Analysis of differential gene expression and transcript usage in hippocampus of Apoe null mutant mice: Implications for Alzheimer's disease.

A dataset of single-nucleus RNA sequencing (snRNAseq) data was analyzed using Seurat, Sierra, and Ingenuity Pathway Analysis (IPA) programs to assess differentially expressed genes (DEGs) and differential transcript usage (DTU) in mouse hippocampal cell types. Seurat identified DEGs between the wild type (WT) and Apoe knockout (EKO) mice. IPA identified 11 statistically significant canonical pathways in >1 cell type. Sierra identified Sipa1l1 with DTU between WT and EKO samples. Analysis of the Sipa1l1 peak region identified an alternative non-canonical polyadenylation signal and a putative cytoplasmic polyadenylation element. APOE regulation of gene transcription and co-transcriptional RNA processing may underlie Alzheimer's disease.

Single Nucleus Transcriptome Data from Alzheimer's Disease Mouse Models Yield New Insight into Pathophysiology.

BACKGROUND: 5XFAD humanized mutant mice and Trem2 knockout (T2KO) mice are two mouse models relevant to the study of Alzheimer's disease (AD)-related pathology. OBJECTIVE: To determine hippocampal transcriptomic and polyadenylation site usage alterations caused by genetic mutations engineered in 5XFAD and T2KO mice. METHODS: Employing a publicly available single-nucleus RNA sequencing dataset, we used Seurat and Sierra analytic programs to identify differentially expressed genes (DEGs) and differential transcript usage (DTU), respectively, in hippocampal cell types from each of the two mouse models. We analyzed cell type-specific DEGs further using Ingenuity Pathway Analysis (IPA). RESULTS: We identified several DEGs in both neuronal and glial cell subtypes in comparisons of wild type (WT) versus 5XFAD and WT versus T2KO mice, including Ttr, Fth1, Pcsk1n, Malat1, Rpl37, Rtn1, Sepw1, Uba52, Mbp, Arl6ip5, Gm26917, Vwa1, and Pgrmc1. We also observed DTU in common between the two comparisons in neuronal and glial subtypes, specifically in the genes Prnp, Rbm4b, Pnisr, Opcml, Cpne7, Adgrb1, Gabarapl2, Ubb, Ndfip1, Car11, and Stmn4. IPA identified three statistically significant canonical pathways that appeared in multiple cell types and that overlapped between 5XFAD and T2KO comparisons to WT, including 'FXR/RXR Activation', 'LXR/RXR Activation', and 'Acute Phase Response Signaling'. CONCLUSION: DEG, DTU, and IPA findings, derived from two different mouse models of AD, highlight the importance of energy imbalance and inflammatory processes in specific hippocampal cell types, including subtypes of neurons and glial cells, in the development of AD-related pathology. Additional studies are needed to further characterize these findings.

Neonatal Opioid Withdrawal Syndrome (NOWS): A Transgenerational Echo of the Opioid Crisis.

The incidence of neonatal opioid withdrawal syndrome (NOWS) has increased substantially in the setting of the opioid epidemic, a major public health problem in the United States. At present, NOWS has commonly used assessment and treatment protocols, but new protocols have questioned old practices. However, because of limited access to opioid use disorder (OUD) treatment and socioeconomic factors, many pregnant (and postpartum) women with OUD do not receive treatment. The pathophysiology of NOWS is not completely understood, although limited research studies have been conducted in humans and animals to better understand its etiology. Moreover, there is evidence that epigenetic and genetic factors play a role in the development of NOWS, but further study is needed. Animal models have suggested that there are deleterious effects of in utero opioid exposure later in life. Clinical research has revealed the harmful long-term sequelae of NOWS, with respect to cognitive function and childhood development. Many psychiatric disorders begin during adolescence, so as infants born with NOWS approach adolescence, additional clinical and molecular studies are warranted to identify biologic and psychosocial risk factors and long-term effects of NOWS. Additionally, access to specialized OUD treatment for pregnant women must be more readily available in the United States, especially in rural areas.

Inherited L1 Retrotransposon Insertions Associated With Risk for Schizophrenia and Bipolar Disorder.

Studies of the genetic heritability of schizophrenia and bipolar disorder examining single nucleotide polymorphisms (SNPs) and copy number variations have failed to explain a large portion of the genetic liability, resulting in substantial missing heritability. Long interspersed element 1 (L1) retrotransposons are a type of inherited polymorphic variant that may be associated with risk for schizophrenia and bipolar disorder. We performed REBELseq, a genome wide assay for L1 sequences, on DNA from male and female persons with schizophrenia and controls (n = 63 each) to identify inherited L1 insertions and validated priority insertions. L1 insertions of interest were genotyped in DNA from a replication cohort of persons with schizophrenia, bipolar disorder, and controls (n = 2268 each) to examine differences in carrier frequencies. We identified an inherited L1 insertion in ARHGAP24 and a quadallelic SNP (rs74169643) inside an L1 insertion in SNTG2 that are associated with risk for developing schizophrenia and bipolar disorder (all odds ratios ~1.2). Pathway analysis identified 15 gene ontologies that were differentially affected by L1 burden, including multiple ontologies related to glutamatergic signaling and immune function, which have been previously associated with schizophrenia. These findings provide further evidence supporting the role of inherited repetitive genetic elements in the heritability of psychiatric disorders.

Association between polymorphisms in the metallophosphoesterase (MPPE1) gene and bipolar disorder.

Genetic linkage studies in bipolar disorder (BPD) suggest that a susceptibility locus exists on chromosome 18p11. The metallophosphoesterase (MPPE1) gene maps to this region. Dysregulation of protein phosphorylation and subsequent abnormal cellular signaling has been postulated to be involved in neuropsychiatric disorders thus making MPPE1 a plausible biological candidate gene for BPD. In this study, we hypothesized that genetic variation in the MPPE1 gene contributes to BPD. We tested this hypothesis by genotyping four SNPs (rs871044; rs3974590; rs593713; rs602201) in BPD patients (n = 570) and healthy controls (n = 725). Genotypes and allele frequencies were compared between groups using Chi square contingency analysis. Linkage disequilibrium (LD) between markers was calculated and estimated haplotype frequencies were compared between groups. Single marker analysis revealed an association of rs3974590 with BPD (P = 0.009; permutation corrected P = 0.046). Haplotype analysis did not show any significant association with disease after permutation correction. Our results provide evidence of an association between a polymorphism in the MPPE1 gene and BPD. Additional studies are necessary to confirm and elucidate the role of MPPE1 as a susceptibility gene for BPD on chromosome 18p.

Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome.

Long interspersed element-1 retrotransposons (LINE-1 or L1) are ∼6 kb mobile DNA elements implicated in the origins of many Mendelian and complex diseases. The actively retrotransposing L1s are mostly limited to the L1 human specific (L1Hs) transcriptional active (Ta) subfamily. In this manuscript, we present REBELseq as a method for the construction of Ta subfamily L1Hs-enriched next-generation sequencing libraries and bioinformatic identification. REBELseq was performed on DNA isolated from NeuN+ neuronal nuclei from postmortem brain samples of 177 individuals and empirically-driven bioinformatic and experimental cutoffs were established. Putative L1Hs insertions passing bioinformatics cutoffs were experimentally validated. REBELseq reliably identified both known and novel Ta subfamily L1Hs insertions distributed throughout the genome. Differences in the proportion of individuals possessing a given reference or non-reference retrotransposon insertion were identified. We conclude that REBELseq is an unbiased, whole genome approach to the amplification and detection of Ta subfamily L1Hs retrotransposons.

Genetic variants in the cocaine- and amphetamine-regulated transcript gene (CARTPT) and cocaine dependence.

Dopaminergic brain systems have been implicated to play a major role in drug reward, thus making genes involved in these circuits plausible candidates for susceptibility to substance use disorders. The cocaine- and amphetamine-regulated transcript peptide (CARTPT) is involved in reward and feeding behavior and has functional characteristics of an endogenous psychostimulant. In this study we tested the hypothesis that variation in the CARTPT gene increases susceptibility to cocaine dependence in individuals of African descent. Genotypes of three HapMap tagging SNPs (rs6894758; rs11575893; rs17358300) across the CARTPT gene region were obtained in cocaine dependent individuals (n=348) and normal controls (n=256). All subjects were of African descent. There were no significant differences in allele, genotype or haplotype frequencies between cases and controls for any of the tested SNPs. Our results do not support an association of the CARTPT gene with cocaine dependence; however, additional studies using larger samples, comprehensive SNP coverage, and different populations are necessary to conclusively rule out CARTPT as a contributing factor in the etiology of cocaine dependence.

Association between polymorphisms in the vesicular monoamine transporter 1 gene (VMAT1/SLC18A1) on chromosome 8p and schizophrenia.

Linkage studies have suggested a susceptibility locus for schizophrenia (SZ) exists on chromosome 8p21-22. The vesicular monoamine transporter 1 gene (VMAT1), also known as SLC18A1, maps to this SZ susceptibility locus. Vesicular monoamine transporters are involved in the presynaptic vesicular packaging of monoamine neurotransmitters, which have been postulated to play a role in the etiology of SZ. Variations in the VMAT1 gene might affect transporter function and/or expression, and might be involved in the etiology of SZ. Genotypes of 62 patients with SZ and 188 control subjects were obtained for 4 missense single nucleotide polymorphisms (Thr4Pro, Thr98Ser, Thr136Ile, Val392Leu) and 2 noncoding single nucleotide polymorphisms (rs988713, rs2279709). All cases and controls were of European descent. The frequency of the minor allele of the Thr4Pro polymorphism was significantly increased in SZ patients when compared to controls (p = 0.0140; d.f. = 1; OR = 1.69; 95% CI = 1.11-2.57). Assuming a recessive mode of inheritance, the frequency of homozygote 4Pro carriers was significantly increased in the SZ patients when compared to controls (24 vs. 8%, respectively; p = 0.0006; d.f. = 1; OR = 3.74; 95% CI = 1.703-8.21). Haplotype analysis showed nominal significance for an individual risk haplotype (p = 0.013); however, after permutation correction, the global p value did not attain a statistically significant level (p = 0.07). Results suggest that variations in the VMAT1 gene may confer susceptibility to SZ in patients of European descent. Further studies are necessary to confirm this effect, and to elucidate the role of VMAT1 in central nervous system physiology and possible involvement in the genetic origins of SZ.

Analysis of variations in the NAPG gene on chromosome 18p11 in bipolar disorder.

OBJECTIVE: A number of studies have implicated the chromosome 18p11 region as a susceptibility region for bipolar disorder. The gene encoding gamma-SNAP (NAPG), one of three soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment proteins (SNAPs), is located in the 18p11 region and is thought to play a role in cellular processes required for neurotransmission in the central nervous system. The purpose of this study is to investigate whether polymorphisms in the human NAPG gene contribute to the etiology of bipolar disorder. METHODS: To test this hypothesis, we used a case-control design in which the genotype and allele frequencies for five single-nucleotide polymorphisms in the human NAPG gene were compared between individuals with a diagnosis of type I bipolar disorder (n=460) and control individuals (n=191). RESULTS: The genotype results indicate that three of the single-nucleotide polymorphisms in the NAPG gene, rs2290279 (P=0.027), rs495484 (P=0.044) and rs510110 (P=0.046), show a nominal, statistically significant association with bipolar disorder at the genotype frequency level. CONCLUSIONS: The results of this study suggest that polymorphisms in the human NAPG gene may represent risk factors for the development of bipolar disorder, but before such a role can be established, the results of this study must be confirmed in additional populations of bipolar disorder patients and controls.

No association between polymorphisms in the prostate apoptosis factor-4 gene and cocaine dependence.

OBJECTIVE: A number of studies have investigated the genes underlying dopamine, serotonin, and glutamine neurotransmitter systems in order to find a genetic basis for the pathology of cocaine dependence. The gene that encodes the prostate apoptosis factor-4 (Par-4) protein is located in the 12q21 region and has been shown to directly interact with the D2 dopamine receptor and through such interaction is thought to directly affect the activity of D2 receptors. The aim of this study is to investigate whether polymorphisms in the human Par-4 gene contribute to the etiology of cocaine dependence. METHODS: To test this hypothesis, we used a case-control design in which the genotype and allele frequencies for five single nucleotide polymorphisms in the human Par-4 gene were compared between cocaine-dependent individuals (n=172) and controls (n=92) of African descent. RESULTS: The genotype results failed to detect any associations between polymorphisms in the Par-4 gene and the cocaine-dependent phenotype. CONCLUSIONS: The results of this study suggest that variations in the human Par-4 gene are unlikely to play a major role in the pathophysiology of cocaine dependence. This study, however, should be repeated in larger cocaine-dependent and control populations to determine that this is indeed the case.

Association of a polymorphism in the Homer1 gene with cocaine dependence in an African American population.

OBJECTIVE: While twin and adoption studies have demonstrated that up to 70% of the risk for becoming addicted to cocaine is due to genetic factors, identifying specific genes involved in the development or progression of cocaine dependence has been difficult. The purpose of this study is to determine whether single-nucleotide polymorphisms in the Homer1 and Homer2 genes associate with the cocaine-dependent phenotype in an African American population. METHODS: This study utilized a case-control design in which the genotype and allele frequencies for four single-nucleotide polymorphisms in the Homer1 gene and three single-nucleotide polymorphisms in the Homer2 gene were compared between African American individuals with a diagnosis of cocaine dependence (n=170) and African American individuals with no history of substance abuse (n=90). RESULTS: The data indicate that one single-nucleotide polymorphism, rs6871510, located in intron 1 of the Homer1 gene significantly (P=0.029) associates with cocaine dependence at the genotype level, and trends toward a significant association at the allele frequency level (chi=2.62, df=1, P=0.106, OR=1.71). None of the single-nucleotide polymorphisms analyzed in the Homer2 gene associates with cocaine dependence. CONCLUSIONS: The results of this study suggest that a polymorphism in the Homer1 gene, rs6871510, is a potential risk factor for the development of cocaine dependence in an African American population, whereas polymorphisms in the Homer2 gene are not.

Association analysis between polymorphisms in the myo-inositol monophosphatase 2 (IMPA2) gene and bipolar disorder.

Linkage studies in bipolar disorder (BPD) suggest that a susceptibility locus exists on chromosome 18p11. The myo-inositol monophosphatase 2 gene (IMPA2) maps to this genomic region. Myo-inositol monophosphatase dephosphorylates inositol monophosphate, regenerating free inositol. Lithium, a common treatment for BPD, has been shown to inhibit IMPA2 activity and decrease levels of inositol. It is hypothesized that lithium conveys its therapeutic effect for BPD patients partially through inositol regulation. Hence, dysfunction of inositol caused by IMPA2 irregularity may contribute to the pathophysiology of BPD. In this study, we hypothesize that genetic variations in the IMPA2 gene contributes to increased susceptibility to BPD. We tested this hypothesis by genotyping 9 SNPs (rs1787984; rs585247; rs3974759; rs650727; rs589247; rs669838; rs636173; rs3786285; rs613993) in BPD patients (n=556) and controls (n=735). Genotype and allele frequencies were compared between groups using Chi square contingency analysis. Linkage disequilibrium (LD) between markers was calculated and estimated haplotype frequencies were compared between groups. Single marker analysis revealed several associations between IMPA2 variations and BPD, which were subsequently rendered non-significant after correction for multiple testing. Although our study did not show strong support for an association between the tested IMPA2 polymorphisms and susceptibility to BPD, additional larger studies are necessary to comprehensively investigate a role of the IMPA2 gene in the pathophysiology of BPD.

Association between polymorphisms in the vesicle-associated membrane protein-associated protein A (VAPA) gene on chromosome 18p and bipolar disorder.

Linkage studies in bipolar disorder (BPD) suggest that a susceptibility locus exists on chromosome 18p11. The vesicle-associated membrane protein-associated protein A (VAPA) gene maps to this region. VAPA interacts with presynaptic proteins and is necessary for vesicular neurotransmission. Dysregulation of synaptic neurotransmission might contribute to the pathophysiology of BPD. In this study, we hypothesize that genetic variations in the VAPA gene contribute to BPD. We tested this hypothesis by genotyping 6 SNPs (rs494015; rs29193; rs29162; rs29145; rs29067; rs29066) in BPD patients (n = 570) and healthy controls (n = 730). Genotype and allele frequencies were compared between groups using Chi square contingency analysis. Linkage disequilibrium (LD) between markers was calculated and estimated haplotype frequencies were compared between groups. Single marker analysis revealed an association of rs29067 and rs29066 with BPD; however, after permutation correction, only rs29066 showed a trend towards an allelic association (P = 0.066). Haplotype analysis did not show any significant association with disease after permutation correction. Our results provide suggestive evidence of an association between SNPs in the 3'UTR of the VAPA gene and BPD. Interestingly, these SNPs are in close proximity to the microsatellite marker D18S464, which showed significant signals in previous linkage studies of BPD. Additional studies are necessary to confirm and elucidate the role of VAPA as a susceptibility gene for BPD on chromosome 18p.

Association analysis of the pituitary adenylate cyclase-activating polypeptide (PACAP/ADCYAP1) gene in bipolar disorder.

BACKGROUND: Linkage studies in bipolar disorder (BPD) suggest that a susceptibility locus exists on chromosome 18p11. The pituitary adenylate cyclase-activating polypeptide/adenylate cyclase-activating polypeptide 1 (pituitary) (PACAP/ADCYAP1) gene maps to this region. PACAP is a neuropeptide involved in neurotransmission in both the peripheral nervous system and central nervous system and is required for catecholamine secretion. Animal models of PACAP mutations show remarkable behavioral defects, including hyperactivity and increased exploratory behavior. OBJECTIVE: In this study we tested the hypothesis that genetic variations in the human PACAP gene contribute to BPD. METHODS: Genotyping of seven single nucleotide polymorphisms (rs1893154; rs2846811; rs8192595; rs2856966; rs928978; rs2231187; rs1610037) was performed in BPD patients (n=570) and healthy controls (n=710). Genotypes and allele frequencies were compared between groups using chi contingency analysis. Linkage disequilibrium between markers was calculated and estimated haplotype frequencies were compared between groups. MAIN RESULTS: There were no significant differences between groups on the allele, genotype or haplotype level for any of the tested single nucleotide polymorphisms. CONCLUSION: Our results provide no evidence of an association of the PACAP gene with BPD in this group of patients and controls. Additional studies are necessary to elucidate the BPD susceptibility locus on chromosome 18p.

Association between the catechol-O-methyltransferase Val158Met polymorphism and cocaine dependence.

Dopaminergic brain systems have been documented to have a major role in drug reward, thus making genes involved in these circuits plausible candidates for susceptibility to substance use disorders. The catechol-O-methyltransferase (COMT) is involved in the degradation of catecholamines and a functional polymorphism (Val158Met) has been suggested to influence enzyme activity. In this study we hypothesize that genetic variation in the COMT gene contributes to increased risk for cocaine dependence. Cocaine-dependent individuals (n=330) and screened unaffected normal controls (n=255) were genotyped for three SNPs in the COMT gene (rs737865, rs4680 (Val158Met), rs165599). All cases and controls were of African descent. Genotype and allele frequencies differed significantly for the Val158Met polymorphism between cases (f(Met)=35%) and controls (f(Met)=27%) (p=0.004; corrected p=0.014; OR 1.44; 95% CI 1.12-1.86). Haplotype analysis showed a significant association for a two-marker haplotype rs737865-Val158Met (p=0.005). Results suggest that variation in COMT increases risk for cocaine dependence. The low enzyme activity 158Met allele or haplotypes containing this variant might have functional effects on dopamine-derived reward processes and cortical functions resulting in increased susceptibility for cocaine dependence. Additional studies are required to elucidate the role of COMT in the pathophysiology of substance use disorders.

Analysis of variations in the tryptophan hydroxylase-2 (TPH2) gene in cocaine dependence.

While the exact physiological mechanisms underlying cocaine dependence remain unclear, a growing body of evidence indicates a role for the serotonergic neurotransmitter system in the pathology of this substance use disorder. The focus of the present study is to determine whether genetic variation in the tryptophan hydroxylase-2 (TPH2) gene, which encodes the enzyme responsible for synthesis of the majority of the serotonin contained in neurons of the central nervous system, contributes to the pathophysiology of cocaine dependence. To examine this hypothesis, we used a case-control study design in which the genotype and allele distributions for six single nucleotide polymorphisms (SNPs) in the TPH2 gene were compared between cocaine-dependent (n = 299) and control individuals (n = 208) of African descent. The results indicate that none of the SNPs in the TPH2 gene examined in this study associate with the cocaine-dependent phenotype. This work suggests that variations in the TPH2 gene are not a risk factor for the development of cocaine dependence, but these findings require confirmation in larger, independent samples of cocaine-dependent and control subjects.

Confirmation of the association between a polymorphism in the promoter region of the prodynorphin gene and cocaine dependence.

The endogenous opioid system has been shown to have a role in the biological processes involved in addiction to numerous drugs of abuse including cocaine. It has recently been reported that the variable nucleotide tandem repeat (VNTR) polymorphism in the 5' promoter region of the prodynorphin gene, which encodes the precursor for three endogenous opioid peptides, is associated with the cocaine dependent phenotype. In order to confirm this finding, we genotyped the prodynorphin promoter polymorphism in cocaine dependent (n = 167) and control (n = 88) individuals of African descent. The results from this experiment indicate a statistically significant (chi2 = 5.64, OR = 1.59, P = 0.018) association between the prodynorphin promoter VNTR polymorphism and the cocaine dependent phenotype. In contrast to previous work showing increased risk conferred by one or two copies of the prodynorphin VNTR, the genotyping results from this study indicate that persons with three or four copies of this polymorphism are more likely to become cocaine dependent. This disparity suggests that the prodynorphin promoter VNTR may not be the functional polymorphism associating with the cocaine dependent phenotype. It is possible that different alleles of the prodynorphin promoter VNTR in the independent populations used for this and the previous study may be in linkage disequilibrium with a yet to be identified functional polymorphism in this gene.