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The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).

Type 2 diabetes (T2D) has been associated with a large number of genomic loci, many of which encompass multiple genes without a definitive causal gene. This complexity has hindered efforts to clearly identify functional candidate genes and interpret their role in mediating susceptibility to disease. Here we examined the relevance of individual genes found at T2D-associated loci by assessing their potential contribution to a phenotype relevant to the disease state: production and maintenance of β-cell mass. Using transgenic zebrafish in which β-cell mass could be rapidly visualized in vivo, we systematically suppressed the expression of orthologs of genes found at T2D-associated genomic loci. Overall, we tested 67 orthologs, many of which had no known relevance to β-cell mass, at 62 human T2D-associated loci, including eight loci with multiple candidate genes. In total we identified 25 genes that were necessary for proper β-cell mass, providing functional evidence for their role in a physiological phenotype directly related to T2D. Of these, 16 had not previously been implicated in the regulation of β-cell mass. Strikingly, we identified single functional candidate genes at the majority of the loci for which multiple genes were analyzed. Further investigation into the contribution of the 25 genes to the adaptive capacity of β-cells suggested that the majority of genes were not required for glucose-induced expansion of β-cell mass but were significantly necessary for the regeneration of β-cells. These findings suggest that genetically programmed deficiencies in β-cell mass may be related to impaired maintenance. Finally, we investigated the relevance of our findings to human T2D onset in diabetic individuals from the Old Order Amish and found that risk alleles in β-cell mass genes were associated with significantly younger age of onset and lower body mass index. Taken together, our study offers a functional approach to assign relevance to genes at T2D-associated loci and offers experimental evidence for the defining role of β-cell mass maintenance in genetic susceptibility to T2D onset.

Background/Introduction: Using whole exome sequencing we identified a truncating mutation in the KIAA2022 gene in a child with mental retardation and antibody-negative diabetes. The KIAA2022 gene encodes a large protein of 1516 amino acids, which has no significant homology with other known proteins. KIAA2022 has already been associated to X-linked mental retardation. In this study we analyze the potential contribution of KIAA2022 to diabetes. Methods: Whole exome capture sequencing was performed using the Illumina HiSeq instrument. We performed qRT-PCR and immunohistochemistry. We used the INS-1E cell line for in vitro and zebrafish and mice for in vivo knock down studies. Results: We confirmed mRNA and protein expression of KIAA2022 in murine and human islets by qRT-PCR and immunohistochemistry. KIAA2022 partially co-stained with insulin in pancreatic sections. In vitro experiments with INS-1E cells showed that KIAA2022 is highly expressed during cell division and after stress induction by H202. To characterize KIAA2022 function in vivo, we performed knock down studies in zebrafish and found a decrease of the beta cell mass by 40% compared to wild type. The beta cell expansion rate, assessed by culturing zebrafish embryos with and without glucose, lead to a 30% decrease of the expansion rate in the knock-down animals in comparison to the control group. We further used the CRISPR Cas9 technology to disrupt the KIAA2022 gene in mice. The knockout mice showed smaller islets and DNA damage signals in islets in comparison to the control mice. The i.p. glucose tolerance tests at twelve weeks of age showed glucose intolerance in mutant in comparison to wild type mice. Conclusion: In vitro experiments using INS-1E cells suggest that KIAA2022 may play a role in cell division and in DNA damage signaling or repair. The KIAA2022 knock down experiments lead to a decrease of the beta cell mass and glucose-induced expansion rate in zebrafish and DNA damage signals in beta cells of mutant mice as well as glucose intolerance. Disclosure C. Stekelenburg: None. J. Blouin: None. F. Santoni: None. E.A. O'Hare: None. N.A. Zaghloul: None. V. Schwitzgebel: None.

Next-generation sequencing (NGS) and expression technologies were utilized to investigate the genes and sequence elements in a 586 kb region of chicken chromosome 1 associated with the autosomal recessive diplopodia-1 (dp-1) mutation. This mutation shows a syndromic phenotype similar to known human developmental abnormalities (e.g., cleft palate, polydactyly, omphalocele [exposed viscera]). Toward our goal to ascertain the variant responsible, the entire 586 kb region was sequenced following utilization of a specifically designed capture array and to confirm/validate fine-mapping results. Bioinformatic analyses identified a total of 6142 sequence variants, which included SNPs, indels, and gaps. Of these, 778 SNPs, 146 micro-indels, and 581 gaps were unique to the UCD-Dp-1.003 inbred congenic line; those found within exons and splice sites were studied for contribution to the mutant phenotype. Upon further validation with additional mutant samples, a smaller subset (of variants [51]) remains linked to the mutation. Additionally, utilization of specific samples in the NGS technology was advantageous in that fine-mapping methodologies eliminated an additional 326 kb of sequence information on chromosome 1. Predicted and confirmed protein-coding genes within the smaller 260 kb region were assessed for their developmental expression patterns over several stages of early embryogenesis in regions/tissues of interest (e.g., digits, craniofacial region). Based on these results and known function in other vertebrates, 2 genes within 5 kb of each other, MRE11 and GPR83, are proposed as high-priority candidates for the dp-1 mutation.

Background: Elevated levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor for cardiovascular disease via its contribution to the development and progression of atherosclerotic lesions. Although the genetic basis of LDL-C has been studied extensively, currently known genetic variants account for only ≈20% of the variation in LDL-C levels. Methods: Through an array-based association analysis in 1102 Amish subjects, we identified a variant strongly associated with LDL-C levels. Using a combination of genetic analyses, zebrafish models, and in vitro experiments, we sought to identify the causal gene driving this association. Results: We identified a founder haplotype associated with a 15 mg/dL increase in LDL-C on chromosome 5. After recombination mapping, the associated region contained 8 candidate genes. Using a zebrafish model to evaluate the relevance of these genes to cholesterol metabolism, we found that expression of the transcribed pseudogene, APOOP1 , increased LDL-C and vascular plaque formation. Conclusions: Based on these data, we propose that APOOP1 regulates levels of LDL-C in humans, thus identifying a novel mechanism of lipid homeostasis.

It is now over 10 years since the first avian genome [International Chicken Genome Sequencing Consortium, 2004] and the first complete avian karyotype [Masabanda et al., 2004] were both published; however, until 2014, avian cytogenetics has focused heavily on descriptive studies [e.g. Griffin et al., 2007, 2008; Skinner et al., 2009; Volker et al., 2010] with less attention to its functional relevance. Last year, however, saw 2 landmark efforts in the chromosomal studies of birds: a special issue of Chromosome Research in April and the announcement of recently completed sequences of multiple new avian genomes in Science and the BMC journals (taking the total number sequenced to over 50) in December. Studying the chromosomes of birds is, perhaps for the first time, telling us more about avian biology, function and evolution than it ever has... Conclusions. The most recent advances in avian cytogenetics have culminated in great promise not only for the study of bird karyotypes, but also for providing insight into the mechanisms of chromosome evolution in general. New avenues for investigation include gene regulation; for instance, it will become necessary to map accurately the physical location of polyadenylation and transcription start sites, important reference points that define promoters and post-transcriptional regulation. It will also become possible to sequence full-length transcripts, to allow accurate identification of alternate splicing events and their controlling elements. The ENCODE (Encyclopedia of DNA Elements) project has helped to define functional elements of the human genome, including those aforementioned as well as other chromatin signals, e.g. active chromatin, enhancers, insulators, methylation domains, etc. An effort of agENCODE is underway to include agriculturally important birds such as chicken, turkey, duck, quail, and perhaps ostrich. The study of cytogenetics will be essential here in helping to define higher-order structures in nuclear organization that show regulatory interactions within and between chromosomes. Finally, reconstruction of evolutionary events allows us to study genome organization and function not only in extant but, by extrapolation, in extinct species also. Reconstruction of avian-reptilian ancestral karyotypes will allow us to define chromosomal rearrangements in long-dead species that have captured the public imagination. Here be dragons!

Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype description, establishing the autosomal recessive pattern of Mendelian inheritance and placing the mutation on an inbred genetic background to create the congenic line UCD Wingless-2.331. The research described in this paper employed the complementary tools of breeding, genetics, and genomics to map the chromosomal location of the mutation and successively narrow the size of the region for analysis of the causative element. Specifically, the wg-2 mutation was initially mapped to a 7 Mb region of chromosome 12 using an Illumina 3 K SNP array. Subsequent SNP genotyping and exon sequencing combined with analysis from improved genome assemblies narrowed the region of interest to a maximum size of 227 kb. Within this region, 3 validated and 3 predicted candidate genes are found, and these are described. The wg-2 mutation is a valuable resource to contribute to an improved understanding of the developmental pathways involved in chicken and avian limb development as well as serving as a model for human development, as the resulting syndrome shares features with human congenital disorders.

talpid(2) is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid(3), talpid(2) has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the etiology of the craniofacial phenotype of this mutant. We confirmed that primary cilia were disrupted in talpid(2) mutants. Molecularly, we found disruptions in Hedgehog signaling. Post-translational processing of GLI2 and GLI3 was aberrant in the developing facial prominences. Although both GLI2 and GLI3 processing were disrupted in talpid(2) mutants, only GLI3 activator levels were significantly altered in the nucleus. Through additional fine mapping and whole-genome sequencing, we determined that the talpid(2) phenotype was linked to a 1.4 Mb region on GGA1q that contained the gene encoding the ciliary protein C2CD3. We cloned the avian ortholog of C2CD3 and found its expression was ubiquitous, but most robust in the developing limbs and facial prominences. Furthermore, we found that C2CD3 is localized proximal to the ciliary axoneme and is important for docking the mother centriole to the ciliary vesicle and cell membrane. Finally, we identified a 19 bp deletion in talpid(2) C2CD3 that produces a premature stop codon, and thus a truncated protein, as the likely causal allele for the phenotype. Together, these data provide insight into the cellular, molecular and genetic etiology of the talpid(2) phenotype. Our data suggest that, although the talpid(2) and talpid(3) mutations affect a common ciliogenesis pathway, they are caused by mutations in different ciliary proteins that result in differences in craniofacial phenotype.

Primary cilia and their anchoring basal bodies are important regulators of a growing list of signaling pathways. Consequently, dysfunction in proteins associated with these structures results in perturbation of the development and function of a spectrum of tissue and cell types. Here, we review the role of cilia in mediating the development and function of the pancreas. We focus on ciliary regulation of major pathways involved in pancreatic development, including Shh, Wnt, TGF-β, Notch, and fibroblast growth factor. We also discuss pancreatic phenotypes associated with ciliary dysfunction, including pancreatic cysts and defects in glucose homeostasis, and explore the potential role of cilia in such defects.

This study evaluated the neural basis of verbal working memory (WM) function in a group of 20 children and adolescents with fetal alcohol spectrum disorders (FASDs) and 20 typically developing comparison participants using functional magnetic resonance imaging (fMRI). Both groups showed prominent activation in the frontal-parietal-cerebellar network known to be important for verbal WM. Despite equivalent behavioral performance between groups, alcohol-exposed individuals showed increased activation relative to typically developing individuals in left dorsal frontal and left inferior parietal cortices, and bilateral posterior temporal regions during verbal WM. These effects remained even when group differences on IQ were statistically controlled. This pattern of increased activation coupled with equivalent behavioral performance between groups suggests that individuals with FASD recruit a more extensive network of brain regions during verbal WM relative to typically developing individuals. These findings may suggest that frontal-parietal processing during verbal WM is less efficient in alcohol-exposed individuals.

Down syndrome is characterized by disproportionately severe impairments of speech and language, yet little is known about the neural underpinnings of these deficits. We compared fMRI activation patterns during passive story listening in 9 young adults with Down syndrome and 9 approximately age-matched, typically developing controls. The typically developing group exhibited greater activation than did the Down syndrome group in classical receptive language areas (superior and middle temporal gyri) for forward > backward speech; the Down syndrome group exhibited greater activation in cingulate gyrus, superior and inferior parietal lobules, and precuneus for both forward speech > rest and backward speech > rest. The Down syndrome group showed almost no difference in activation patterns between the language (forward speech) and nonlanguage (backward speech) conditions.

The effects of prenatal exposure to methamphetamine on neurocognitive skills are not well understood. Chang and colleagues documented lower scores on measures of verbal memory, spatial memory, attention, and visual motor integration in methamphetamine-exposed children compared to unexposed participants (1). However, the largest reported sample of prenatal MA exposure reported that 49% of participating methamphetamine users also used alcohol during pregnancy, and only 10% used methamphetamine alone (2). These high rates of co-exposure to alcohol during pregnancy render it difficult to separate effects of methamphetamine exposure from alcohol exposure. Heavy prenatal alcohol exposure is known to detrimentally affect verbal memory (3). Verbal memory encompasses learning and recall of verbal information from long-term storage. Verbal learning is an important cognitive skill because it underlies basic academic skills such as vocabulary acquisition. The verbal nature of classroom instruction implicates that deficits in verbal learning may detrimentally influence achievement in other areas such as math and science. Three independent research groups have shown that children with prenatal exposure to alcohol perform more poorly on both immediate and delayed recall of verbal information compared to unexposed participants (4-6). All three studies concluded that problems of verbal memory were specific to encoding or learning of new information, and retention of information was not different between exposed and unexposed groups after accounting for initial encoding differences (4-6). Consistent with these behavioral findings, we have recently shown that unexposed control subjects activate left medial temporal structures important for verbal memory more than children with prenatal alcohol exposure during a verbal memory task using functional magnetic resonance imaging (fMRI) (7). Examination of prenatal methamphetamine exposure presents significant challenges given that a significant number of individuals with methamphetamine exposure have concomitant alcohol exposure. For this reason, we have recently attempted to address the specificity of methamphetamine exposure on neurocognitive performance by comparing a methamphetamine plus alcohol-exposed group of children to an alcohol-exposed only group and an unexposed control group. Both exposed groups performed more poorly than the unexposed control group on measures of verbal memory; however, there was no difference between our methamphetamine plus alcohol-exposed and our alcohol exposure only groups (8). These behavioral data suggested that decreased verbal memory performance in children exposed to both methamphetamine and alcohol may be due to the effects of alcohol rather than to the effects of methamphetamine. A limitation with behavioral performance data is that they cannot reveal whether neurocognitive systems engaged to achieve task completion are similar in children with different exposure histories. A similar level of performance can be achieved via engagement of different neurocognitive systems. For example, Brown and colleagues showed that children and adults engage different brain regions on word generation tasks despite equivalent levels of performance (9). Among atypically developing populations (e.g., dyslexia), different patterns of activation between the studied population and normally developing controls have been interpreted as effortful compensatory mechanisms and/or functional disruptions in the studied population (10, 11). To date, no studies have evaluated the effects of prenatal methamphetamine exposure on brain activation patterns. In the present exploratory study, we asked whether children with histories of prenatal exposure to methamphetamine plus alcohol engaged different neurocognitive systems during a verbal memory task in comparison to unexposed controls and children with exposure to alcohol only. The alcohol-exposed group is a necessary control given that the majority of our methamphetamine-exposed participants also have concomitant alcohol exposure. The term Fetal Alcohol Spectrum Disorders (FASD) has been proposed to capture the continuum of effects attributable to prenatal alcohol exposure (12, 13). We followed the Washington diagnostic system (12), under which the FASD umbrella term includes Fetal Alcohol Syndrome (FAS) and Partial Fetal Alcohol Syndrome (PFAS) on the more severe end of the spectrum where facial dysmorphology is prominent. Those who do not meet criteria for FAS or PFAS have less prominent facial dysmorphology and physical evidence of brain damage, and have been referred to as Alcohol-Related Neurobehavioral Disorder (ARND). Individuals in the ARND category also show impaired cognitive performance across multiple domains, suggesting that alcohol teratogenesis on brain development exists in the absence of facial dysmorphology (14). In order to minimize the possibility that heavy alcohol exposure effects may mask more subtle effects of methamphetamine exposure, we excluded from both the methamphetamine-exposed and the alcohol-exposed only groups those who met criteria for FAS and PFAS, the most severe manifestations of prenatal alcohol exposure. If methamphetamine has a specific effect on the development of the verbal memory system or interacts with alcohol in influencing the development of this system, then the methamphetamine-exposed children are expected to show a different pattern of activation during our verbal memory task compared to both the unexposed and the alcohol-exposed only groups.

Dynamic changes in brain structure, activation, and cognitive abilities co-occur during development, but little is known about how changes in brain structure relate to changes in cognitive function or brain activity. By using cortical pattern matching techniques to correlate cortical gray matter thickness and functional brain activity over the entire brain surface in 24 typically developing children, we integrated structural and functional magnetic resonance imaging data with cognitive test scores to identify correlates of mature performance during orthographic processing. Fast-naming individuals activated the right fronto-parietal attention network in response to novel fonts more than slow-naming individuals, and increased activation of this network was correlated with more mature brain morphology in the same fronto-parietal region. These relationships remained even after effects of age or general cognitive ability were statistically controlled. These results localized cortical regions where mature morphology corresponds to mature patterns of activation, and may suggest a role for experience in mediating brain structure-activation relationships.

Rare genetic syndromes characterized by early-onset type 2 diabetes have revealed the importance of pancreatic β-cells in genetic susceptibility to diabetes. However, the role of genetic regulation of β-cells in disorders that are also characterized by highly penetrant obesity, a major additional risk factor, is unclear. In this study, we investigated the contribution of genes associated with two obesity ciliopathies, Bardet-Biedl Syndrome and Alstrom Syndrome, to the production and maintenance of pancreatic β-cells. Using zebrafish models of these syndromes, we identified opposing effects on production of β-cells. Loss of the Alstrom gene, alms1, resulted in a significant decrease in β-cell production whereas loss of BBS genes, bbs1 or bbs4, resulted in a significant increase. Examination of the regulatory program underlying β-cell production suggested that these effects were specific to β-cells. In addition to the initial production of β-cells, we observed significant differences in their continued maintenance. Under prolonged exposure to high glucose conditions, alms1-deficient β-cells were unable to continually expand as a result of decreased proliferation and increased cell death. Although bbs1-deficient β-cells were similarly susceptible to apoptosis, the overall maintenance of β-cell number in those animals was sustained likely due to increased proliferation. Taken together, these findings implicate discrepant production and maintenance of β-cells in the differential susceptibility to diabetes found between these two genetic syndromes.

Although there is a documented association between plasma nicotine levels and smoking behavior, recent studies indicate that denicotinized cigarettes reduced craving and symptoms of tobacco withdrawal. Denicotinized cigarettes (that deliver tar but insignificant amounts of nicotine) and conventional cigarettes were compared in a within-subject spaced smoking study. In six sessions, subjects (n=10) smoked denicotinized cigarettes or conventional cigarettes every 30, 60 or 240 min (8, 4 or 1 cigarette(s)). EEG effects of the last cigarette of each session were deduced by comparisons with EEG recordings collected before smoking. Conventional cigarettes increased spectral edge EEG frequency, decreased θ power and increased β1 power. Denicotinized cigarettes decreased spectral frequency. The EEG effects of both cigarettes depended upon the recentness of smoking. The results indicate that nicotine delivery, recentness and the process of smoking importantly influence the EEG; other, non-nicotine components of tobacco smoke may also exert EEG effects.

Neuroscience can be used as a tool to inspire an interest in science in school children as well as to provide teaching experience to college students.

Relational reasoning, or the ability to identify and consider relationships between multiple mental representations, is a fundamental component of high-level cognition (Robin and Holyoak, 1995). The capacity to reason with relations enables abstract thought and may be at the core of what makes human cognition unique (Penn et al., 2008). This capacity improves throughout childhood and adolescence (Ferrer et al., 2009). Here, we sought to better understand the neural mechanisms that support its emergence. We have hypothesized previously, based on fMRI research in adults, that (1) inferior parietal lobe (IPL) plays a central role in representing relationships between mental representations (first-order relations) and (2) rostrolateral prefrontal cortex (RLPFC) integrates inputs from IPL to build second-order relational structures (i.e., relations between relations). In the present study, we examined fMRI and cortical thickness data from 85 children and adolescents (ages 6–18 years). Participants performed a relational matching task in which they viewed arrays of four visual stimuli and determined whether two stimuli shared a particular feature (a first-order relational judgment) or whether two pairs of stimuli matched according to the same feature (a second-order relational judgment). fMRI results provide evidence for increased functional selectivity across ages 6–18 years in RLPFC and IPL. Specifically, young children engaged RLPFC and IPL indiscriminately for first-order and second-order relational judgments, and activation for first-order relations diminished with age whereas activation for second-order relations stayed elevated. Examination of cortical thickness revealed that increased functional selectivity in RLPFC could be partly accounted for by cortical thinning in IPL.

We examined functional MRI activation patterns corresponding to verbal paired associate learning in a group of 11 children with heavy prenatal alcohol exposure compared with 16 typically developing children. Among the typically developing children, prominent activation was observed in the left medial temporal lobe, left dorsal frontal lobe and bilateral posterior temporal cortices during learning and recall. Analyses revealed significantly less activation in left medial and posterior temporal regions and significantly more activation in right dorsal frontal cortex in the alcohol-exposed children relative to controls, even when group differences in memory test performance were statistically controlled. These results may indicate an increased reliance on frontal memory systems in the children with heavy prenatal alcohol exposure, perhaps compensating for dysfunctional medial temporal memory systems. Our findings are consistent with neuropsychological and structural imaging studies, and provide the first evidence for brain activation abnormalities, independent of group performance differences, during verbal learning and recall in children with heavy prenatal alcohol exposure.

Prenatal exposure to alcohol can result in neuroanatomical and neurocognitive deficits. High-resolution magnetic resonance imaging, surface-based image analytic methods, and neuropsychological measures were used to characterize the cerebellar vermis and to evaluate potential cognitive correlates of vermal morphology in 21 children and adolescents with prenatal alcohol exposure and 21 normally developing individuals. Alcohol-exposed individuals showed statistically significant reductions in the midline sagittal areas of the anterior vermis and posterior-inferior vermis, and significant displacement of the anterior and posterior-inferior vermal regions. Anterior vermal dysmorphology was negatively correlated with verbal learning and memory performance within the alcohol-exposed group. These observations expand on previous reports of cerebellar abnormalities in prenatal alcohol exposure, in that they localize the specific pattern of cerebellar vermal dysmorphology.