Jessica Ezzell Hunter

Evaluating gene expression in C57BL/6J and DBA/2J mouse striatum using RNA-Seq and microarrays.

C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, ‘digital mRNA counting’ is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation.

A BAC transgenic mouse model reveals neuron subtype-specific effects of a Generalized Epilepsy with Febrile Seizures Plus (GEFS+) mutation

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability.

An Epilepsy Mutation in the Sodium Channel SCN1A That Decreases Channel Excitability

Mutations in three voltage-gated sodium channel genes, SCN1A, SCN2A, and SCN1B, and two GABAA receptor subunit genes, GABRG2 and GABRD, have been identified in families with generalized epilepsy with febrile seizures plus (GEFS+). A novel mutation, R859C, in the Nav1.1 sodium channel was identified in a four-generation, 33-member Caucasian family with a clinical presentation consistent with GEFS+. The mutation neutralizes a positively charged arginine in the domain 2 S4 voltage sensor of the Nav1.1 channel α subunit. This residue is conserved in mammalian sodium channels as well as in sodium channels from lower organisms. When the mutation was placed in the rat Nav1.1 channel and expressed in Xenopus oocytes, the mutant channel displayed a positive shift in the voltage dependence of sodium channel activation, slower recovery from slow inactivation, and lower levels of current compared with the wild-type channel. Computational analysis suggests that neurons expressing the mutant channel have higher thresholds for firing a single action potential and for firing multiple action potentials, along with decreased repetitive firing. Therefore, this mutation should lead to decreased neuronal excitability, in contrast to most previous GEFS+ sodium channel mutations, which have changes predicted to increase neuronal firing.

Epidemiology of fragile X syndrome: a systematic review and meta-analysis.

Prevalence estimates for fragile X syndrome vary considerably. This systematic review and meta‐analysis was conducted to provide an accurate prevalence estimate for this disorder using primary publications in PubMed, Embase, and the Cochrane library. Data were pooled using Bayesian fixed‐effects and random‐effects models. Primary analyses assessed the frequency of the full mutation and premutation in males and females in the total population (no bias against individuals with intellectual disability) and in female carriers of the premutation in normal populations (biased against individuals with intellectual disability), based on diagnosis by polymerase chain reaction or Southern blotting. A sensitivity analysis included studies using any diagnostic testing method and conference abstracts. Sixty‐eight recorded observations provided data for the primary (56 observations) and sensitivity (12 observations) analysis. Using the random‐effects model, frequency of the full mutation was 1.4 (95% CI: 0.1–3.1) per 10,000 males and 0.9 (95% CI: 0.0–2.9) per 10,000 females (1:7,143 and 1:11,111, respectively) in the total population. The premutation frequency was 11.7 (95% CI: 6.0–18.7) per 10,000 males and 34.4 (95% CI: 6.3–83.3) per 10,000 for females (1:855 and 1:291, respectively) in the total population. The prevalence of female carriers of the premutation in the normal population was 34.4 (95% CI: 8.9–60.3) per 10,000, or 1:291. Sensitivity analyses resulted in similar prevalence estimates but with wider heterogeneity. Prevalence estimates for the full mutation from this meta‐analysis are lower than those in previous reviews of fragile X syndrome epidemiological data.

Co-occurring diagnoses among FMR1 premutation allele carriers.

Hunter JE, Rohr JK, Sherman SL. Co‐occurring diagnoses among FMR1 premutation allele carriers.

Investigation of Phenotypes Associated with Mood and Anxiety Among Male and Female Fragile X Premutation Carriers

The fragile X disorder spectrum, due to a CGG expansion in FMR1, includes fragile X syndrome (>200 repeats) and the premutation-associated disorders of ovarian insufficiency and tremor/ataxia syndrome (~55–199 repeats). Altered neurobehavioral profiles including variation of phenotypes associated with mood and anxiety may be expected among younger premutation carriers given this spectrum of disorders. However, previous studies have produced conflicting findings, providing the motivation to examine these phenotypes further. We investigated measures of mood and anxiety in 119 males and 446 females age 18–50 ascertained from families with a history of fragile X syndrome and from the general population. Scores were analyzed using a linear model with repeat length as the main predictor, adjusting for potential confounders. Repeat length was not associated with anxiety, but was marginally associated with depression and negative affect in males and negative affect only in females. These results suggest that premutation carriers may be at risk for emotional morbidity; however, phenotypic differences were subtle and of small effect size.

Is there evidence for neuropsychological and neurobehavioral phenotypes among adults without FXTAS who carry the FMR1 premutation? A review of current literature

Abstract: Carriers of expanded, but unmethylated, premutation alleles of the fragile X mental retardation gene are at risk for a late-onset tremor/ataxia syndrome, mostly affecting men over age 50. However, the general neuropsychological and neurobehavioral impact of carrying a premutation allele in younger adults not affected by the tremor/ataxia syndrome remains unclear. Past studies have utilized varying study designs resulting in inconsistent conclusions. To better understand the current evidence of the influence of the premutation on such traits in adult carriers, we reviewed the literature and identified 16 studies that met conservative inclusion criteria, including molecular measures of the fragile X mental retardation gene CGG triplet repeat length and standard measures of neurobehavioral and neurocognitive phenotypes. A review of these studies is presented to assess the evidence for possible premutation-associated neuropsychological deficits among adult men and women who do not meet diagnostic criteria of the tremor/ataxia syndrome. Results of these studies, and possible reasons for inconsistent conclusions, are discussed. The primary conclusion from this review is the need for further research using a standard protocol in a large multisite project to ensure the necessary sample size.

Fragile X-associated primary ovarian insufficiency: evidence for additional genetic contributions to severity

The fragile X mental retardation gene (FMR1) contains a CGG repeat sequence in its 5′ untranslated region that can become unstable and expand in length from generation to generation. Alleles with expanded repeats in the range of ∼55–199, termed premutation alleles, are associated with an increased risk for fragile‐X‐associated primary ovarian insufficiency (FXPOI). However, not all women who carry the premutation develop FXPOI. To determine if additional genes could explain variability in onset and severity, we used a random‐effects Cox proportional hazards model to analyze age at menopause on 680 women from 225 families who have a history of fragile X syndrome and 321 women from 219 families from the general population. We tested for the presence of a residual additive genetic effect after adjustment for FMR1 repeat length, race, smoking, body mass index, and method of ascertainment. Results showed significant familial aggregation of age at menopause with an estimated additive genetic variance of 0.55–0.96 depending on the parameterization of FMR1 repeat size and definition of age at menopause (P‐values ranging between 0.0002 and 0.0027). This is the first study to analyze familial aggregation of FXPOI. This result is important for proper counseling of women who carry FMR1 premutation alleles and for guidance of future studies to identify additional genes that influence ovarian insufficiency. Genet. Epidemiol. 2008.

A standardized, evidence-based protocol to assess clinical actionability of genetic disorders associated with genomic variation.

Purpose:Genome and exome sequencing can identify variants unrelated to the primary goal of sequencing. Detecting pathogenic variants associated with an increased risk of a medical disorder enables clinical interventions to improve future health outcomes in patients and their at-risk relatives. The Clinical Genome Resource, or ClinGen, aims to assess clinical actionability of genes and associated disorders as part of a larger effort to build a central resource of information regarding the clinical relevance of genomic variation for use in precision medicine and research.Methods:We developed a practical, standardized protocol to identify available evidence and generate qualitative summary reports of actionability for disorders and associated genes. We applied a semiquantitative metric to score actionability.Results:We generated summary reports and actionability scores for the 56 genes and associated disorders recommended by the American College of Medical Genetics and Genomics for return as secondary findings from clinical genome-scale sequencing. We also describe the challenges that arose during the development of the protocol that highlight important issues in characterizing actionability across a range of disorders.Conclusion:The ClinGen framework for actionability assessment will assist research and clinical communities in making clear, efficient, and consistent determinations of actionability based on transparent criteria to guide analysis and reporting of findings from clinical genome-scale sequencing.Genet Med 18 12, 1258–1268.

Capturing the fragile X premutation phenotypes: a collaborative effort across multiple cohorts

OBJECTIVE To capture the neuropsychological profile among male carriers of the FMR1 premutation allele (55-200 CGG repeats) who do not meet diagnostic criteria for the late-onset fragile X-associated tremor/ataxia syndrome, FXTAS. METHOD We have initiated a multicenter collaboration that includes 3 independent cohorts, totaling 100 carriers of the premutation and 216 noncarriers. The initial focus of this collaboration has been on executive function. Four executive function scores are shared among the 3 cohorts (Controlled Oral Word Association Test, Stroop Color-Word Test, and Wechsler backward digit span and letter-number sequencing) whereas additional executive function scores are available for specific cohorts (Behavior Dyscontrol Scale, Hayling Sentence Completion Test Part B, and Wisconsin Card Sorting Test). Raw scores were analyzed by using statistical models that adjust for cohort-specific effects as well as age and education. RESULTS Carriers scored significantly lower compared to noncarriers on the Stroop Color-Word Test (p = .01), Hayling Sentence Completion Test Part B (p < .01), and Behavioral Dyscontrol Scale (p = .03), with the Hayling displaying a significant age-related decline (p = .01), as assessed by an age and repeat length-group interaction. Follow-up analysis of the collective data did not identify any specific age groups or repeat length ranges (i.e., low premutation = 55-70 repeats, midpremutation = 71-100 repeats, high premutation = 101-199 repeats) that were associated with an increased risk of executive function deficits. CONCLUSIONS Preliminary analyses do not indicate global executive function impairment among male carriers without FXTAS compared to noncarriers. However, impairment in inhibitory capacity may be present among a subset of carriers, though the risk factors for this group do not appear to be related to age or repeat length.