PDF file - 23K, Summary of Interactions for sterol pathway genes

PDF file - 1.2MB, Fig. S1. Validation of the sterol pathway targeting siRNA. Fig. S2. Silencing of SC4MOL increased erlotinib-induced apoptosis. Fig. S3. Metabolic effects of SC4MOL silencing and sterols supplementation. Fig. S4. Sterol pathway interactions. Fig. S5. Effects of SC4MOL silencing on EGFR synthesis and I-125 EGF internalization and degradation. Fig. S6. Effects of sterol pathway genes silencing on EGFR endosomal traffic. Fig. S7. Effects of SC4MOL silencing on EGFR ubiquitin conjugation and accumulation in late endosomes. Fig. S8. Supporting data for Figure 5. Fig. S9. Supporting data for Figure 7A. Fig. S10. Analysis of NSDHL expression in malignant and benign epithelial tissues

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.

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.

Guidance from the original American College of Medical Genetics and Genomics (ACMG) Policy Statement on incidental findings in 2013 established that clinical laboratories performing exome or genome sequencing (ES/GS) should report known pathogenic (KP) or expected pathogenic (EP) variants in a defined set of genes considered medically actionable, even when unrelated to the primary medical reason for testing. 1. Green R.C. et al. ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing. 1:CAS:528:DC%2BC3sXhtVKku73K 23788249 3727274 10.1038/gim.2013.73Genet. Med. 2013; 15: 565-574 Google Scholar Subsequently, the ACMG updated the terminology used to describe these types of findings to align with nomenclature recommendations from the Presidential Commission on Bioethical Issues that defined “secondary findings” (SF) as variants that are actively sought in genes that are part of a defined list, as opposed to genomic variants found incidentally or accidentally. 2. Kalia S.S. et al. 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. 27854360 10.1038/gim.2016.190Genet. Med. 2017; 19: 249-255 Google Scholar In a survey of ACMG members, more than 90% of respondents supported a minimum gene list of SFs that would be updated and refined over time. 3. Scheuner M.T. et al. Reporting genomic secondary findings: ACMG members weigh in. 25394173 10.1038/gim.2014.165Genet. Med. 2015; 17: 27-35 Google Scholar Recognizing this need, the ACMG Board of Directors (BOD) created the ACMG Secondary Findings Maintenance Working Group (SFWG) in 2014 to define and implement a process for updating the SF list. Shortly thereafter, we established a mechanism for ACMG members to submit nominations to add or remove genes from the list through a nomination form found on the ACMG website. 4. ACMG. Secondary findings nomination form. https://www.acmg.net/PDFLibrary/Secondary-Findings-Panel-Nomination-Form.pdf (2021). Google Scholar The 2017 update added four new genes, removed one gene, and rejected one nominated gene from inclusion in the list. Adoption of clinical ES/GS since then has expanded even further. 2. Kalia S.S. et al. 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. 27854360 10.1038/gim.2016.190Genet. Med. 2017; 19: 249-255 Google Scholar The ACMG SFWG and the BOD agree that timely updates to the SF list are increasingly important, which has led to the decision to separate the ACMG SFWG policy update discussed here from the updated ACMG SF gene list. The SFWG will update this general policy statement as needed, but on a less frequent basis, generally every 3–4 years, and the gene list will be updated on an annual basis, with a goal of publishing the updated list each year in January.

ABSTRACTIndividuals with autism spectrum disorder (ASD) or related neurodevelopmental disorders (NDDs) often carry disruptive mutations in genes that are depleted of functional variation in the broader population. We build upon this observation and exome sequencing from 154,842 individuals to explore the allelic diversity of rare protein-coding variation contributing risk for ASD and related NDDs. Using an integrative statistical model, we jointly analyzed rare protein-truncating variants (PTVs), damaging missense variants, and copy number variants (CNVs) derived from exome sequencing of 63,237 individuals from ASD cohorts. We discovered 71 genes associated with ASD at a false discovery rate (FDR) ≤ 0.001, a threshold approximately equivalent to exome-wide significance, and 183 genes at FDR ≤ 0.05. Associations were predominantly driven by de novo PTVs, damaging missense variants, and CNVs: 57.4%, 21.2%, and 8.32% of evidence, respectively. Though fewer in number, CNVs conferred greater relative risk than PTVs, and repeat-mediated de novo CNVs exhibited strong maternal bias in parent-of-origin (e.g., 92.3% of 16p11.2 CNVs), whereas all other CNVs showed a paternal bias. To explore how genes associated with ASD and NDD overlap or differ, we analyzed our ASD cohort alongside a developmental delay (DD) cohort from the deciphering developmental disorders study (DDD; n=91,605 samples). We first reanalyzed the DDD dataset using the same models as the ASD cohorts, then performed joint analyses of both cohorts and identified 373 genes contributing to NDD risk at FDR ≤ 0.001 and 662 NDD risk genes at FDR ≤ 0.05. Of these NDD risk genes, 54 genes (125 genes at FDR ≤ 0.05) were unique to the joint analyses and not significant in either cohort alone. Our results confirm overlap of most ASD and DD risk genes, although many differ significantly in frequency of mutation. Analyses of single-cell transcriptome datasets showed that genes associated predominantly with DD were strongly enriched for earlier neurodevelopmental cell types, whereas genes displaying stronger evidence for association in ASD cohorts were more enriched for maturing neurons. The ASD risk genes were also enriched for genes associated with schizophrenia from a separate rare coding variant analysis of 121,570 individuals, emphasizing that these neuropsychiatric disorders share common pathways to risk.

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.

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.

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.

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 ). 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. 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. We find associations between ASD phenotypes and regulatory variants in catecholamine biosynthesis genes. This work may help guide future genetics studies related to ASD.

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.

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.

Objective It is recognised that 5%-10% of children with macrocephaly and autism spectrum disorder (ASD) and/or intellectual disability (ID) have a heterozygous pathogenic mutation in the PTEN tumour suppressor gene that is associated with PTEN hamartoma tumour syndrome. However, the clinical features and course in children with a pathogenic PTEN mutation are unclear and have not been well documented.Study objectives We undertook a retrospective chart review of children (<18 years) with pathogenic PTEN mutations to ascertain clinical findings, clinical course and possible outcomes.Results Clinical and molecular data were collected and analysed for 47 patients with PTEN mutation from 38 eligible families. Macrocephaly (average head circumference of + 5.7 SD) with developmental delay, ID and/or ASD were the most common presenting signs/symptoms (66%). Clinical features included dermatological findings (66%), gastrointestinal (GI) symptoms (34%), ASD diagnosis (50%), abnormal brain imaging (53% of those examined) and abnormal thyroid imaging (26%).Conclusions This is the largest survey of clinical features in children with PTEN pathogenic mutations to date. It confirms earlier reports of increased rates of neurodevelopmental disorders. Dermatological, GI and thyroid abnormalities are age dependent and may not be present at the time of diagnosis, requiring regular monitoring and medical surveillance. Early paediatric diagnosis is important for institution of medical and developmental surveillance as well as for testing other at-risk family members.

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.

The introduction of diagnostic clinical genome and exome sequencing (CGES) is changing the scope of practice for clinical geneticists. Many large institutions are making a significant investment in infrastructure and technology, allowing clinicians to access CGES, especially as health-care coverage begins to extend to clinically indicated genomic sequencing-based tests. Translating and realizing the comprehensive clinical benefits of genomic medicine remain a key challenge for the current and future care of patients. With the increasing application of CGES, it is necessary for geneticists and other health-care providers to understand its benefits and limitations in order to interpret the clinical relevance of genomic variants identified in the context of health and disease. New, collaborative working relationships with specialists across diverse disciplines (e.g., clinicians, laboratorians, bioinformaticians) will undoubtedly be key attributes of the future practice of clinical genetics and may serve as an example for other specialties in medicine. These new skills and relationships will also inform the development of the future model of clinical genetics training curricula. To address the evolving role of the clinical geneticist in the rapidly changing climate of genomic medicine, two Clinical Genetics Think Tank meetings were held that brought together physicians, laboratorians, scientists, genetic counselors, trainees, and patients with experience in clinical genetics, genetic diagnostics, and genetics education. This article provides recommendations that will guide the integration of genomics into clinical practice.Genet Med 18 11, 1075-1084.

Our previous studies have shown that inhibition of 3-hydroxy-3-methyl-glutaryl-CoA reductase, a rate-limiting enzyme in the early stages of cholesterol synthesis, perturbs epidermal permeability barrier function. But inherited or acquired blockade of distal steps in the cholesterol synthetic pathway also result in syndromic forms of ichthyosis, likely associated with, and/or driven by a barrier abnormality. Yet, whether the dermatoses is due to reduced cholesterol production and/or the accumulation of toxic metabolic precursors is not clear. Mutations that result in reduced function of the gene that encodes 3b-hydroxysteroid-Δ8, Δ7-isomerase, an enzyme late in the cholesterol synthetic pathway, cause mosaic patchy epidermal hyperkeratinization. We determined here whether epidermal functions are altered in 3b-hydroxysteroid-Δ8, Δ7-isomerase deficient mice (TD), an analogue for CHILD syndrome in humans vs. their wild type controls. Basal transepidermal water loss (TEWL), stratum corneum hydration and skin surface pH were measured with respective probes connected to an MPA5 skin physiology monitor. Epidermal permeability barrier homeostasis was assessed by measuring permeability barrier recovery 2 and 4 hours after acute barrier disruption by repeated tape-stripping. Additionally, differences in inflammatory responses were assessed in an irritant dermatitis model, induced by a single topical application of 12-0-tetradecanoylphorbol-13-acetate (TPA). Rather than the expected compromise in functions, our results showed that TD mice exhibit accelerated permeability barrier recovery. Yet, in comparison with wild type controls, TD mice showed growth retardation and significant reductions in ear thickness following TPA or oxazolone challenge, further suggestive of a barrier abnormality. Together, these results suggest that the competent barrier of TD mice reflects an ongoing compensatory response to a chronic underlying barrier abnormality.

NSDHL is a 3β-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.