomalies, and tumor predisposition. METHODS: Literature and new cases classified as Group I (pre-HRAS), Group II (HRAS confirmed), and Group III (HRAS confirmed in natural history study, plus three contributed cases). RESULTS: Polyhydramnios occurred in most (mean 79%) pregnancies of cases in Groups I (98), II (107), and III (17); advanced paternal age and prematurity were noted in approximately half. Less frequent were nuchal thickening, ascites, shortened long bones, abnormal hand posture, ventriculomegaly, macrosomia, and macrocephaly. Fetal arrhythmia occurred in nine cases (six supraventricular or unspecified tachycardia, one unspecified arrhythmia, and two premature atrial contractions, PACs); excluding three new cases and two with PACs, the estimated prenatal frequency is 4/222 (2%). CONCLUSION: Costello syndrome can be suspected prenatally when polyhydramnios is accompanied by nuchal thickening, hydrops, shortened long bones, abnormal hand posture, ventriculomegaly, large size, and macrocephaly, and especially fetal atrial tachycardia. Consideration should be given for timely prenatal diagnostic studies for confirmative HRAS gene mutations and for maternal treatment of serious fetal arrhythmia.

Gene domain-specific DNA methylation episignatures highlight distinct molecular entities of ADNP syndrome.

BACKGROUND: ADNP syndrome is a rare Mendelian disorder characterized by global developmental delay, intellectual disability, and autism. It is caused by truncating mutations in ADNP, which is involved in chromatin regulation. We hypothesized that the disruption of chromatin regulation might result in specific DNA methylation patterns that could be used in the molecular diagnosis of ADNP syndrome. RESULTS: We identified two distinct and partially opposing genomic DNA methylation episignatures in the peripheral blood samples from 22 patients with ADNP syndrome. The "epi-ADNP-1" episignature included ~ 6000 mostly hypomethylated CpGs, and the "epi-ADNP-2" episignature included ~ 1000 predominantly hypermethylated CpGs. The two signatures correlated with the locations of the ADNP mutations. Epi-ADNP-1 mutations occupy the N- and C-terminus, and epi-ADNP-2 mutations are centered on the nuclear localization signal. The episignatures were enriched for genes involved in neuronal system development and function. A classifier trained on these profiles yielded full sensitivity and specificity in detecting patients with either of the two episignatures. Applying this model to seven patients with uncertain clinical diagnosis enabled reclassification of genetic variants of uncertain significance and assigned new diagnosis when the primary clinical suspicion was not correct. When applied to a large cohort of unresolved patients with developmental delay (N = 1150), the model predicted three additional previously undiagnosed patients to have ADNP syndrome. DNA sequencing of these subjects, wherever available, identified pathogenic mutations within the gene domains predicted by the model. CONCLUSIONS: We describe the first Mendelian condition with two distinct episignatures caused by mutations in a single gene. These highly sensitive and specific DNA methylation episignatures enable diagnosis, screening, and genetic variant classifications in ADNP syndrome.

Clinical genetic testing for patients with autism spectrum disorders.

BACKGROUND: Multiple lines of evidence indicate a strong genetic contribution to autism spectrum disorders (ASDs). Current guidelines for clinical genetic testing recommend a G-banded karyotype to detect chromosomal abnormalities and fragile X DNA testing, but guidelines for chromosomal microarray analysis have not been established. PATIENTS AND METHODS: A cohort of 933 patients received clinical genetic testing for a diagnosis of ASD between January 2006 and December 2008. Clinical genetic testing included G-banded karyotype, fragile X testing, and chromosomal microarray (CMA) to test for submicroscopic genomic deletions and duplications. Diagnostic yield of clinically significant genetic changes was compared. RESULTS: Karyotype yielded abnormal results in 19 of 852 patients (2.23% [95% confidence interval (CI): 1.73%-2.73%]), fragile X testing was abnormal in 4 of 861 (0.46% [95% CI: 0.36%-0.56%]), and CMA identified deletions or duplications in 154 of 848 patients (18.2% [95% CI: 14.76%-21.64%]). CMA results for 59 of 848 patients (7.0% [95% CI: 5.5%-8.5%]) were considered abnormal, which includes variants associated with known genomic disorders or variants of possible significance. CMA results were normal in 10 of 852 patients (1.2%) with abnormal karyotype due to balanced rearrangements or unidentified marker chromosome. CMA with whole-genome coverage and CMA with targeted genomic regions detected clinically relevant copy-number changes in 7.3% (51 of 697) and 5.3% (8 of 151) of patients, respectively, both higher than karyotype. With the exception of recurrent deletion and duplication of chromosome 16p11.2 and 15q13.2q13.3, most copy-number changes were unique or identified in only a small subset of patients. CONCLUSIONS: CMA had the highest detection rate among clinically available genetic tests for patients with ASD. Interpretation of microarray data is complicated by the presence of both novel and recurrent copy-number variants of unknown significance. Despite these limitations, CMA should be considered as part of the initial diagnostic evaluation of patients with ASD.

The natural history of trisomy 12p.

Trisomy of the short arm of chromosome 12 is a rare chromosomal anomaly, with an estimated incidence of 1/50,000 births. It may present as a pure trisomy (complete or incomplete), as mosaic trisomy, or with other chromosomal abnormalities. Little is known from prior reports about the natural history and life expectancy of these individuals. In this study we describe the long-term outcome and the differences between patients with mosaic trisomy 12p compared to patients with complete trisomy. We present a series of 16 patients with trisomy 12p; 6 of them are older than 10 years. Most patients were born at term with normal or above normal birth weight. Seven were born with congenital anomalies, but no single anomaly was present in more than one individual. A clear and consistent dysmorphic facial pattern was apparent in all of the subjects. Most patients over 7 years old had a seizure disorder. All individuals exhibited developmental delay with speech affected more severely than motor skills. Six patients were described as "being social." Six had severe behavioral problems, and seven had significant sleep disturbances. Facial features of the three adult patients were different than the younger individuals. We show here that the outcome for patients with mosaic trisomy 12p is better than the outcome in complete trisomy 12p or in trisomy 12p with other chromosomal anomalies. We also provide recommendations for the long-term follow-up of patients with trisomy 12p.