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CRISPR/Cas9 gene editing is an emerging therapeutic modality that shows promise in Huntington's disease (HD) and spinocerebellar ataxia (SCA) mouse models. However, advancing CRISPR-based therapies requires methods to fully define in vivo editing outcomes. Here, we use polymerase-free, targeted long-read nanopore sequencing and evaluate single- and dual-gRNA AAV-CRISPR editing of human ATXN2 in transgenic mouse models of spinocerebellar ataxia type 2 (SCA2). Unbiased high sequencing coverage showed 10-25% editing. Along with intended edits there was AAV integration, 1-2% of which contained the entire AAV genome and were largely unmethylated. Greater than 150 kb deletions at target loci and rearrangements of the transgenic allele (1%) were also found. In contrast, PCR-based nanopore sequencing showed bias for partial AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. Cumulatively this work defines the spectrum of outcomes of CRISPR editing in mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.

RNA interference (RNAi) for spinocerebellar ataxia type 1 can prevent and reverse behavioral deficits and neuropathological readouts in mouse models, with safety and benefit lasting over many months. The RNAi trigger, expressed from adeno-associated virus vectors (AAV.miS1), also corrected misregulated microRNAs (miRNA) such as miR150. Subsequently, we showed that the delivery method was scalable, and that AAV.miS1 was safe in short-term pilot nonhuman primate (NHP) studies. To advance the technology to patients, investigational new drug (IND)-enabling studies in NHPs were initiated. After AAV.miS1 delivery to deep cerebellar nuclei, we unexpectedly observed cerebellar toxicity. Both small-RNA-seq and studies using AAVs devoid of miRNAs showed that this was not a result of saturation of the endogenous miRNA processing machinery. RNA-seq together with sequencing of the AAV product showed that, despite limited amounts of cross-packaged material, there was substantial inverted terminal repeat (ITR) promoter activity that correlated with neuropathologies. ITR promoter activity was reduced by altering the miS1 expression context. The surprising contrast between our rodent and NHP findings highlight the need for extended safety studies in multiple species when assessing new therapeutics for human application.

BACKGROUND The diagnosis of 22q11 deletion syndrome (22q11DS) is often delayed or missed due to the wide spectrum of clinical involvement ranging from mild to severe, often life-threatening conditions. A delayed diagnosis can lead to life-long health issues that could be ameliorated with early intervention and treatment. Owing to the high impact of 22q11DS on public health, propositions have been made to include 22q11DS in newborn screening panels; however, the method of choice for detecting 22q11DS, fluorescent in situ hybridization, requires specialized equipment and is cumbersome for most laboratories to implement as part of their routine screening. We sought to develop a new genetic screen for 22q11DS that is rapid, cost-effective, and easily used by laboratories currently performing newborn screening. METHODS We evaluated the accuracy of multiplex droplet digital PCR (ddPCR) in the detection of copy number of 22q11DS by screening samples from 26 patients with 22q11DS blindly intermixed with 1096 blood spot cards from the general population (total n = 1122). RESULTS Multiplex ddPCR correctly identified all 22q11DS samples and distinguished between 1.5- and 3-Mb deletions, suggesting the approach is sensitive and specific for the detection of 22q11DS. CONCLUSIONS These data demonstrate the utility of multiplex ddPCR for large-scale population-based studies that screen for 22q11DS. The use of samples from blood spot cards suggests that this approach has promise for newborn screening of 22q11DS, and potentially for other microdeletion syndromes, for which early detection can positively impact clinical outcome for those affected.

ObjectivesSeveral neurotransmitters involved in brain development are altered in fragile X syndrome (FXS), the most common monogenic cause of autism spectrum disorder (ASD). Serotonin plays a vital role in synaptogenesis and postnatal brain development. Deficits in serotonin synthesis and abnormal neurogenesis were shown in young children with autism, suggesting that treating within the first years of life with a selective serotonin reuptake inhibitor might be the most effective time. In this study we aimed to identify molecular biomarkers involved in the serotonergic pathway that could predict the response to sertraline treatment in young children with FXS.MethodsGenotypes were determined for several genes involved in serotonergic pathway in 51 children with FXS, ages 24-72months. Correlations between genotypes and deviations from baseline in primary and secondary outcome measures were modeled using linear regression models.ResultsA significant association was observed between a BDNF polymorphism and improvements for several clinical measures, including the Clinical Global Impression scale (P=0.008) and the cognitive T score (P=0.017) in those treated with sertraline compared to those in the placebo group. Additionally, polymorphisms in the MAOA, Cytochrome P450 2C19 and 2D6, and in the 5-HTTLPR gene showed a significant correlation with some of the secondary measures included in this study.ConclusionThis study shows that polymorphisms of genes involved in the serotonergic pathway could play a potential role in predicting response to sertraline treatment in young children with FXS. Larger studies are warranted to confirm these initial findings.

Background Over 40% of male and ∼16% of female carriers of a premutation FMR1 allele (55–200 CGG repeats) will develop fragile X-associated tremor/ataxia syndrome, an adult onset neurodegenerative disorder, while about 20% of female carriers will develop fragile X-associated primary ovarian insufficiency. Marked elevation in FMR1 mRNA transcript levels has been observed with premutation alleles, and RNA toxicity due to increased mRNA levels is the leading molecular mechanism proposed for these disorders. However, although the FMR1 gene undergoes alternative splicing, it is unknown whether all or only some of the isoforms are overexpressed in premutation carriers and which isoforms may contribute to the premutation pathology. Methods To address this question, we have applied a long-read sequencing approach using single-molecule real-time (SMRT) sequencing and qRT-PCR. Results Our SMRT sequencing analysis performed on peripheral blood mononuclear cells, fibroblasts and brain tissue samples derived from premutation carriers and controls revealed the existence of 16 isoforms of 24 predicted variants. Although the relative abundance of all mRNA isoforms was significantly increased in the premutation group, as expected based on the bulk increase in mRNA levels, there was a disproportionate (fourfold to sixfold) increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14, specifically Iso10 and Iso10b, containing the complete exon 15 and differing only in splicing in exon 17. Conclusions These findings suggest that RNA toxicity may arise from a relative increase of all FMR1 mRNA isoforms. Interestingly, the Iso10 and Iso10b mRNA isoforms, lacking the C-terminal functional sites for fragile X mental retardation protein function, are the most increased in premutation carriers relative to normal, suggesting a functional relevance in the pathology of FMR1 -associated disorders.

It seems obvious to state that schooling makes a difference in individual developmental and life outcomes, and that it strongly influences who will contribute to society at large and what kind of contributions will be made (Lutz & KC, 2011). In developed (i.e., high income) countries (The World Bank, 2013a), it is generally taken for granted that formal schooling is closely linked to both individual (Bronfenbrenner, McClelland, Wethington, Moen, & Ceci, 1996) and social (Glaeser, Laibson, & Sacerdote, 2002) capital—that is, it contributes significantly to an individual’s worth both to him- or herself and to society at large. Correspondingly, schooling is viewed both as the primary developmental task and the main accomplishment of such countries’ young members prior to entry into adulthood. These societies require schooling and stipulate the minimum number of years their youngsters are expected to spend in school; but these requirements and stipulations are not universal. In post-industrial economies, 96% of their school-aged members are engaged in regular or special education, whereas in emerging economies (less and least developed countries) these percentages are about 85% and 65%, respectively (UNICEF, 2009). The role of formal schooling in developing (i.e., low and middle income) economies is much more tangential to both social (Godoy et al., 2008) and individual (Serpell, 1993) capital; it is not uniformly viewed as either a requirement or as an accomplishment, so parents decide which of their children (if any), should or should not go to school (Brock & Levers, 2007). Primarily due to the uncertainty of the role of formal schooling in developing countries (Grigorenko et al., 2001; Grigorenko, Hein, & Reich, in press; Serpell & Jere-Folotiya, 2008), but also due to other characteristics of these societies (e.g., shortage of funds, societal conflict, and economic instability), many children in low and middle income countries are not enrolled in formal education. For example, in 2010, about 132 million children of primary and lower-secondary school age were not being schooled formally (UNICEF, 2013); approximately half of these out-of-school children lived in sub Saharan Africa (UNESCO, 2008). Yet, the impact of schooling, regardless of the magnitude of its main effect, is not homogeneous; individual differences in classrooms among both students and teachers remain the object of investigation of large subfields in psychology and education. To explain the presence of these differences, chiefly, three different hypotheses have been investigated. First, it has been assumed that these differences are due to the impact of underlying genetic factors that influence either general cognitive (e.g., Plomin et al., 2004) or scholastic (e.g., Martin et al., 2011) performance, although recent large-scale re-investigations of the previously published data suggest that, if they exist, these effects are of very small magnitude and cannot, when conceived additively, explain the observed broad range of variability of individual differences in the classroom (Chabris et al., 2012; Rietveld & et al., in press; for intelligence and educational attainment, correspondingly). Second, it has been argued that classroom-based individual differences arise differentially, depending on the quality of instruction, so that genetic influences are pronounced more in higher and less in lower quality classrooms (Taylor, Roehrig, Soden Hensler, Connor, & Schatschneider, 2010). It has also been assumed that genetic factors shape the parameters of brain structure and function (Kwan, Sestan, & Anton, 2012), which, in turn, determine the parameters of basic information processing functions (Rowe et al., 2007); these, in turn, underlie the high (.45–.80) correlations between cognitive and scholastic performance (Luo, Thompson, & Detterman, 2003). It is this third hypothesis that seems to be particularly interesting for an investigation in societies where schooling is not a given, where the impact of schooling can be differential for individuals differentiated by characteristics of basic information processing functions and, in turn, by the parameters of their brain functioning, and, correspondingly, by the variation in the genome which defines these parameters. To quantify the relevant genetic variation, we genotyped selected markers in the catechol-O-methyltransferase (COMT) gene (MIM 116790, 22q11). The enzyme produced by this gene is involved in the degradation of catecholamines (dopamine, epinephrine, and norepinephrine) and, through its participation in dopamine turnover, is related to neural functioning. COMT is said to account for >60% of the dopamine degradation in the prefrontal cortex (PFC); thus, it plays an important role in regulating dopamine levels in the PFC and, correspondingly, in psychological processes, both lower (e.g., reaction and inspection time) and higher (e.g., metacognitive functions) that engage the PFC (Karoum, Chrapusta, & Egan, 1994). Two isoforms of COMT exist: the soluble isoform (S-COMT) and the membrane-bound isoform (MB-COMT). These are regulated by different promoters, are of different lengths (221 and 271 aminoacid residues, respectively), and have different functions. Structural DNA variants within this gene and in its near vicinity within noncoding regions have been associated with individual differences in a number of cognitive and affective processes, indicators of brain activity, and neuropsychiatric conditions (Dickinson & Elvevag, 2009). The pleiotropic nature of the COMT gene action has been related to the complex dynamics of cognitive and effective functions (Mier, Kirsch, & Meyer-Lindenberg, 2010; Papaleo et al., 2008; Tunbridge, Harrison, & Weinberger, 2006). The most studied polymorphic variant of the COMT gene is a single nucleotide change G/A (also known as the rs4680 single nucleotide polymorphism, SNP), resulting in an amino-acid substitution of valine (Val) with methionine (Met) at codon 108 for S-COMT and codon 158 for MB-COMT (Val108/158Met) generating alternative forms of COMT with different functional properties (Lachman et al., 1996). Yet, the gene has other numerous polymorphisms that have been extensively studied in isolation, in combination with the rs4680 polymorphism, and in haplotype structures representing of the gene (Witte & Floel, 2012). Of note also is that the variation in the COMT gene has been previously associated with individual differences in IQ (Payton, 2009), academic attainment (Enoch, Waheed, Harris, Albaugh, & Goldman, 2009; Yeh, Chang, Hu, Yeh, & Lin, 2009), and numerous PFC-rooted processes (Lundwall, Guo, & Dannemiller, 2012; Stormer, Passow, Biesenack, & Li, 2012). Here we investigated the effect of schooling on levels of nonverbal intelligence in a sample of children from a developing country. Our specific hypothesis is that, assuming that schooling is the major causal factor in levels of intelligence around the world, genetic variation (specifically, genetic variation in the COMT gene) might modulate the impact of schooling and help explain, at least partially, the presence of individual differences in classrooms. To verify this hypothesis, we ascertained a sample of children with a graded amount of exposure to schooling and assessed them behaviorally and genotypically.

The CGG repeat within the premutation range in the fragile X mental retardation 1 (FMR1) gene can lead to neurodegenerative disorders and intellectual disabilities. An increase in size upon the transmission from parent to child is more likely to occur for larger alleles and without AGG interruptions. We describe the molecular structure and the transmission of an FMR1 premutation allele in a multigenerational family, identified through newborn screening for fragile X syndrome. Transmission of the premutation allele was traced through five generations in 14 of the 23 individuals who were genotyped through cascade testing. Allele size instability during transmission was observed, but no expansions to a full mutation were detected. Clinical and molecular characterizations of the participants lead to the diagnosis of fragile X-associated tremor ataxia syndrome in one subject identified as a premutation carrier. A gradual small increase in the size of the premutation allele was observed during transmission through five generations. The relative stability is likely due to the presence of two AGGs within the allele. The detection of AGG interruptions within the premutation alleles is important in genetic counseling, to better predict the risk of expansion during transmission from a premutation to a full-mutation allele.

BACKGROUND AND OBJECTIVE: Developmental language disorder (DLD) is a highly prevalent neurodevelopmental disorder associated with negative outcomes in different domains; the etiology of DLD is unknown. To investigate the genetic underpinnings of DLD, we performed genome-wide association and whole exome sequencing studies in a geographically isolated population with a substantially elevated prevalence of the disorder (ie, the AZ sample). METHODS: DNA samples were collected from 359 individuals for the genome-wide association study and from 12 severely affected individuals for whole exome sequencing. Multifaceted phenotypes, representing major domains of expressive language functioning, were derived from collected speech samples. RESULTS: Gene-based analyses revealed a significant association between SETBP1 and complexity of linguistic output (P = 5.47 × 10−7). The analysis of exome variants revealed coding sequence variants in 14 genes, most of which play a role in neural development. Targeted enrichment analysis implicated myocyte enhancer factor–2 (MEF2)-regulated genes in DLD in the AZ population. The main findings were successfully replicated in an independent cohort of children at risk for related disorders (n = 372). CONCLUSIONS: MEF2-regulated pathways were identified as potential candidate pathways in the etiology of DLD. Several genes (including the candidate SETBP1 and other MEF2-related genes) seem to jointly influence certain, but not all, facets of the DLD phenotype. Even when genetic and environmental diversity is reduced, DLD is best conceptualized as etiologically complex. Future research should establish whether the signals detected in the AZ population can be replicated in other samples and languages and provide further characterization of the identified pathway.

Epigenetic mechanisms, including DNA methylation, that underlie neuropsychiatric conditions have become a promising area of research. Most commonly used DNA sources in such studies are peripheral (whole) blood (WB), saliva (SL), and lymphoblastoid cell lines (LCLs); thus, the question of the consistency of DNA methylation patterns in those cells is of particular interest. To investigate this question we performed comparative analyses of methylation patterns in WB, SL, and LCLs derived from the same individuals, using Illumina HumanMethylation27 BeadChip arrays. Our results showed that DNA methylation patterns in SL are relatively consistent with those in WB, whereas the patterns in LCLs are similarly distinct from both WB and SL. The results indicated that due to multiple random and directed changes in DNA methylation throughout cell culturing, LCLs are not a reliable source of DNA for epigenetic studies and should be used with caution when investigating epigenetic mechanisms underlying biological processes.

In both children and adults there is large variability in reading skill, with approximately 5–10% of individuals characterized as having reading disability; these individuals struggle to learn to read despite adequate intelligence and opportunity. Although it is well established that a substantial portion of this variability is attributed to the genetic differences between individuals, specifics of the connections between reading and the genome are not understood. This article presents data that suggest that variation in the COMT gene, which has previously been associated with variation in higher-order cognition, is associated with reading and reading-related skills, both at the level of brain and behavior. In particular, we found that the COMT Val/Met polymorphism at rs4680, which results in the substitution of the ancestral Valine (Val) by Methionine (Met), was associated with better performance on a number of critical reading measures and with patterns of functional neural activation that have been linked to better readers. We argue that this polymorphism, known for its broad effects on cognition, may modulate (likely through frontal lobe function) reading skill.

The ability to accurately predict the likelihood of expansion of the CGG repeats in the FMR1 gene to a full mutation is of critical importance for genetic counseling of women who are carriers of premutation alleles (55–200 CGG repeats) and who are weighing the risk of having a child with fragile X syndrome. The presence of AGG interruptions within the CGG repeat tract is thought to decrease the likelihood of expansion to a full mutation during transmission, thereby reducing risk, although their contribution has not been quantified. We retrospectively analyzed 267 premutation alleles for number and position of AGG interruptions, length of pure CGG repeats, and CGG repeat lengths present in the offspring of the maternal transmissions. In addition, we determined the haplotypes of four markers flanking the 5′-UTR locus in the premutation mothers. We found that the presence of AGG interruptions significantly increased genetic stability, whereas specific haplotypes had a marginal association with transmission instability. The presence of AGG interruptions reduced the risk of transmission of a full mutation for all maternal (premutation) repeat lengths below ~100 CGG repeats, with a differential risk (0 vs. 2 AGG) exceeding 60% for alleles in the 70- to 80-CGG repeat range. Genet Med 2012:14(8):729–736

Objective Variation in the catechol-O-methyltransferase gene (COMT) has been associated with antisocial behavior in populations with attention deficit/hyperactivity disorder (ADHD). This study examined whether COMTwould predict antisocial behavior in a sample with high levels of behavior problems, not necessarily ADHD. In addition, because previous research suggests that COMT may be associated with ADHD in males, association between COMT and ADHD symptoms was examined. Method This study tested whether variation in three polymorphisms of the COMT gene was predictive of symptoms of conduct disorder and ADHD, in a sample of 174 incarcerated Russian adolescent male delinquents. Results The Val allele of the Val 158 Met polymorphism was significantly associated with conduct disorder diagnosis and symptoms, whereas the Met allele was associated with ADHD symptoms. Conclusion The Val 158 Met polymorphism of the COMT gene shows a complex relation to behavior problems, influencing conduct disorder and ADHD symptoms in opposite directions in a high-risk population. Psychiatr Genet 20:20–24 � c 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins. Psychiatric Genetics 2010, 20:20–24

Background Autism spectrum disorders (ASD) are neurodevelopmental disorders of complex etiology, with a recognized substantial contribution of heterogeneous genetic factors; one of the core features of ASD is a lack of affiliative behaviors. Methods On the basis of the existing literature, in this study we examined the hypothesis of allelic associations between genetic variants in six genes involved in control of maternal and affiliative behaviors ( OXT, OXTR, PRL, PRLR , D β H , and FOSB ). One hundred and seventy-seven probands with ASD from 151 families ( n = 527) were assessed with a set of related instruments capturing multiple facets of ASD. Multivariate and univariate phenotypes were constructed from these assessments and subjected to genetic linkage and association analyses with PBAT and FBAT software. Results The resulting pattern of findings, in general, confirmed the hypotheses of the significance of the genes involved in the development of affiliative behaviors in the manifestation of ASD ( p values ranging from .000005 to .05); statistically speaking, the strongest results were obtained for allelic associations with the PRL, PRLR , and OXTR genes. Conclusions These preliminary data provide additional support for the hypothesis that the allelic variants of genes necessary for the development of species-typical affiliative behaviors are associated with ASD. Independent replication of these findings is needed and studies of other genes associated with affiliative behaviors are indicated.

Objective: Fragile X syndrome (FXS) and tuberous sclerosis (TSC) are genetic disorders that result in intellectual disability and an increased prevalence of autism spectrum disorders (ASD). While the clinical presentation of each disorder is distinct, the molecular causes are linked to a disruption in the mTORC1 (mammalian Target of Rapamycin Complex 1) and ERK1/2 (Extracellular signal- Regulated Kinase) signaling pathways. Methods: We assessed the clinical and molecular characteristics of an individual seen at the UC Davis MIND Institute with a diagnosis of FXS and TSC. Clinical evaluation of physical, behavioral, and cognitive impairments were performed. Additionally, total and phosphorylated proteins along the mTORC1 and ERK1/2 pathways were measured in primary fibroblast cell lines from the proband. Results: In this case the phenotypic effects that result in a human with both FXS and TSC are shown to be severe. Changes in mTORC1 and ERK1/2 signaling proteins and global protein synthesis were not found to be noticeably different between four cohorts (typically developing, FMR1 full mutation, FMR1 full mutation and TSC1 loss of function mutation, and TSC1 loss of function mutation); however cohort sizes prevented stringent comparisons. Conclusion: It has previously been suggested that disruption of the mTORC1 pathway was reciprocal in TSC and FXS double knock-out mouse models so that the regulation of these pathways were more similar to wild-type mice compared to mice harboring a Fmr1-/y or Tsc2-/+ mutation alone. However, in this first reported case of a human with a diagnosis of both FXS and TSC, substantial clinical impairments, as a result of these two disorders were observed. Differences in the mTORC and ERK1/2 pathways were not clearly established when compared between individuals with either disorder, or both.

The CGG trinucleotide repeat within the FMR1 gene is associated with multiple clinical disorders, including fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome. Differences in the distribution and prevalence of CGG repeat length and of AGG interruption patterns have been reported among different populations and ethnicities. In this study we characterized the AGG interruption patterns within 3,065 normal CGG repeat alleles from nine world populations including Australia, Chile, United Arab Emirates, Guatemala, Indonesia, Italy, Mexico, Spain, and United States. Additionally, we compared these populations with those previously reported, and summarized the similarities and differences. We observed significant differences in AGG interruption patterns. Frequencies of longer alleles, longer uninterrupted CGG repeat segments and alleles with greater than 2 AGG interruptions varied between cohorts. The prevalence of fragile X syndrome and FMR1 associated disorders in various populations is thought to be affected by the total length of the CGG repeat and may also be influenced by the AGG distribution pattern. Thus, the results of this study may be important in considering the risk of fragile X-related conditions in various populations.

Expansions of more than 200 CGG repeats (full mutation) in the FMR1 gene give rise to fragile X syndrome (FXS) through a process that generally involves hypermethylation of the FMR1 promoter region and gene silencing, resulting in absence of expression of the encoded protein, FMRP. However, mosaicism with alleles differing in size and extent of methylation often exist within or between tissues of individuals with FXS. In the current work, CGG-repeat lengths and methylation status were assessed for eighteen individuals with FXS, including 13 mosaics, for which peripheral blood cells (PBMCs) and primary fibroblast cells were available. Our results show that for both PBMCs and fibroblasts, FMR1 mRNA and FMRP expression are directly correlated with the percent of methylation of the FMR1 allele. In addition, Full Scale IQ (FSIQ) scores were inversely correlated with the percent methylation and positively correlated with higher FMRP expression. These latter results point toward a positive impact on cognition for full mutation, methylation mosaics with lower methylation compared to individuals with fully methylated, full mutation alleles. However, we did not observe a significant reduction in the number of seizures, nor in the severity of hyperactivity or autism spectrum disorder, among individuals with mosaic genotypes in the presentation of FXS.These observations suggest that low, but non-zero expression of FMRP may be sufficient to positively impact cognitive function in individuals with FXS, with methylation mosaicism (lowered methylation fraction) contributing to a more positive clinical outcome.

Genetic factors are integral to the understanding of the etiology of antisocial behaviour, as evidenced by family and twin studies that indicate a heritability of at least 50% (Moffitt, 2005; Viding, Larsson, & Jones, 2008). Quantitative genetic studies indicate that genetic and environmental influences on the emergence of antisocial behaviour appear remarkably similar in their magnitude despite different approaches, assessment methods, age of assessment, or the gender of the participants (Rhee & Waldman, 2002; Rhee & Waldman, 2011; Lahey & Waldman, 2012). In addition, many risk factors that are traditionally thought to be environmental may also reflect genetic vulnerability (Moffitt, 2005). Over the past decade, it was widely expected that the genetic basis of common disorders would be resolved by genome-wide association studies (GWAS)--large-scale studies in which the entire genome is covered by genetic markers. Although recent GWAS have given us a clearer picture of the allelic architecture of genetic susceptibility for some pediatric-onset disorders including type I diabetes, the bulk of heritable variance remains unexplained for many pediatric-onset neuropsychiatric disorders including antisocial behaviour and substance use disorders. Information about gene function has led to the identification of a number of relevant candidate systems of genes (CSG) that influence antisocial (delinquent, criminal, and substance use) behaviours. The CSG approach provides the advantages of hypothesis-driven research, while mitigating some limitations of a candidate gene approach that usually targets only one or a small number of loci. Although a CSG approach is a viable alternative to both a candidate gene and GWAS studies, it still requires a very large number of subjects to identify allelic variations that by themselves account for a very small proportion of the phenotypic variance (Maher et al., 2011; Grigorenko et al., 2010; Yrigollen et al., 2008; Greenwood et al., 2011). While CSG studies examine related families of genes, the analytical approach often focuses on only one allelic variant at a time. Several studies have demonstrated that multiple measurements of genes are needed to fully realize the association between the variance in genes to phenotypic variation (Burt & Mikolajewski, 2008; Grigorenko et al, 2010). Thus, whereas positive CSG studies allow inferences to be made concerning the probable presence of gene-gene interactions, they do not permit an accurate estimation of the phenotypic variance explained since the variance accounted for by gene-gene interactions is unlikely to be simply additive. Indeed, very few approaches, if any, are currently available to assess systematically the presence of a ‘shared’ variance across genetic risk alleles with regard to a particular phenotype of interest. The application of a latent variable approach to genetic data should facilitate the identification of allelic variants that collectively contribute to particular phenotypic outcome. This approach has been widely used in economics and psychology to study observable behavior that may be influenced by unobservable constructs that are not directly measurable (Avery, 1979; Bollen, 2002). A clear advantage of using a latent variable analysis is that it reduces the dimensionality of data so that a large number of observable variables can be aggregated in a model to represent a single underlying factor when there are strong relationships among variables. The application of latent variable analyses to genetic data is novel. Because observed variables that have no correlation cannot result in a latent construct, the identification of a latent genetic construct provides a potential path to discover a ‘shared’ variance across genetic risk alleles. This shared variance could be additive, multiplicative or interactive. However, this latent variable approach is limited by its inability to specify the biological nature of the gene-gene interactions that underlie this shared variance. Consequently, we have chosen to refer to the “co-action” of allelic variants (additive, multiplicative, and other) in describing our findings. The choice of the genetic loci to be assessed is critically important. Several studies have reported associations between antisocial behaviour and drug use and genetic variants associated with dopaminergic and serotoninergic pathways (Chambers et al., 2001; Bierut et al., 2002; Solanto, 2002; Kim-Cohen et al., 2006; Moffitt, 2005; Blum et al., 2012; McCrory et al., 2012; Moul et al., 2013). We also selected genes associated with affiliative behaviors and stress response. The rationale for including in the CSG genetic loci implicated in affiliative behaviours is based, in part, on studies that report strong associations between inconsistent, harsh, or abusive disciplinary practices by parents and child rule-breaking behaviours, delinquency, and aggression (Stouthamer-Loeber et al., 2001; Stanger et al., 2004). Childhood conduct problems have also been associated with a lack of parental involvement, a lack of parental warmth and parent–child conflict (Burt et al., 2005; Caspi et al., 2004; Stanger et al., 2004). Dopaminergic pathways are also critically involved in affiliative (e.g., maternal care) and other reward-related behaviours (Depue & Collins, 1999; Mileva-Seitz et al., 2012). The rationale for including in the CSG genetic loci implicated in stress response is based on studies that have reported an association between cortisol levels and aggression in adolescents (Gao et al., 2009; Poustka et al., 2010; Matthys et al., 2013). In addition, the neural substrates of affiliation (parenting behaviours) are closely interrelated to the stress response, salience, and reward pathways (Leckman & Herman, 2002; Lim & Young, 2006; Gordon et al., 2011). The goals of this preliminary study were: (1) to construct a latent phenotypic antisocial variable using all, or a subset, of 15 indicators of delinquency, antisocial behaviour and drug use collected as part of the 15 year follow-up of a randomized trial of a prenatal and infancy nurse-home visitation program in Elmira, New York (Olds et al., 1998); (2) to define a novel CSG that includes genetic loci that have been implicated in affiliative behaviours and stress response as delinquency and drug use; (3) to carry out initial univariate analyses of each of the informative single nucleotide polymorphisms (SNPs) within the CSG; (4) to perform a quasi-bootstrapping procedure to construct an initial latent genetic variable; (5) to refine and extend the latent genetic variable by reassessing each of the remaining informative SNPs one at a time leading to a final model where both latent genetic and phenotypic variables are incorporated simultaneously; (6) to determine if there is a continuous relationship between the number of risk alleles and scores from the latent antisocial variable; and (7) to identify biological pathways and processes that are over-represented by all of the genes in the CSG that were implicated in this analysis. A flow chart of these steps and procedures is presented in Figure 1. Figure 1 Flow Chart

We investigated the effect of AGG interruptions on fragile X repeat instability upon transmission of fragile X intermediate and small premutation alleles with 45–69 CGG repeats. The FMR1 repeat structure was determined for 375 mothers, 48 fathers, and 538 offspring (457 maternal and 81 paternal transmissions) using a novel PCR assay to determine repeat length and AGG interruptions. The number of AGG interruptions and the length of uninterrupted CGG repeats at the 3′ end were correlated with repeat instability on transmission. Maternal alleles with no AGGs conferred the greatest risk for unstable transmissions. All nine full mutation expansions were inherited from maternal alleles with no AGGs. Furthermore, the magnitude of repeat expansion was larger for alleles lacking AGG interruptions. Transmissions from paternal alleles with no AGGs also exhibited greater instability than those with one or more AGGs. Our results demonstrate that characterization of the AGG structure within the FMR1 repeat allows more accurate risk estimates of repeat instability and expansion to full mutations for intermediate and small premutation alleles. © 2013 Wiley Periodicals, Inc.

We report the clinical and cytogenetic findings on a male child with developmental language disorder, no physical abnormalities, and a balanced t(10;15)(q24.1;q21.1) translocation. As the child's parents are unavailable for investigations, it is unclear whether the translocation is inherited or de novo. Fluorescence in situ hybridization (FISH) analyses were carried out using specific RP11-BAC clones mapping near 15q21.1 and 10q24.1 to refine the location of the breakpoints. The breakpoint on 15q21.1 interrupts the SEMA6D gene and the breakpoint on 10q24.1 is located between the ENTPD1 and CCNJ genes. The SEMA6D gene was further investigated in samples of individuals with developmental language disorders and controls; this investigation offered further evidence of the involvement of SEMA6D with developmental language disorders.

Fragile X associated disorders are caused by a premutation allele in the fragile X mental retardation 1 gene (FMR1) and are hypothesized to result from the toxic effect of elevated levels of expanded FMR1 transcripts. Increased levels of FMR1 mRNA have indeed been reported in premutation carriers; however the mechanism by which expanded alleles lead to elevated levels of FMR1 mRNA in premutation carriers is unknown. Within the CGG repeat tract AGG interruptions are found, generally 1-3 present in normal/intermediate alleles (6-54 CGG repeats) and usually 0-1 in premutation alleles (55-200 CGG repeats). They are present at specific locations, generally occurring after 9 or 10 uninterrupted CGG repeats [(CGG)(9)AGG(CGG)(9)AGG(CGG)(n)]. We evaluated both the number of AGG interruptions and the resulting length of the uninterrupted 3' CGG repeat pure tract in premutation alleles derived from two large cohorts of male and female carriers to determine whether the presence of AGG interruptions or the length of a pure stretch of CGG repeats influence the levels of FMR1 mRNA in blood. Our findings indicate that neither the number of AGG interruptions, nor their position along the CGG tract have a significant affect on mRNA levels in premutation carriers. We also, as expected based on previous findings, observed a highly significant correlation between CGG repeat number (as both total length and length of pure CGG stretch) and FMR1 mRNA expression levels, in both males and females. Importantly, we did not observe any significant difference in FMR1 mRNA levels in premutation carriers based on age.

We provide an illustration of an application of the elastic net to a large number of common genetic variants in the context of the search for the genetic bases of an endophenotype conceivably related to individual differences in learning. GABA concentration in the occipital cortex, a critical area for reading, was obtained in a group (n = 76) of children aged 6–10 years. Two extreme groups, high and low, were selected for genotyping with the 650Y Illumina array chip (Ilmn650Y). An elastic net approach was applied to the resulting SNP dataset; 100 SNPs were identified for each chromosome as “interesting” based on having the highest absolute value coefficients. The analyses highlighted chromosomes 15 and 20, which contained 55 candidate genes. The STRING partner analyses of the associated proteins pointed to a number of related genes, most notably, GABA and NTRK receptors.

:::::::::::::::: :::::::::::: A number of dopamine-related genes have been implicated in the etiology of violent behavior and conduct problems. Of these genes, the ones that code for the enzymes that influence the turnover of dopamine (DA) have received the most attention. In this study, we investigated 12 genetic polymorphisms in four genes involved with DA functioning (COMT, MAOA and MAOB, and DbH) in 179 incarcerated male Russian adolescents and two groups of matched controls: boys without criminal records referred to by their teachers as (a) ‘‘troubled-behavior-free’’ boys, n 5 182; and (b) ‘‘troubled-behavior’’ boys, n 5 60. The participants were classified as (1) being incarcerated or not, (2) having the DSM-IV diagnosis of conduct disorder (CD) or not, and (3) having committed violent or nonviolent crimes (for the incarcerated individuals only). The findings indicate that, although no single genetic variant in any of the four genes differentiated individuals in the investigated groups, various linear combinations (i.e., haplotypes) and nonlinear combinations (i.e., interactions between variants within and across genes) of genetic variants resulted in informative and robust classifications for two of the three groupings. These combinations of genetic variants differentiated individuals in incarceration vs. nonincarcerated and CD vs. no-CD groups; no informative combinations were established consistently for the grouping by crime within the incarcerated individuals. This study underscores the importance of considering multiple rather than single markers within candidate genes and their additive and interactive combinations, both with themselves and with nongenetic indicators, while attempting to understand the genetic background of such complex behaviors as serious conduct problems. Aggr. Behav. 36:158–176, 2010. r 2010 Wiley-Liss, Inc.

Objective Sodium-dependent and chloride-dependent gamma-aminobutyric acid (GABA) transporter 1 (SLC6A1) is the target of a number of drugs of clinical importance and is a major determinant of synaptic GABA concentrations. We resequenced the human SLC6A1 gene previously and discovered a novel 21 bp insertion in the predicted promoter region that creates a second tandem copy of the sequence. Here we sought to determine the functional relevance of this variation. Methods We used reporter assays, mobility shift assays, quantitative PCR, and proteomics methods as well as postmortem expression analysis for this work. Results Reporter assays showed that the insertion allele significantly increases promoter activity in multiple cell lines. The zinc finger transcription factor ZNF148 was found to significantly transactivate the promoter and increase expression when overexpressed but could not account for the differences in activity between the two alleles of the promoter. Copy number of the insertion sequence was associated with exponentially increasing activity of a downstream promoter, suggesting that the insertion sequence has enhancer activity when present in multiple copies. SLC6A1 promoter genotype was found to predict SLC6A1 RNA expression in human postmortem hippocampal samples. These results suggest that the insertion polymorphism leads to increased SLC6A1 promoter activity because, in part, of creation of an enhancer element when present as multiple copies. Genotyping individuals from Tanzania in this study suggested that the insertion allele has its origin in Africa. Conclusion On account of the effect of the insertion on promoter activity, this relatively common polymorphism may prove useful in predicting clinical response to pharmacological modulators of SLC6A1 as well as GABAergic function in individuals of African descent.

OBJECTIVE. Autistic spectrum disorders are childhood neurodevelopmental disorders characterized by social and communicative impairment and repetitive and stereotypical behavior. Macrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity that promotes monocyte/macrophage-activation responses by increasing the expression of Toll-like receptors and inhibiting activation-induced apoptosis. On the basis of results of previous genetic linkage studies and reported altered innate immune response in autism spectrum disorder, we hypothesized that MIF could represent a candidate gene for autism spectrum disorder or its diagnostic components.METHODS. Genetic association between autism spectrum disorder and MIF was investigated in 2 independent sets of families of probands with autism spectrum disorder, from the United States (527 participants from 152 families) and Holland (532 participants from 183 families). Probands and their siblings, when available, were evaluated with clinical instruments used for autism spectrum disorder diagnoses. Genotyping was performed for 2 polymorphisms in the promoter region of the MIF gene in both samples sequentially. In addition, MIF plasma analyses were conducted in a subset of Dutch patients from whom plasma was available.RESULTS. There were genetic associations between known functional polymorphisms in the promoter for MIF and autism spectrum disorder–related behaviors. Also, probands with autism spectrum disorder exhibited higher circulating MIF levels than did their unaffected siblings, and plasma MIF concentrations correlated with the severity of multiple autism spectrum disorder symptoms.CONCLUSIONS. These results identify MIF as a possible susceptibility gene for autism spectrum disorder. Additional research is warranted on the precise relationship between MIF and the behavioral components of autism spectrum disorder, the mechanism by which MIF contributes to autism spectrum disorder pathogenesis, and the clinical use of MIF genotyping.

Previous research has generated examples of how genetic and environmental factors can interact to create risk for psychopathology. Using a gene-by-environment (G X E) interaction design, we tested whether three polymorphisms in the dopamine transporter gene (DAT1, also referred to as SLC6A3, located at 5p15.33) interacted with maternal parenting style to predict first-onset episodes of depression. Participants were male adolescents (N = 176) recruited from a juvenile detention center in northern Russia. As hypothesized, one of the polymorphisms (rs40184) moderated the effect of perceived maternal rejection on the onset of major depressive disorder, as well as on suicidal ideation. Further, this G X E interaction was specific to depression; it did not predict clinically significant anxiety. These results highlight the need for further research investigating the moderating effects of dopaminergic genes on depression.

It is established that reading and reading-related processes are heritable; genes thus play an important role in the foundation of individual differences in reading. In this article, we focus on one facet of reading–comprehension. Comprehension is a higher order cognitive skill that requires many other cognitive processes for it to unfold completely and successfully. One such process is executive functioning, which has been associated with genetic variation in the catechol-O-methyltransferase (COMT) gene. Genotypes and haplotypes of four single nucleotide polymorphisms in COMT were investigated in 179 incarcerated adolescent delinquents. Four hierarchical logistic regression models predicting the presence/absence of comprehension difficulties were fitted to the data; genetic variation in COMT and the presence/absence of maternal rejection were investigated as main effects and as effects acting interactively. Three out of four interaction terms were found to be important predictors of individual differences in comprehension. These findings were supported by the results of the haplotype analyses, in which the four investigated polymorphisms were considered simultaneously.

Background The presence of AGG interruptions in the CGG repeat locus of the fragile X mental retardation 1 (FMR1) gene decreases the instability of the allele during transmission from parent to child, and decreases the risk of expansion of a premutation allele to a full mutation allele (the predominant cause of fragile X syndrome) during maternal transmission. Methods To strengthen recent findings on the utility of AGG interruptions in predicting instability or expansion to a full mutation of FMR1 CGG repeat alleles, we assessed the outcomes of 108 intermediate (also named gray zone) and 710 premutation alleles that were transmitted from parent to child, and collected from four international clinical sites. We have used the results to revise our initial model that predicted the risk of a maternal premutation allele expanding to a full mutation during transmission and to test the effect of AGG interruptions on the magnitude of expanded allele instability of intermediate or premutation alleles that did not expand to a full mutation. Results Consistent with previous studies, the number of AGG triplets that interrupts the CGG repeat locus was found to influence the risk of allele instability, including expansion to a full mutation. The total length of the CGG repeat allele remains the best predictor of instability or expansion to a full mutation, but the number of AGG interruptions and, to a much lesser degree, maternal age are also factors when considering the risk of transmission of the premutation allele to a full mutation. Conclusions Our findings demonstrate that a model with total CGG length, number of AGG interruptions, and maternal age is recommended for calculating the risk of expansion to a full mutation during maternal transmission. Taken together, the results of this study provide relevant information for the genetic counseling of female premutation carriers, and improve the current predictive models which calculate risk of expansion to a full mutation using only total CGG repeat length.