Patterns of Cytogenomic Findings from a Case Series of Recurrent Pregnancy Loss Provide Insight into the Extent of Genetic Defects Causing Miscarriages.

Background A retrospective study was performed to evaluate the patterns of cytogenomic findings detected from a case series of products of conception (POC) in recurrent pregnancy loss (RPL) over a 16-year period from 2007 to 2023. Results This case series of RPL was divided into a single analysis (SA) group of 266 women and a consecutive analysis (CA) group of 225 women with two to three miscarriages analyzed. Of the 269 POC from the SA group and the 469 POC from the CA group, a spectrum of cytogenomic abnormalities of simple aneuploidies, compound aneuploidies, polyploidies, and structural rearrangements/pathogenic copy number variants (pCNVs) were detected in 109 (41%) and 160 cases (34%), five (2%) and 11 cases (2%), 35 (13%) and 36 cases (8%), and 10 (4%) and 19 cases (4%), respectively. Patterns with recurrent normal karyotypes, alternating normal and abnormal karyotypes, and recurrent abnormal karyotypes were detected in 74 (33%), 71 (32%), and 80 (35%) of consecutive miscarriages, respectively. Repeat aneuploidies of monosomy X and trisomy 16, triploidy, and tetraploidy were detected in nine women. Conclusions A comparable spectrum of cytogenomic abnormalities was noted in the SA and CA groups of RPL. A skewed likelihood of 2/3 for recurrent normal and abnormal karyotypes and 1/3 for alternating normal and abnormal karyotypes in consecutive miscarriages was observed. Routine cytogenetic analysis should be performed for consecutive miscarriages. Further genomic sequencing to search for detrimental and embryonic lethal variants causing miscarriages and pathogenic variants inducing aneuploidies and polyploidies should be considered for RPL with recurrent normal and abnormal karyotypes.

Antifungal and elicitor activities of p-hydroxybenzoic acid for the control of aflatoxigenic Aspergillus flavus in kiwifruit.

Aspergillus flavus not only reduces kiwifruit production but also synthesizes carcinogenic aflatoxins, resulting in a relevant threat to human health. p-Hydroxybenzoic acid (pHBA) is one of the most abundant phenolics in kiwifruit. In this study, pHBA was found to reduce A. flavus mycelial growth by blocking the fungal mitotic exit network (MEN) and cytokinesis and to inhibit the biosynthesis of aflatoxins B1 and B2. The application of pHBA promoted the accumulation of endogenous pHBA and induced oxidative stress in A. flavus-infected kiwifruit, resulting in an increase in H2O2 content and catalase (CAT) and superoxide dismutase (SOD) activities. Preventive and curative treatments with 5 mM pHBA reduced A. flavus advancement by 46.1% and 68.0%, respectively. Collectively, the antifungal and elicitor properties of pHBA were examined for the first time, revealing new insights into the role of pHBA in the defense response of kiwifruit against A. flavus infection.

SHANK3 in vagal sensory neurons regulates body temperature, systemic inflammation, and sepsis.

Excessive inflammation has been implicated in autism spectrum disorder (ASD), but the underlying mechanisms have not been fully studied. SHANK3 is a synaptic scaffolding protein and mutations of SHANK3 are involved in ASD. Shank3 expression in dorsal root ganglion sensory neurons also regulates heat pain and touch. However, the role of Shank3 in the vagus system remains unknown. We induced systemic inflammation by lipopolysaccharide (LPS) and measured body temperature and serum IL-6 levels in mice. We found that homozygous and heterozygous Shank3 deficiency, but not Shank2 and Trpv1 deficiency, aggravates hypothermia, systemic inflammation (serum IL-6 levels), and sepsis mortality in mice, induced by lipopolysaccharide (LPS). Furthermore, these deficits can be recapitulated by specific deletion of Shank3 in Nav1.8-expressing sensory neurons in conditional knockout (CKO) mice or by selective knockdown of Shank3 or Trpm2 in vagal sensory neurons in nodose ganglion (NG). Mice with Shank3 deficiency have normal basal core temperature but fail to adjust body temperature after perturbations with lower or higher body temperatures or auricular vagus nerve stimulation. In situ hybridization with RNAscope revealed that Shank3 is broadly expressed by vagal sensory neurons and this expression was largely lost in Shank3 cKO mice. Mechanistically, Shank3 regulates the expression of Trpm2 in NG, as Trpm2 but not Trpv1 mRNA levels in NG were significantly reduced in Shank3 KO mice. Our findings demonstrated a novel molecular mechanism by which Shank3 in vagal sensory neurons regulates body temperature, inflammation, and sepsis. We also provided new insights into inflammation dysregulation in ASD.

D-bifunctional protein deficiency caused by splicing variants in a neonate with severe peroxisomal dysfunction and persistent hypoglycemia.

D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient's father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.

TET1-mediated DNA hydroxymethylation regulates adult remyelination in mice.

The mechanisms regulating myelin repair in the adult central nervous system (CNS) are unclear. Here, we identify DNA hydroxymethylation, catalyzed by the Ten-Eleven-Translocation (TET) enzyme TET1, as necessary for myelin repair in young adults and defective in old mice. Constitutive and inducible oligodendrocyte lineage-specific ablation of Tet1 (but not of Tet2), recapitulate this age-related decline in repair of demyelinated lesions. DNA hydroxymethylation and transcriptomic analyses identify TET1-target in adult oligodendrocytes, as genes regulating neuro-glial communication, including the solute carrier (Slc) gene family. Among them, we show that the expression levels of the Na+/K+/Cl- transporter, SLC12A2, are higher in Tet1 overexpressing cells and lower in old or Tet1 knockout. Both aged mice and Tet1 mutants also present inefficient myelin repair and axo-myelinic swellings. Zebrafish mutants for slc12a2b also display swellings of CNS myelinated axons. Our findings suggest that TET1 is required for adult myelin repair and regulation of the axon-myelin interface.

The role of Hipk2-p53 pathways in arsenic-induced autistic behaviors: A translational study from rats to humans.

Previous studies have associated the risk of autism spectrum disorder (ASD) with increased exposures to metals and metalloids such as arsenic. In this study, we used an animal-to-human translational strategy to identify key molecular changes that potentially mediated the effects of arsenic exposures on ASD development. In a previously established rat model, we have induced autistic behaviors in rat pups with gestational arsenic exposures (10 and 45 µg/L As2O3 in drinking water). Neuronal apoptosis and the associated epigenetic dysregulations in frontal cortex were assayed to screen potential mediating pathways, which were subsequently validated with qPCR, western blotting, and immunohistochemistry analyses. Furthermore, the identified pathway, along with serum levels of 26 elements including arsenic, were characterized in a case-control study with 21 ASD children and 21 age-matched healthy controls. In animals, we found that arsenic exposures caused difficulties of social interaction and increased stereotypic behaviors in a dose-dependent manner, accompanied by increased neuronal apoptosis and upregulation of Hipk2-p53 pathway in the frontal cortex. In humans, we found that serum levels of Hipk2 and p53 were 24.7 (95%CI: 8.5 to 43.4) % and 23.7 (95%CI: 10.5 to 38.5) % higher in ASD children than in healthy controls. ASD children had significantly higher serum levels of 15 elements, among which arsenic, silicon, strontium, and vanadium were positively associated with both Hipk2 and p53. Results from both the rat arsenic exposure and human case-control studies suggest a likely role of Hipk2-p53 pathway in ASD development induced by exposures to environmental pollutants such as arsenic.

Subacute Neuropsychiatric Syndrome in Girls With SHANK3 Mutations Responds to Immunomodulation.

Phenotypic and biological characterization of rare monogenic disorders represents 1 of the most important avenues toward understanding the mechanisms of human disease. Among patients with SH3 and multiple ankyrin repeat domains 3 (SHANK3) mutations, a subset will manifest neurologic regression, psychosis, and mood disorders. However, which patients will be affected, when, and why are important unresolved questions. Authors of recent studies suggest neuronal SHANK3 expression is modulated by both inflammatory and hormonal stimuli. In this case series, we describe 4 independent clinical observations of an immunotherapy responsive phenotype of peripubertal-onset neuropsychiatric regression in 4 girls with pathogenic SHANK3 mutations. Each child exhibited a history of stable, mild-to-moderate lifelong developmental disability until 12 to 14 years of age, at which time each manifested a similar, subacute-onset neurobehavioral syndrome. Symptoms included mutism, hallucinations, insomnia, inconsolable crying, obsessive-compulsive behaviors, loss of self-care, and urinary retention and/or incontinence. Symptoms were relatively refractory to antipsychotic medication but improved after immunomodulatory treatment. All 4 patients exhibited chronic relapsing courses during a period of treatment and follow-up ranging from 3 to 6 years. Two of the 4 girls recovered their premorbid level of functioning. We briefly review the scientific literature to offer a conceptual and molecular framework for understanding these clinical observations. Future clinical and translational investigations in this realm may offer insights into mechanisms and therapies bridging immune function and human behavior.

Epigenetic dysregulation of Oxtr in Tet1-deficient mice has implications for neuropsychiatric disorders.

OXTR modulates a variety of behaviors in mammals, including social memory and recognition. Genetic and epigenetic dysregulation of OXTR has been suggested to be implicated in neuropsychiatric disorders, including autism spectrum disorder (ASD). While the involvement of DNA methylation is suggested, the mechanism underlying epigenetic regulation of OXTR is largely unknown. This has hampered the experimental design and interpretation of the results of epigenetic studies of OXTR in neuropsychiatric disorders. From the generation and characterization of a new line of Tet1 mutant mice - by deleting the largest coding exon 4 (Tet1Δe4) - we discovered for the first time to our knowledge that Oxtr has an array of mRNA isoforms and a complex transcriptional regulation. Select isoforms of Oxtr are significantly reduced in the brain of Tet1Δe4-/- mice. Accordingly, CpG islands of Oxtr are hypermethylated during early development and persist into adulthood. Consistent with the reduced express of OXTR, Tet1Δe4-/- mice display impaired maternal care, social behavior, and synaptic responses to oxytocin stimulation. Our findings elucidate a mechanism mediated by TET1 protein in regulating Oxtr expression by preventing DNA hypermethylation of Oxtr. The discovery of epigenetic dysregulation of Oxtr in TET1-deficient mouse brain supports the necessity of a reassessment of existing findings and a value of future studies of OXTR in neuropsychiatric disorders.

Further evidence for the involvement of EFL1 in a Shwachman-Diamond-like syndrome and expansion of the phenotypic features.

Recent evidence has implicated EFL1 in a phenotype overlapping Shwachman-Diamond syndrome (SDS), with the functional interplay between EFL1 and the previously known causative gene SBDS accounting for the similarity in clinical features. Relatively little is known about the phenotypes associated with pathogenic variants in the EFL1 gene, but the initial indication was that phenotypes may be more severe, when compared with SDS. We report a pediatric patient who presented with a metaphyseal dysplasia and was found to have biallelic variants in EFL1 on reanalysis of trio whole-exome sequencing data. The variant had not been initially reported because of the research laboratory's focus on de novo variants. Subsequent phenotyping revealed variability in her manifestations. Although her metaphyseal abnormalities were more severe than in the original reported cohort with EFL1 variants, the bone marrow abnormalities were generally mild, and there was equivocal evidence for pancreatic insufficiency. Despite the limited number of reported patients, variants in EFL1 appear to cause a broader spectrum of symptoms that overlap with those seen in SDS. Our report adds to the evidence of EFL1 being associated with an SDS-like phenotype and provides information adding to our understanding of the phenotypic variability of this disorder. Our report also highlights the value of exome data reanalysis when a diagnosis is not initially apparent.

Systematic reconstruction of autism biology from massive genetic mutation profiles.

Autism spectrum disorder (ASD) affects 1% of world population and has become a pressing medical and social problem worldwide. As a paradigmatic complex genetic disease, ASD has been intensively studied and thousands of gene mutations have been reported. Because these mutations rarely recur, it is difficult to (i) pinpoint the fewer disease-causing versus majority random events and (ii) replicate or verify independent studies. A coherent and systematic understanding of autism biology has not been achieved. We analyzed 3392 and 4792 autism-related mutations from two large-scale whole-exome studies across multiple resolution levels, that is, variants (single-nucleotide), genes (protein-coding unit), and pathways (molecular module). These mutations do not recur or replicate at the variant level, but significantly and increasingly do so at gene and pathway levels. Genetic association reveals a novel gene + pathway dual-hit model, where the mutation burden becomes less relevant. In multiple independent analyses, hundreds of variants or genes repeatedly converge to several canonical pathways, either novel or literature-supported. These pathways define recurrent and systematic ASD biology, distinct from previously reported gene groups or networks. They also present a catalog of novel ASD risk factors including 118 variants and 72 genes. At a subpathway level, most variants disrupt the pathway-related gene functions, and in the same gene, they tend to hit residues extremely close to each other and in the same domain. Multiple interacting variants spotlight key modules, including the cAMP (adenosine 3',5'-monophosphate) second-messenger system and mGluR (metabotropic glutamate receptor) signaling regulation by GRKs (G protein-coupled receptor kinases). At a superpathway level, distinct pathways further interconnect and converge to three biology themes: synaptic function, morphology, and plasticity.

Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.

The importance of managing the patient and not the gene: expanded phenotype of GLE1-associated arthrogryposis.

GLE1 encodes a protein important for mRNA export and appears to play roles in translation initiation and termination as well. Pathogenic variants in GLE1 mutations have been associated with lethal contracture syndrome and lethal arthrogryposis with anterior horn cell disease; phenotypes reported in individuals include fetal akinesia and a severe form of motor neuron disease, typically presenting with prenatal symptoms and perinatal lethality. In this article, we identified biallelic missense mutations in GLE1 by trio whole-exome sequencing in an individual affected with congenital motor weakness and contractures as well as feeding and respiratory difficulties. Muscle biopsy was consistent with anterior horn cell disease and supported the pathogenicity of the sequence variants. Importantly, this individual survived past the perinatal period with respiratory support and currently demonstrates age-appropriate cognition and slow but steady motor developmental progress. We propose that pathogenic variants in GLE1 can be associated with a nonperinatal lethal motor phenotype, and affected individuals can demonstrate motor skill progression, unlike prototypical anterior horn cell diseases such as spinal muscular atrophy.

Neonatal nonepileptic myoclonus is a prominent clinical feature of KCNQ2 gain-of-function variants R201C and R201H.

OBJECTIVE: To analyze whether KCNQ2 R201C and R201H variants, which show atypical gain-of-function electrophysiologic properties in vitro, have a distinct clinical presentation and outcome. METHODS: Ten children with heterozygous, de novo KCNQ2 R201C or R201H variants were identified worldwide, using an institutional review board (IRB)-approved KCNQ2 patient registry and database. We reviewed medical records and, where possible, interviewed parents and treating physicians using a structured, detailed phenotype inventory focusing on the neonatal presentation and subsequent course. RESULTS: Nine patients had encephalopathy from birth and presented with prominent startle-like myoclonus, which could be triggered by sound or touch. In seven patients, electroencephalography (EEG) was performed in the neonatal period and showed a burst-suppression pattern. However, myoclonus did not have an EEG correlate. In many patients the paroxysmal movements were misdiagnosed as seizures. Seven patients developed epileptic spasms in infancy. In all patients, EEG showed a slow background and multifocal epileptiform discharges later in life. Other prominent features included respiratory dysfunction (perinatal respiratory failure and/or chronic hypoventilation), hypomyelination, reduced brain volume, and profound developmental delay. One patient had a later onset, and sequencing indicated that a low abundance (~20%) R201C variant had arisen by postzygotic mosaicism. SIGNIFICANCE: Heterozygous KCNQ2 R201C and R201H gain-of-function variants present with profound neonatal encephalopathy in the absence of neonatal seizures. Neonates present with nonepileptic myoclonus that is often misdiagnosed and treated as seizures. Prognosis is poor. This clinical presentation is distinct from the phenotype associated with loss-of-function variants, supporting the value of in vitro functional screening. These findings suggest that gain-of-function and loss-of-function variants need different targeted therapeutic approaches.

Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is an imprinting disorder caused by a deficiency of paternally expressed gene(s) in the 15q11-q13 chromosomal region. The regulation of imprinted gene expression in this region is coordinated by an imprinting center (PWS-IC). In individuals with PWS, genes responsible for PWS on the maternal chromosome are present, but repressed epigenetically, which provides an opportunity for the use of epigenetic therapy to restore expression from the maternal copies of PWS-associated genes. Through a high-content screen (HCS) of >9,000 small molecules, we discovered that UNC0638 and UNC0642-two selective inhibitors of euchromatic histone lysine N-methyltransferase-2 (EHMT2, also known as G9a)-activated the maternal (m) copy of candidate genes underlying PWS, including the SnoRNA cluster SNORD116, in cells from humans with PWS and also from a mouse model of PWS carrying a paternal (p) deletion from small nuclear ribonucleoprotein N (Snrpn (S)) to ubiquitin protein ligase E3A (Ube3a (U)) (mouse model referred to hereafter as m+/pΔS-U). Both UNC0642 and UNC0638 caused a selective reduction of the dimethylation of histone H3 lysine 9 (H3K9me2) at PWS-IC, without changing DNA methylation, when analyzed by bisulfite genomic sequencing. This indicates that histone modification is essential for the imprinting of candidate genes underlying PWS. UNC0642 displayed therapeutic effects in the PWS mouse model by improving the survival and the growth of m+/pΔS-U newborn pups. This study provides the first proof of principle for an epigenetics-based therapy for PWS.

Isoform Switch of TET1 Regulates DNA Demethylation and Mouse Development.

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. However, it remains elusive how exactly they target substrates and execute oxidation. Interestingly, we found that, in mice, the full-length TET1 isoform (TET1e) is restricted to early embryos, embryonic stem cells (ESCs), and primordial germ cells (PGCs). By contrast, a short isoform (TET1s) is preferentially expressed in somatic cells, which lacks the N terminus including the CXXC domain, a DNA-binding module that often recognizes CpG islands (CGIs) where TET1 predominantly occupies. Unexpectedly, TET1s can still bind CGIs despite the fact that its global chromatin binding is significantly reduced. Interestingly, global chromatin binding, but not targeted binding at CGIs, is correlated with TET1-mediated demethylation. Finally, mice with exclusive expression of Tet1s failed to erase imprints in PGCs and displayed developmental defects in progeny. These data show that isoform switch of TET1 regulates epigenetic memory erasure and mouse development.

The E3-ligase E6AP Represses Breast Cancer Metastasis via Regulation of ECT2-Rho Signaling.

Metastatic disease is the major cause of breast cancer-related death and despite many advances, current therapies are rarely curative. Tumor cell migration and invasion require actin cytoskeletal reorganization to endow cells with capacity to disseminate and initiate the formation of secondary tumors. However, it is still unclear how these migratory cells colonize distant tissues to form macrometastases. The E6-associated protein, E6AP, acts both as an E3 ubiquitin-protein ligase and as a coactivator of steroid hormone receptors. We report that E6AP suppresses breast cancer invasiveness, colonization, and metastasis in mice, and in breast cancer patients, loss of E6AP associates with poor prognosis, particularly for basal breast cancer. E6AP regulates actin cytoskeletal remodeling via regulation of Rho GTPases, acting as a negative regulator of ECT2, a GEF required for activation of Rho GTPases. E6AP promotes ubiquitination and proteasomal degradation of ECT2 for which high expression predicts poor prognosis in breast cancer patients. We conclude that E6AP suppresses breast cancer metastasis by regulating actin cytoskeleton remodeling through the control of ECT2 and Rho GTPase activity. These findings establish E6AP as a novel suppressor of metastasis and provide a compelling rationale for inhibition of ECT2 as a therapeutic approach for patients with metastatic breast cancer. Cancer Res; 76(14); 4236-48. ©2016 AACR.

Adult surgical experience with Loeys-Dietz syndrome.

BACKGROUND: Loeys-Dietz syndrome (LDS) results from mutations in receptors for the cytokine transforming growth factor-ß leading to aggressive aortic pathology sometimes accompanied by specific phenotypic features including bifid uvula, hypertelorism, cleft palate, and generalized arterial tortuosity. We reviewed our adult surgical experience with LDS in order to validate current recommendations regarding management of this newly described disease. METHODS: All adult (≥ 18 years old) patients with LDS undergoing surgical treatment at a single referral institution from September 1999 to May 2013 were retrospectively reviewed. RESULTS: Eleven adult LDS patients were identified by clinical criteria and genotyping. Seven (64%) experienced acute type A dissection at some point in their lives. All eventually required aortic root replacement, and 73% required multiple vascular surgical interventions. Over a mean follow-up of 65 ± 49 months, 2.8 cardiovascular procedures per patient were performed. In patients with type A dissection, a mean of 3.4 operations were performed versus 1.8 operations for patients without dissection. Total aortic replacement was required in 5 patients (45%) and 2 (18%) required neurosurgical intervention for cerebrovascular pathology. There was 1 late death from infectious complications, and no deaths from vascular catastrophe. CONCLUSIONS: These results confirm the aggressive nature of LDS aortic pathology. However, the improved survival compared with earlier LDS reports suggest that aggressive treatment strategies may alter outcomes and improve the natural history of this syndrome.

Overview of mouse models of autism spectrum disorders.

This overview describes many well characterized mouse models of autism spectrum disorders (ASDs). Mouse models considered here were selected because they are examples of genetically engineered models where human genetic evidence supports a causative relationship between the targeted mutation and the behavioral phenotype. As the ASD diagnosis is based primarily on behavioral evaluations in humans in the domains of social interaction, communication, and restricted interests, the murine phenotypes analogous to human autistic behaviors are highlighted for the different models and behaviors. Although genetically engineered mouse models with good construct and face validity are valuable for identifying and defining underlying pathophysiological mechanisms and for developing potential therapeutic interventions for the human condition, the translational value of various rodent behavioral assays remains a subject of debate. Significant challenges associated with modeling ASDs in rodents because of the clinical and molecular heterogeneity that characterize this disorder are also considered.

Epigenetic dysregulation of SHANK3 in brain tissues from individuals with autism spectrum disorders.

The molecular basis for the majority of cases of autism spectrum disorders (ASD) remains unknown. We tested the hypothesis that ASD have an epigenetic cause by performing DNA methylation profiling of five CpG islands (CGI-1 to CGI-5) in the SHANK3 gene in postmortem brain tissues from 54 ASD patients and 43 controls. We found significantly increased overall DNA methylation (epimutation) in three intragenic CGIs (CGI-2, CGI-3 and CGI-4). The increased methylation was clustered in the CGI-2 and CGI-4 in ∼15% of ASD brain tissues. SHANK3 has an extensive array of mRNA splice variants resulting from combinations of five intragenic promoters and alternative splicing of coding exons. Altered expression and alternative splicing of SHANK3 isoforms were observed in brain tissues with increased methylation of SHANK3 CGIs in ASD brain tissues. A DNA methylation inhibitor modified the methylation of CGIs and altered the isoform-specific expression of SHANK3 in cultured cells. This study is the first to find altered methylation patterns in SHANK3 in ASD brain samples. Our finding provides evidence to support an alternative approach to investigating the molecular basis of ASD. The ability to alter the epigenetic modification and expression of SHANK3 by environmental factors suggests that SHANK3 may be a valuable biomarker for dissecting the role of gene and environment interaction in the etiology of ASD.