David M. Margulies

Genetic Misdiagnoses and the Potential for Health Disparities

BACKGROUND For more than a decade, risk stratification for hypertrophic cardiomyopathy has been enhanced by targeted genetic testing. Using sequencing results, clinicians routinely assess the risk of hypertrophic cardiomyopathy in a patients relatives and diagnose the condition in patients who have ambiguous clinical presentations. However, the benefits of genetic testing come with the risk that variants may be misclassified. METHODS Using publicly accessible exome data, we identified variants that have previously been considered causal in hypertrophic cardiomyopathy and that are overrepresented in the general population. We studied these variants in diverse populations and reevaluated their initial ascertainments in the medical literature. We reviewed patient records at a leading genetic-testing laboratory for occurrences of these variants during the near-decade-long history of the laboratory. RESULTS Multiple patients, all of whom were of African or unspecified ancestry, received positive reports, with variants misclassified as pathogenic on the basis of the understanding at the time of testing. Subsequently, all reported variants were recategorized as benign. The mutations that were most common in the general population were significantly more common among black Americans than among white Americans (P<0.001). Simulations showed that the inclusion of even small numbers of black Americans in control cohorts probably would have prevented these misclassifications. We identified methodologic shortcomings that contributed to these errors in the medical literature. CONCLUSIONS The misclassification of benign variants as pathogenic that we found in our study shows the need for sequencing the genomes of diverse populations, both in asymptomatic controls and the tested patient population. These results expand on current guidelines, which recommend the use of ancestry-matched controls to interpret variants. As additional populations of different ancestry backgrounds are sequenced, we expect variant reclassifications to increase, particularly for ancestry groups that have historically been less well studied. (Funded by the National Institutes of Health.).

High-throughput sequencing of mGluR signaling pathway genes reveals enrichment of rare variants in autism.

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism.

Will disruptive mood dysregulation disorder reduce false diagnosis of bipolar disorder in children

Margulies DM, Weintraub S, Basile J, Grover PJ, Carlson GA. Will disruptive mood dysregulation disorder reduce false diagnosis of bipolar disorder in children? Bipolar Disord 2012: 14: 488–496.

Comparative RNA editing in autistic and neurotypical cerebella.

Adenosine-to-inosine (A-to-I) RNA editing is a neurodevelopmentally regulated epigenetic modification shown to modulate complex behavior in animals. Little is known about human A-to-I editing, but it is thought to constitute one of many molecular mechanisms connecting environmental stimuli and behavioral outputs. Thus, comprehensive exploration of A-to-I RNA editing in human brains may shed light on gene–environment interactions underlying complex behavior in health and disease. Synaptic function is a main target of A-to-I editing, which can selectively recode key amino acids in synaptic genes, directly altering synaptic strength and duration in response to environmental signals. Here, we performed a high-resolution survey of synaptic A-to-I RNA editing in a human population, and examined how it varies in autism, a neurodevelopmental disorder in which synaptic abnormalities are a common finding. Using ultra-deep (>1000 × ) sequencing, we quantified the levels of A-to-I editing of 10 synaptic genes in postmortem cerebella from 14 neurotypical and 11 autistic individuals. A high dynamic range of editing levels was detected across individuals and editing sites, from 99.6% to below detection limits. In most sites, the extreme ends of the population editing distributions were individuals with autism. Editing was correlated with isoform usage, clusters of correlated sites were identified, and differential editing patterns examined. Finally, a dysfunctional form of the editing enzyme adenosine deaminase acting on RNA B1 was found more commonly in postmortem cerebella from individuals with autism. These results provide a population-level, high-resolution view of A-to-I RNA editing in human cerebella and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism.

Mutation of KCNJ8 in a patient with Cantú syndrome with unique vascular abnormalities - support for the role of K(ATP) channels in this condition.

KCNJ8 (NM_004982) encodes the pore forming subunit of one of the ATP-sensitive inwardly rectifying potassium (KATP) channels. KCNJ8 sequence variations are traditionally associated with J-wave syndromes, involving ventricular fibrillation and sudden cardiac death. Recently, the KATP gene ABCC9 (SUR2, NM_020297) has been associated with the multi-organ disorder Cantú syndrome or hypertrichotic osteochondrodysplasia (MIM 239850) (hypertrichosis, macrosomia, osteochondrodysplasia, and cardiomegaly). Here, we report on a patient with a de novo nonsynonymous KCNJ8 SNV (p.V65M) and Cantú syndrome, who tested negative for mutations in ABCC9. The genotype and multi-organ abnormalities of this patient are reviewed. A careful screening of the KATP genes should be performed in all individuals diagnosed with Cantú syndrome and no mutation in ABCC9.

Callous-Unemotional traits, proactive aggression, and treatment outcomes of aggressive children with attention-deficit/hyperactivity disorder

OBJECTIVE Stimulant treatment improves impulse control among children with attention-deficit/hyperactivity disorder (ADHD). Decreased aggression often accompanies stimulant pharmacotherapy, suggesting that impulsiveness is integral to aggressive behavior in these children. However, children with high callous-unemotional (CU) traits and proactive aggression may benefit less from ADHD pharmacotherapy, because their aggressive behavior seems more purposeful and deliberate. This studys objective was to determine whether pretreatment CU traits and proactive aggression affect treatment outcomes among aggressive children with ADHD receiving stimulant monotherapy. METHOD We implemented a stimulant optimization protocol with 160 children 6 to 13 years of age (mean [SD] age of 9.31 [2.02] years; 78.75% male) with ADHD, oppositional defiant or conduct disorder, and significant aggressive behavior. Family-focused behavioral intervention was provided concurrently. The primary outcome was the Retrospective Modified Overt Aggression Scale. The Antisocial Process Screening Device and the Aggression Scale, also completed by parents, measured CU traits and proactive aggression, respectively. Analyses examined moderating effects of CU traits and proactive aggression on outcomes. RESULTS In all, 82 children (51%) experienced remission of aggressive behavior. Neither CU traits nor proactive aggression predicted remission (CU traits: odds ratio [OR] = 0.94, 95% CI = 0.80-1.11; proactive aggression, OR = 1.05, 95% CI = 0.86-1.29). Children whose overall aggression remitted showed decreases in CU traits (effect size = -0.379, 95% CI = -0.60 to -0.16) and proactive aggression (effect size = -0.463, 95% CI = -0.69 to -0.23). CONCLUSIONS Findings suggest that pretreatment CU traits and proactive aggression do not forecast worse outcomes for aggressive children with ADHD receiving optimized stimulant pharmacotherapy. With such treatment, CU traits and proactive aggression may decline alongside other behavioral improvements. Clinical trial registration information--Medication Strategies for Treating Aggressive Behavior in Youth With Attention Deficit Hyperactivity Disorder; http://clinicaltrials.gov/; NCT00228046; and Effectiveness of Combined Medication Treatment for Aggression in Children With Attention Deficit With Hyperactivity Disorder (The SPICY Study); http://clinicaltrials.gov/; NCT00794625.

iPSC-derived neurons as a higher-throughput readout for autism: Promises and pitfalls

The elucidation of disease etiologies and establishment of robust, scalable, high-throughput screening assays for autism spectrum disorders (ASDs) have been impeded by both inaccessibility of disease-relevant neuronal tissue and the genetic heterogeneity of the disorder. Neuronal cells derived from induced pluripotent stem cells (iPSCs) from autism patients may circumvent these obstacles and serve as relevant cell models. To date, derived cells are characterized and screened by assessing their neuronal phenotypes. These characterizations are often etiology-specific or lack reproducibility and stability. In this review, we present an overview of efforts to study iPSC-derived neurons as a model for autism, and we explore the plausibility of gene expression profiling as a reproducible and stable disease marker.

Liquid risperidone in the treatment of rages in psychiatrically hospitalized children with possible bipolar disorder.

OBJECTIVE To examine the safety and efficacy of liquid risperidone to reduce duration of rages in children with severe mood dysregulation (SMD) or possible bipolar disorder (BP). METHOD The sample included 151 consecutive admissions of 5-12 year old children to a psychiatric inpatient unit. Diagnostic information and history of prior rage outbursts were obtained at admission. In hospital, a first rage was treated with seclusion. If a second rage occurred, the child was offered liquid risperidone to help him/her regain control. Durations of unmedicated and last medicated rage were compared. Rage frequency in children with SMD and several definitions of BP were compared. RESULTS Although 82 of 151 admissions were prompted by rages, rages occurred during only 49 hospitalizations and occurred more than once in only 24. In 16 multiply medicated children, duration of rages dropped from a baseline of 44.4 +/- 20.2 min to 25.6 +/- 12.5 min at the childs last dose. Neither SMD nor any definition of BP influenced rage response in this small sample. The average liquid risperidone dose was 0.02 mg/kg. All but two children also took atypical antipsychotics daily. In the evaluation of medicated rage episodes with standard rating scales, no extrapyramidal side effects, akathisia, or abnormal involuntary movements were observed, and the rate of sedation/sleepiness (7/67 = 10.4%) was similar and not significantly different from that observed during nonmedicated episodes (8/46 = 17.4%). CONCLUSIONS Liquid risperidone may be a safe and effective way to shorten the duration of rage episodes regardless of diagnosis. However, definitive conclusions cannot be drawn in the absence of a placebo control as children were also receiving other behavioral and psychopharmacologic treatments.

The sensitivity of exome sequencing in identifying pathogenic mutations for LGMD in the United States

The current study characterizes a cohort of limb-girdle muscular dystrophy (LGMD) in the United States using whole-exome sequencing. Fifty-five families affected by LGMD were recruited using an institutionally approved protocol. Exome sequencing was performed on probands and selected parental samples. Pathogenic mutations and cosegregation patterns were confirmed by Sanger sequencing. Twenty-two families (40%) had novel and previously reported pathogenic mutations, primarily in LGMD genes, and also in genes for Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital myopathy, myofibrillar myopathy, inclusion body myopathy and Pompe disease. One family was diagnosed via clinical testing. Dominant mutations were identified in COL6A1, COL6A3, FLNC, LMNA, RYR1, SMCHD1 and VCP, recessive mutations in ANO5, CAPN3, GAA, LAMA2, SGCA and SGCG, and X-linked mutations in DMD. A previously reported variant in DMD was confirmed to be benign. Exome sequencing is a powerful diagnostic tool for LGMD. Despite careful phenotypic screening, pathogenic mutations were found in other muscle disease genes, largely accounting for the increased sensitivity of exome sequencing. Our experience suggests that broad sequencing panels are useful for these analyses because of the phenotypic overlap of many neuromuscular conditions. The confirmation of a benign DMD variant illustrates the potential of exome sequencing to help determine pathogenicity.

Prevalence and Treatment Outcomes of Persistent Negative Mood Among Children with Attention-Deficit/Hyperactivity Disorder and Aggressive Behavior.

OBJECTIVE Diagnostic criteria for disruptive mood dysregulation disorder (DMDD) require 1) periodic rageful outbursts and 2) disturbed mood (anger or irritability) that persists most of the time in between outbursts. Stimulant monotherapy, methodically titrated, often culminates in remission of severe aggressive behavior, but it is unclear whether those with persistent mood symptoms benefit less.This study examined the association between the presence of persistent mood disturbances and treatment outcomes among children with attention-deficit/hyperactivity disorder (ADHD) and periodic aggressive, rageful outbursts. METHODS Within a cohort of children with ADHD and aggressive behavior (n = 156), the prevalence of persistent mood symptoms was evaluated at baseline and after completion of a treatment protocol that provided stimulant monotherapy and family-based behavioral treatment (duration mean [SD] = 70.04 [37.83] days). The relationship of persistent mood symptoms on posttreatment aggressive behavior was assessed, as well as changes in mood symptoms. RESULTS Aggressive behavior and periodic rageful outbursts remitted among 51% of the participants. Persistent mood symptoms at baseline did not affect the odds that aggressive behavior would remit during treatment. Reductions in symptoms of sustained mood disturbance accompanied reductions in periodic outbursts. Children who at baseline had high irritability but low depression ratings showed elevated aggression scores at baseline and after treatment; however, they still displayed large reductions in aggression. CONCLUSIONS Among aggressive children with ADHD, aggressive behaviors are just as likely to decrease following stimulant monotherapy and behavioral treatment among those with sustained mood symptoms and those without. Improvements in mood problems are evident as well. Therefore, the abnormalities in persistent mood described by DMDDs criteria do not contraindicate stimulant therapy as initial treatment among those with comorbid ADHD. Rather, substantial improvements may be anticipated, and remission of both behavioral and mood symptoms seems achievable for a proportion of patients. TRIAL REGISTRATION ClinicalTrials.gov (U.S.); IDs: NCT00228046 and NCT00794625; www.clinicaltrials.gov.