Rare coding variation provides insight into the genetic architecture and phenotypic context of autism.

Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of protein-truncating variants (PTVs), missense variants and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 genes associated with ASD at false discovery rate (FDR) ≤ 0.001 (185 at FDR ≤ 0.05). De novo PTVs, damaging missense variants and CNVs represented 57.5%, 21.1% and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD) (n = 91,605) yielded 373 genes associated with ASD/DD at FDR ≤ 0.001 (664 at FDR ≤ 0.05), some of which differed in relative frequency of mutation between ASD and DD cohorts. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells, whereas genes showing stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophrenia-associated genes, emphasizing that these neuropsychiatric disorders may share common pathways to risk.

Phenotypic Spectrum in a Family Sharing a Heterozygous KCNQ3 Variant.

BACKGROUND AND PURPOSE: Mutations in KCNQ3 have classically been associated with benign familial neonatal and infantile seizures and more recently identified in patients with neurodevelopmental disorders and abnormal electroencephalogram (EEG) findings. We present 4 affected patients from a family with a pathogenic mutation in KCNQ3 with a unique constellation of clinical findings. METHODS: A family of 3 affected siblings and mother sharing a KCNQ3 pathogenic variant are described, including clinical history, genetic results, and EEG and magnetic resonance imaging (MRI) findings. RESULTS: This family shows a variety of clinical manifestations, including neonatal seizures, developmental delays, autism spectrum disorder, and anxiety. One child developed absence epilepsy, 2 children have infrequent convulsive seizures that have persisted into childhood, and their parent developed adult-onset epilepsy. An underlying c.1091G>A (R364H) variant in KCNQ3 was found in all affected individuals. CONCLUSIONS: The phenotypic variability of KCNQ3 channelopathies continues to expand as more individuals and families are described, and the variant identified in this family adds to the understanding of the manifestations of KCNQ3-related disorders.

CNTN6 copy number variations: Uncertain clinical significance in individuals with neurodevelopmental disorders.

Copy number variations (CNVs) of the CNTN6 gene - a member of the contactin gene superfamily - have been previously proposed to have an association with neurodevelopmental and autism spectrum disorders. However, no functional evidence has been provided to date and phenotypically normal and mildly affected carriers complicate the interpretation of this aberration. In view of conflicting reports on the pathogenicity of CNVs involving CNTN6 and association with different phenotypes, we, independently, evaluated clinical features of nineteen patients with detected CNV of CNTN6 as part of their clinical microarray analysis at Children's Mercy and Nationwide Children's Hospitals for the period of 2008-2015. The clinical presentations of these patients were variable making it difficult to establish genotype-phenotype correlations. CNVs were inherited in six patients. For thirteen patients, inheritance pattern was not established due to unavailability of parental samples for testing. In three cases CNV was inherited from a healthy parent and in three cases from a parent with neurodevelopmental symptoms. Of the nineteen patients, four had a separate genetic abberation in addition to CNV of the CNTN6 that could independently explain their respective phenotypes. Separately, CNTN6 sequencing was performed on an autism spectrum disorder (ASD) research cohort of 94 children from 80 unrelated families. We found no difference in frequency of rare coding variants between the cohort of patients and controls. We conclude that CNVs involving CNTN6 alone seem to be most likely a neutral variant or a possible modifier rather than a disease-causing variant. Patients with CNVs encompassing CNTN6 could benefit from additional genetic testing since a clinical diagnosis due to a CNV of CNTN6 alone is still questionable.

Testing genetic modifiers of behavior and response to atomoxetine in autism spectrum disorder with ADHD.

BACKGROUND: Frequent non-pathogenic genetic variants may act as moderators of phenotypic severity for complex disorders such as autism spectrum disorder (ASD). We previously identified polymorphisms affecting mRNA expression of candidate genes, including tryptophan hydroxylase 2 (TPH2), dopamine beta hydroxylase (DBH), and dopamine transporter (DAT, SLC6A3). METHOD: We compare genotypes and (1) clinical response to atomoxetine, (2) scores from the Autism Diagnostic Interview-Revised (ADI-R), and (3) severity of Attention Deficit Hyperactivity Disorder (ADHD) symptoms in a cohort of patients with ASD from multiple study sites. RESULTS: There was no association between CYP2D6 metabolizer status and atomoxetine response. TPH2 rs7305115 genotype was associated with ADI-R Restrictive/Repetitive Behavior score (p=0.03). DBH rs1611115 genotype was associated with ADI-R Social score (p=0.002) and Restrictive/Repetitive Behavior score (p=0.04). The DAT intron 8 5/6 repeat was associated with ADHD symptoms (ABC Hyperactivity p=0.01 and SNAP ADHD p=0.03), replicating a previous finding. CONCLUSIONS: We find associations between ASD phenotypes and regulatory variants in catecholamine biosynthesis genes. This work may help guide future genetics studies related to ASD.

Language Regression in an Atypical SLC6A1 Mutation.

Recent technological advances in exome sequencing or targeted gene sequencing with epilepsy panels have allowed clinicians to better understand the pathogenesis and clinical presentation of children with epilepsy. We present a child with a SLC6A1 mutation with language delay and autistic spectrum disorder and remind the reader that the identification of specific mutations in these conditions increase the likelihood of identification of potential therapeutic targets.

Severe neurocognitive and growth disorders due to variation in THOC2, an essential component of nuclear mRNA export machinery.

Highly conserved TREX-mediated mRNA export is emerging as a key pathway in neuronal development and differentiation. TREX subunit variants cause neurodevelopmental disorders (NDDs) by interfering with mRNA export from the cell nucleus to the cytoplasm. Previously we implicated four missense variants in the X-linked THOC2 gene in intellectual disability (ID). We now report an additional six affected individuals from five unrelated families with two de novo and three maternally inherited pathogenic or likely pathogenic variants in THOC2 extending the genotypic and phenotypic spectrum. These comprise three rare missense THOC2 variants that affect evolutionarily conserved amino acid residues and reduce protein stability and two with canonical splice-site THOC2 variants that result in C-terminally truncated THOC2 proteins. We present detailed clinical assessment and functional studies on a de novo variant in a female with an epileptic encephalopathy and discuss an additional four families with rare variants in THOC2 with supportive evidence for pathogenicity. Severe neurocognitive features, including movement and seizure disorders, were observed in this cohort. Taken together our data show that even subtle alterations to the canonical molecular pathways such as mRNA export, otherwise essential for cellular life, can be compatible with life, but lead to NDDs in humans.

Human CRMP4 mutation and disrupted Crmp4 expression in mice are associated with ASD characteristics and sexual dimorphism.

Autism spectrum disorders (ASD) are more common among boys than girls. The mechanisms responsible for ASD symptoms and their sex differences remain mostly unclear. We previously identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sex-different expression during sexual differentiation of the hypothalamic sexually dimorphic nucleus. This study investigated the relationship between the sex-different development of autistic features and CRMP4 deficiency. Whole-exome sequencing detected a de novo variant (S541Y) of CRMP4 in a male ASD patient. The expression of mutated mouse CRMP4 S540Y, which is homologous to human CRMP4 S541Y, in cultured hippocampal neurons derived from Crmp4-knockout (KO) mice had increased dendritic branching, compared to those transfected with wild-type (WT) Crmp4, indicating that this mutation results in altered CRMP4 function in neurons. Crmp4-KO mice showed decreased social interaction and several alterations of sensory responses. Most of these changes were more severe in male Crmp4-KO mice than in females. The mRNA expression levels of some genes related to neurotransmission and cell adhesion were altered in the brain of Crmp4-KO mice, mostly in a gender-dependent manner. These results indicate a functional link between a case-specific, rare variant of one gene, Crmp4, and several characteristics of ASD, including sexual differences.

Bedside Back to Bench: Building Bridges between Basic and Clinical Genomic Research.

Genome sequencing has revolutionized the diagnosis of genetic diseases. Close collaborations between basic scientists and clinical genomicists are now needed to link genetic variants with disease causation. To facilitate such collaborations, we recommend prioritizing clinically relevant genes for functional studies, developing reference variant-phenotype databases, adopting phenotype description standards, and promoting data sharing.

Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics.

Disclaimer: These recommendations are designed primarily as an educational resource for medical geneticists and other healthcare providers to help them provide quality medical services. Adherence to these recommendations is completely voluntary and does not necessarily assure a successful medical outcome. These recommendations should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed toward obtaining the same results. In determining the propriety of any specific procedure or test, the clinician should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. Clinicians are encouraged to document the reasons for the use of a particular procedure or test, whether or not it is in conformance with this statement. Clinicians also are advised to take notice of the date this statement was adopted and to consider other medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.To promote standardized reporting of actionable information from clinical genomic sequencing, in 2013, the American College of Medical Genetics and Genomics (ACMG) published a minimum list of genes to be reported as incidental or secondary findings. The goal was to identify and manage risks for selected highly penetrant genetic disorders through established interventions aimed at preventing or significantly reducing morbidity and mortality. The ACMG subsequently established the Secondary Findings Maintenance Working Group to develop a process for curating and updating the list over time. We describe here the new process for accepting and evaluating nominations for updates to the secondary findings list. We also report outcomes from six nominations received in the initial 15 months after the process was implemented. Applying the new process while upholding the core principles of the original policy statement resulted in the addition of four genes and removal of one gene; one gene did not meet criteria for inclusion. The updated secondary findings minimum list includes 59 medically actionable genes recommended for return in clinical genomic sequencing. We discuss future areas of focus, encourage continued input from the medical community, and call for research on the impact of returning genomic secondary findings.Genet Med 19 2, 249-255.

Hematopoietic cell transplantation for a child with OSTM1 osteopetrosis.

HCT prior to onset of neurologic symptoms in children with OSTM1 osteopetrosis does not halt neurologic progression.

Endogenous Sterol Metabolites Regulate Growth of EGFR/KRAS-Dependent Tumors via LXR.

Meiosis-activating sterols (MAS) are substrates of SC4MOL and NSDHL in the cholesterol pathway and are important for normal organismal development. Oncogenic transformation by epidermal growth factor receptor (EGFR) or RAS increases the demand for cholesterol, suggesting a possibility for metabolic interference. To test this idea in vivo, we ablated Nsdhl in adult keratinocytes expressing KRAS(G12D). Strikingly, Nsdhl inactivation antagonized the growth of skin tumors while having little effect on normal skin. Loss of Nsdhl induced the expression of ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, reduced the expression of low-density lipoprotein receptor (LDLR), decreased intracellular cholesterol, and was dependent on the liver X receptor (LXR) α. Importantly, EGFR signaling opposed LXRα effects on cholesterol homeostasis, whereas an EGFR inhibitor synergized with LXRα agonists in killing cancer cells. Inhibition of SC4MOL or NSDHL, or activation of LXRα by sterol metabolites, can be an effective strategy against carcinomas with activated EGFR-KRAS signaling.

Analysis of hedgehog signaling in cerebellar granule cell precursors in a conditional Nsdhl allele demonstrates an essential role for cholesterol in postnatal CNS development.

NSDHL is a 3ß-hydroxysterol dehydrogenase that is involved in the removal of two C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in the gene encoding the enzyme are responsible for the X-linked, male lethal mouse mutations bare patches and striated, as well as most cases of human CHILD syndrome. Rare, hypomorphic NSDHL mutations are also associated with X-linked intellectual disability in males with CK syndrome. Since hemizygous male mice with Nsdhl mutations die by midgestation, we generated a conditional targeted Nsdhl mutation (Nsdhl(tm1.1Hrm)) to investigate the essential role of cholesterol in the early postnatal CNS. Ablation of Nsdhl in radial glia using GFAP-cre resulted in live-born, normal appearing affected male pups. However, the pups develop overt ataxia by postnatal day 8-10 and die shortly thereafter. Histological abnormalities include progressive loss of cortical and hippocampal neurons, as well as deficits in the proliferation and migration of cerebellar granule precursors and subsequent massive apoptosis of the cerebellar cortex. We replicated the granule cell precursor proliferation defect in vitro and demonstrate that it results from defective signaling by SHH. Furthermore, this defect is almost completely rescued by supplementation of the culture media with exogenous cholesterol, while methylsterol accumulation above the enzymatic block appears to be associated with increased cell death. These data support the absolute requirement for cholesterol synthesis in situ once the blood-brain-barrier forms and cholesterol transport to the fetus is abolished. They further emphasize the complex ramifications of cholesterogenic enzyme deficiency on cellular metabolism.

Reporting genomic secondary findings: ACMG members weigh in.

PURPOSE: The aim of this study was to survey American College of Medical Genetics and Genomics members about secondary findings from clinical genome-scale sequencing. METHODS: A Web-based survey was mailed to 1,687 members of the American College of Medical Genetics and Genomics. Exploratory factor analysis identified underlying factors assessed by survey items. Linear regression assessed associations between factor scores and respondent characteristics. RESULTS: The response rate was 29%. Four factors explained 51% of the survey variance: best practices, patient preferences, guidance, and informed consent. Most agreed with "best practice" items describing seeking and reporting of secondary findings as consistent with medical standards, having sufficient evidence, and, for adults, the benefits generally outweighing potential harms. There was lack of agreement regarding benefits versus harms for children and impact on health-care resources. The majority agreed that patient preferences should be considered, including ability to opt out, and that informed consent was feasible and critical. Characteristics significantly associated with factor scores included country of residence, sequencing experience, and years in practice. CONCLUSION: The American College of Medical Genetics and Genomics should update a list of genes to be assessed when clinical genome-scale sequencing is performed. Informed consent is necessary, and reporting of secondary findings should be optional. Research on implementation of secondary findings reporting is needed.

ERCC6 dysfunction presenting as progressive neurological decline with brain hypomyelination.

Mutations in ERCC6 are associated with growth failure, intellectual disability, neurological dysfunction and deterioration, premature aging, and photosensitivity. We describe siblings with biallelic ERCC6 mutations (NM_000124.2:c. [543+4delA];[2008C>T]) and brain hypomyelination, microcephaly, cognitive decline, and skill regression but without photosensitivity or progeria. DNA repair assays on cultured skin fibroblasts confirmed a defect of transcription-coupled nucleotide excision repair and increased ultraviolet light sensitivity. This report expands the disease spectrum associated with ERCC6 mutations.

Family-based clinical associations and functional characterization of the serotonin 2A receptor gene (HTR2A) in autism spectrum disorder.

The serotonin 2A receptor gene (HTR2A) harbors two functional single nucleotide polymorphisms (SNPs) that are frequent in populations of African and European descent; rs6311, which affects mRNA expression, and rs6314, which changes the amino acid sequence of the encoded protein and affects the signaling properties of the receptor. Multiple clinical associations support a role for these SNPs in cognitive and neuropsychiatric phenotypes, although studies in autism spectrum disorder (ASD) remain equivocal. Here, we tested transmission disequilibrium of rs6311 and rs6314 in a cohort of 158 ASD trios (simplex and multiplex), observing significant under-transmission of the minor "A" allele of rs6311 to offspring with ASD (permuted P = 0.0004). Consistent with our previous findings in the dorsolateral prefrontal cortex of unaffected individuals, rs6311/A decreases expression of HTR2A mRNA with an extended 5' untranslated region (UTR) in the frontopolar cortex in brain samples from 54 ASD patients and controls. Interpreting the clinical results in the context of our mRNA expression analysis, we speculate that any risk associated with rs6311 is conferred by greater expression of the long 5'UTR mRNA isoform. The current study corroborates earlier associations between rs6311 and ASD in a family study, supporting the hypothesis that rs6311 plays a modulatory role in ASD risk.

Analysis of two candidate genes for Basan syndrome.

Basan syndrome is an extremely rare ectodermal dysplasia with autosomal dominant inheritance and variable expressivity. The etiology of Basan syndrome remains unknown. To identify the Basan syndrome gene, we sequenced keratin 14 (KRT14) and SMARCAD1 in a previously unreported kindred with the disease. Sequencing of the coding regions and splice junctions of KRT14 and SMARCAD1 was performed using PCR-amplified genomic DNA isolated from blood or saliva and standard PCR protocols. In vitro functional studies were performed for a variant identified in SMARCAD1. While direct sequencing of KRT14 failed to reveal any likely pathogenic sequence alterations or splice site variants, a heterozygous splicing variant (c.378+3A>T) that segregated with the disease was identified in the skin-specific isoform of SMARCAD1. In vitro studies failed to demonstrate a splicing defect in SMARCAD1. We screened two candidate genes for Basan syndrome in a 3-generation pedigree. The skin-specific isoform of SMARCAD1 remains a good candidate for this disease.

Targeting C4-demethylating genes in the cholesterol pathway sensitizes cancer cells to EGF receptor inhibitors via increased EGF receptor degradation.

UNLABELLED: Persistent signaling by the oncogenic EGF receptor (EGFR) is a major source of cancer resistance to EGFR targeting. We established that inactivation of 2 sterol biosynthesis pathway genes, SC4MOL (sterol C4-methyl oxidase-like) and its partner, NSDHL (NADP-dependent steroid dehydrogenase-like), sensitized tumor cells to EGFR inhibitors. Bioinformatics modeling of interactions for the sterol pathway genes in eukaryotes allowed us to hypothesize and then extensively validate an unexpected role for SC4MOL and NSDHL in controlling the signaling, vesicular trafficking, and degradation of EGFR and its dimerization partners, ERBB2 and ERBB3. Metabolic block upstream of SC4MOL with ketoconazole or CYP51A1 siRNA rescued cancer cell viability and EGFR degradation. Inactivation of SC4MOL markedly sensitized A431 xenografts to cetuximab, a therapeutic anti-EGFR antibody. Analysis of Nsdhl-deficient Bpa(1H/+) mice confirmed dramatic and selective loss of internalized platelet-derived growth factor receptor in fibroblasts, and reduced activation of EGFR and its effectors in regions of skin lacking NSDHL. SIGNIFICANCE: This work identifies a critical role for SC4MOL and NSDHL in the regulation of EGFR signaling and endocytic trafficking and suggests novel strategies to increase the potency of EGFR antagonists in tumors.