Human placental lactogen (human chorionic somatomammotropin) and oxytocin during pregnancy: Individual patterns and associations with maternal-fetal attachment, anxiety, and depression.

Previous studies support links among maternal-fetal attachment, psychological symptoms, and hormones during pregnancy and the post-partum period. Other studies connect maternal feelings and behaviors to oxytocin and suggest that an increase in oxytocin during pregnancy may prime maternal-fetal attachment. To date, researchers have not examined a possible association between maternal-fetal attachment with human placental lactogen although animal models are suggestive. In the current study, we sought to describe oxytocin and human placental lactogen levels as related to psychological constructs across pregnancy. Seventy women participated in the study. At each of three time-points (early, mid, and late pregnancy), the women had their blood drawn to assess oxytocin and human placental lactogen levels, and they completed psychological assessments measuring maternal-fetal attachment, anxiety, and depression. Our results indicate that oxytocin levels were statistically similar across pregnancy, but that human placental lactogen significantly increased across pregnancy. Results did not indicate significant associations of within-person (comparing individuals to themselves) oxytocin or human placental lactogen levels with maternal-fetal attachment. Additionally, results did not show between-person (comparing individuals to other individuals) oxytocin or human placental lactogen levels with maternal-fetal attachment. Oxytocin levels were not associated with anxiety; rather the stage of pregnancy moderated the effect of the within-person OT level on depression. Notably, increasing levels of human placental lactogen were significantly associated with increasing levels of both anxiety and depression in between subject analyses. The current study is important because it describes typical hormonal and maternal fetal attachment levels during each stage of pregnancy, and because it suggests an association between human placental lactogen and psychological symptoms during pregnancy. Future research should further elucidate these relationships.

Targeted sequencing for hereditary breast and ovarian cancer in BRCA1/2-negative families reveals complex genetic architecture and phenocopies.

Approximately 20% of breast cancer cases are attributed to increased family risk, yet variation in BRCA1/2 can only explain 20%-25% of cases. Historically, only single gene or single variant testing were common in at-risk family members, and further sequencing studies were rarely offered after negative results. In this study, we applied an efficient and inexpensive targeted sequencing approach to provide molecular diagnoses in 245 human samples representing 134 BRCA mutation-negative (BRCAX) hereditary breast and ovarian cancer (HBOC) families recruited from 1973 to 2019 by Dr. Henry Lynch. Sequencing identified 391 variants, which were functionally annotated and ranked based on their predicted clinical impact. Known pathogenic CHEK2 breast cancer variants were identified in five BRCAX families in this study. While BRCAX was an inclusion criterion for this study, we still identified a pathogenic BRCA2 variant (p.Met192ValfsTer13) in one family. A portion of BRCAX families could be explained by other hereditary cancer syndromes that increase HBOC risk: Li-Fraumeni syndrome (gene: TP53) and Lynch syndrome (gene: MSH6). Interestingly, many families carried additional variants of undetermined significance (VOUSs) that may further modify phenotypes of syndromic family members. Ten families carried more than one potential VOUS, suggesting the presence of complex multi-variant families. Overall, nine BRCAX HBOC families in our study may be explained by known likely pathogenic/pathogenic variants, and six families carried potential VOUSs, which require further functional testing. To address this, we developed a functional assay where we successfully re-classified one family's PMS2 VOUS as benign.

In vitro data suggest a role for PMS2 Kozak sequence mutations in Lynch syndrome risk.

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Heterozygous loss-of-function variants in PMS2 are linked to LS. While these variants are not directly cancer causing, reduced PMS2 function results in the accumulation of somatic variants and increased cancer risk over time due to DNA mismatch repair dysfunction. It is reasonable that other types of genetic variation that impact the expression of PMS2 may also contribute to cancer risk. The Kozak sequence is a highly conserved translation initiation motif among higher eukaryotes and is defined as the nine base pairs upstream of the translation start codon through the first four bases of the translated sequence (5'-[GTT]GCATCCATGG-3'; human PMS2: NM_000535.7). While Kozak sequence variants in PMS2 have been reported in ClinVar in patients with suspected hereditary cancer, all variants upstream of the translation start site are currently classified as variants of undetermined significance (VUSs). We hypothesized that variants significantly disrupting the Kozak sequence of PMS2 would decrease PMS2 protein expression, contributing to increased cancer risk over time. Using a dual-luciferase reporter plasmid and site-directed mutagenesis, we generated the wild-type human PMS2 and the ClinVar VUSs within the PMS2 Kozak sequence. Besides the c.1A>C variant, which is already known to be pathogenic, we implicate six additional variants as American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) pathogenic supporting (PP) variants and classify ten as benign supporting (BP). In summary, we present a method developed for the classification of human PMS2 Kozak sequence variants that can contribute to the re-classification of VUSs identified in patients.

Complete sequence of the closed circular extrachromosomal element of Naegleria pringsheimi De Jonckheere (strain Singh).

The DNA encoding the ribosomal RNA in Naegleria is encoded on closed circular extrachromosomal ribosomal DNA-containing elements (CERE) in the nucleolus. In this report, we describe the sequence of the CERE of Naegleria pringsheimi De Jonckheere (strain Singh).

Complete sequence of the closed circular extrachromosomal element (CERE) of Naegleria australiensis De Jonckheere (strain PP 397).

Ribosomal RNA is not encoded in chromosomal DNA in amoebae of the Naegleria genus but the rRNA genes are located on closed circular extrachromosomal ribosomal DNA (rDNA)-containing elements (CERE). In this report, we describe the sequence of the CERE of Naegleria australiensis De Jonckheere (strain PP397).

GluN2D Subunit in Parvalbumin Interneurons Regulates Prefrontal Cortex Feedforward Inhibitory Circuit and Molecular Networks Relevant to Schizophrenia.

BACKGROUND: Parvalbumin interneuron (PVI) activity synchronizes the medial prefrontal cortex circuit for normal cognitive function, and its impairment may contribute to schizophrenia (SZ). NMDA receptors in PVIs participate in these activities and form the basis for the NMDA receptor hypofunction hypothesis of SZ. However, the role of the GluN2D subunit, which is enriched in PVIs, in regulating molecular networks relevant to SZ is unknown. METHODS: Using electrophysiology and a mouse model with conditional deletion of GluN2D from PVIs (PV-GluN2D knockout [KO]), we examined the cell excitability and neurotransmission in the medial prefrontal cortex. Histochemical, RNA sequencing analysis and immunoblotting were conducted to understand molecular mechanisms. Behavioral analysis was conducted to test cognitive function. RESULTS: PVIs in the medial prefrontal cortex were found to express putative GluN1/2B/2D receptors. In a PV-GluN2D KO model, PVIs were hypoexcitable, whereas pyramidal neurons were hyperexcitable. Excitatory neurotransmission was higher in both cell types in PV-GluN2D KO, whereas inhibitory neurotransmission showed contrasting changes, which could be explained by reduced somatostatin interneuron projections and increased PVI projections. Genes associated with GABA (gamma-aminobutyric acid) synthesis, vesicular release, and uptake as well as those involved in formation of inhibitory synapses, specifically GluD1-Cbln4 and Nlgn2, and regulation of dopamine terminals were downregulated in PV-GluN2D KO. SZ susceptibility genes including Disc1, Nrg1, and ErbB4 and their downstream targets were also downregulated. Behaviorally, PV-GluN2D KO mice showed hyperactivity and anxiety behavior and deficits in short-term memory and cognitive flexibility. CONCLUSIONS: These findings demonstrate that GluN2D in PVIs serves as a point of convergence of pathways involved in the regulation of GABAergic synapses relevant to SZ.

Complete Sequence of the Closed Circular Extrachromosomal Element of Naegleria jadini Willaert and Ray (Strain ITMAP400).

Amoebae of the Naegleria genus carry all ribosome-encoding DNA on closed circular extrachromosomal elements (CERE). We report the sequence of the CERE of Naegleria jadini (strain Willaert and Ray).

KMT5B is required for early motor development.

Disruptive variants in lysine methyl transferase 5B (KMT5B/SUV4-20H1) have been identified as likely-pathogenic among humans with neurodevelopmental phenotypes including motor deficits (i.e., hypotonia and motor delay). However, the role that this enzyme plays in early motor development is largely unknown. Using a Kmt5b gene trap mouse model, we assessed neuromuscular strength, skeletal muscle weight (i.e., muscle mass), neuromuscular junction (NMJ) structure, and myofiber type, size, and distribution. Tests were performed over developmental time (postnatal days 17 and 44) to represent postnatal versus adult structures in slow- and fast-twitch muscle types. Prior to the onset of puberty, slow-twitch muscle weight was significantly reduced in heterozygous compared to wild-type males but not females. At the young adult stage, we identified decreased neuromuscular strength, decreased skeletal muscle weights (both slow- and fast-twitch), increased NMJ fragmentation (in slow-twitch muscle), and smaller myofibers in both sexes. We conclude that Kmt5b haploinsufficiency results in a skeletal muscle developmental deficit causing reduced muscle mass and body weight.

Epidemiologic and Genomic Analysis of the Severe Acute Respiratory Syndrome Coronavirus 2 Epidemic in the Nebraska Region of the United States, March 2020-2021.

COVID-19 emerged at varying intervals in different regions of the United States in 2020. This report details the epidemiologic and genetic evolution of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the first year of the epidemic in the state of Nebraska using data collected from the Creighton Catholic Health Initiatives (CHI) health system. Statistical modelling identified age, gender, and previous history of diabetes and/or stroke as significant risk factors associated with mortality in COVID-19 patients. In parallel, the viral genomes of over 1,000 samples were sequenced. The overall rate of viral variation in the population was 0.07 mutations/day. Genetically, the first 9 months of the outbreak, which include the initial outbreak, a small surge in August and a major outbreak in November 2020 were primarily characterized by B.1. lineage viruses. In early 2021, the United Kingdom variant (B.1.1.7 or alpha) quickly became the dominant variant. Notably, surveillance of non-consensus variants detected B.1.1.7 defining mutations months earlier in Fall 2020. This work provides insights into the regional variance and evolution of SARS-CoV-2 in the Nebraska region during the first year of the pandemic.

Differential effects by sex with Kmt5b loss.

Lysine methyl transferase 5B (KMT5B) has been recently highlighted as a risk gene in genetic studies of neurodevelopmental disorders (NDDs), specifically, autism spectrum disorder (ASD) and intellectual disability (ID); yet, its role in the brain is not known. The goal of this work was to neurodevelopmentally characterize the effect(s) of KMT5B haploinsufficiency using a mouse model. A Kmt5b gene-trap mouse line was obtained from the Knockout Mouse Project. Wild type (WT) and heterozygous (HET) mice were subjected to a comprehensive neurodevelopmental test battery to assess reflexes, motor behavior, learning/memory, social behavior, repetitive movement, and common ASD comorbidities (obsessive compulsion, depression, and anxiety). Given the strong sex bias observed in the ASD patient population, we tested both a male and female cohort of animals and compared differences between genotypes and sexes. HET mice were significantly smaller than WT littermates starting at postnatal day 10 through young adulthood which was correlated with smaller brain size (i.e., microcephaly). This was more severe in males than females. HET male neonates also had delayed eye opening and significantly weaker reflexes than WT littermates. In young adults, significant differences between genotypes relative to anxiety, depression, fear, and extinction learning were observed. Interestingly, several sexually dimorphic differences were noted including increased repetitive grooming behavior in HET females and an increased latency to hot plate response in HET females versus a decreased latency in HET males. LAY SUMMARY: Lysine methyl transferase 5B (KMT5B) has been recently highlighted as a risk gene in neurodevelopmental disorders (NDDs), specifically, autism spectrum disorder (ASD) and intellectual disability (ID); yet its role in the brain is not known. Our study indicates that mice lacking one genomic copy of Kmt5b show deficits in neonatal reflexes, sociability, repetitive stress-induced grooming, changes in thermal pain sensing, decreased depression and anxiety, increased fear, slower extinction learning, and lower body weight, length, and brain size. Furthermore, several outcomes differed by sex, perhaps mirroring the sex bias in ASD.

ATF3 Coordinates Antitumor Synergy between Epigenetic Drugs and Protein Disulfide Isomerase Inhibitors.

Histone deacetylase inhibitors (HDACi) are largely ineffective in the treatment of solid tumors. In this study, we describe a new class of protein disulfide isomerase (PDI) inhibitors that significantly and synergistically enhance the antitumor activity of HDACi in glioblastoma and pancreatic cancer preclinical models. RNA-sequencing screening coupled with gene silencing studies identified ATF3 as the driver of this antitumor synergy. ATF3 was highly induced by combined PDI and HDACi treatment as a result of increased acetylation of key histone lysine residues (acetylated histone 3 lysine 27 and histone 3 lysine 18) flanking the ATF3 promoter region. These chromatin marks were associated with increased RNA polymerase II recruitment to the ATF3 promoter, a synergistic upregulation of ATF3, and a subsequent apoptotic response in cancer cells. The HSP40/HSP70 family genes DNAJB1 and HSPA6 were found to be critical ATF3-dependent genes that elicited the antitumor response after PDI and HDAC inhibition. In summary, this study presents a synergistic antitumor combination of PDI and HDAC inhibitors and demonstrates a mechanistic and tumor suppressive role of ATF3. Combined treatment with PDI and HDACi offers a dual therapeutic strategy in solid tumors and the opportunity to achieve previously unrealized activity of HDACi in oncology. SIGNIFICANCE: This study uses a first-in-class PDI inhibitor entering clinical development to enhance the effects of epigenetic drugs in some of the deadliest forms of cancer.

Bovine HDL and Dual Domain HDL-Mimetic Peptides Inhibit Tumor Development in Mice.

A growing body of literature supports the role of apolipoproteins present in HDL in the treatment of pro-inflammatory diseases including cancer. We examined whether bovine HDL (bHDL) and three dual-domain peptides, namely AEM-28 and its analog AEM-28-2, and HM-10/10, affect tumor growth and development in mouse models of ovarian and colon cancer. We demonstrate that bHDL inhibits mouse colorectal cancer cell line CT26-mediated lung tumor development, and mouse ovarian cancer cell line ID8-mediated tumor burden. We also demonstrate that, although to different degrees, dual-domain peptides inhibit cell viability of mouse and human ovarian and colon cancer cell lines, but not that of normal human colonic epithelial cells or NIH3T3 mouse fibroblasts. Dual-domain peptides administered subcutaneously or in a chow diet decrease CT26 cell-mediated tumor burden, tumor growth, and tumor dissemination in BALB/c mice. Plasma levels of lysophosphatidic acid (LPA) are significantly reduced in mice that received bHDL and the dual-domain peptides, suggesting that reduction by effecting accumulation and/or synthesis of pro-inflammatory lipids may be one of the mechanisms for the inhibition of tumor development by bHDL and the dual-domain peptides. Our studies suggest that therapeutics based on apolipoproteins present in HDL may be novel agents for the treatment of epithelial adenocarcinomas of the ovary and colon.

Endometriosis: A Malignant Fingerprint.

BACKGROUND: Endometriosis is complex, but identifying the novel biomarkers, inflammatory molecules, and genetic links holds the key to the enhanced detection, prediction and treatment of both endometriosis and endometriosis related malignant neoplasia. Here we review the literature relating to the specific molecular mechanism(s) mediating tumorigenesis arising within endometriosis. METHODS: Guidance (e.g. Cochrane) and published studies were identified. The Published studies were identified through PubMed using the systematic review methods filter, and the authors' topic knowledge. These data were reviewed to identify key and relevant articles to create a comprehensive review article to explore the molecular fingerprint associated with in endometriosis-driven tumorigenesis. RESULTS: An important focus is the link between C3aR1, PGR, ER1, SOX-17 and other relevant gene expression profiles and endometriosis-driven tumorigenesis. Further studies should also focus on the combined use of CA-125 with HE-4, and the role for OVA1/MIA as clinically relevant diagnostic biomarkers in the prediction of endometriosis-driven tumorigenesis. CONCLUSIONS: Elucidating endometriosis' molecular fingerprint is to understand the molecular mechanisms that drive the endometriosis-associated malignant phenotype. A better understanding of the predictive roles of these genes and the value of the biomarker proteins will allow for the derivation of unique molecular treatment algorithms to better serve our patients.

Phenotypic and biochemical analysis of an international cohort of individuals with variants in NAA10 and NAA15.

N-alpha-acetylation is one of the most common co-translational protein modifications in humans and is essential for normal cell function. NAA10 encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. The auxiliary and regulatory subunits of the NatA complex are NAA15 and Huntington-interacting protein (HYPK), respectively. Through a genotype-first approach with exome sequencing, we identified and phenotypically characterized 30 individuals from 30 unrelated families with 17 different de novo or inherited, dominantly acting missense variants in NAA10 or NAA15. Clinical features of affected individuals include variable levels of intellectual disability, delayed speech and motor milestones and autism spectrum disorder. Additionally, some subjects present with mild craniofacial dysmorphology, congenital cardiac anomalies and seizures. One of the individuals is an 11-year-old boy with a frameshift variant in exon 7 of NAA10, who presents most notably with microphthalmia, which confirms a prior finding with a single family with Lenz microphthalmia syndrome. Biochemical analyses of variants as part of the human NatA complex, as well as enzymatic analyses with and without the HYPK regulatory subunit, help to explain some of the phenotypic differences seen among the different variants.

Histone 4 Lysine 20 Methylation: A Case for Neurodevelopmental Disease.

Neurogenesis is an elegantly coordinated developmental process that must maintain a careful balance of proliferation and differentiation programs to be compatible with life. Due to the fine-tuning required for these processes, epigenetic mechanisms (e.g., DNA methylation and histone modifications) are employed, in addition to changes in mRNA transcription, to regulate gene expression. The purpose of this review is to highlight what we currently know about histone 4 lysine 20 (H4K20) methylation and its role in the developing brain. Utilizing publicly-available RNA-Sequencing data and published literature, we highlight the versatility of H4K20 methyl modifications in mediating diverse cellular events from gene silencing/chromatin compaction to DNA double-stranded break repair. From large-scale human DNA sequencing studies, we further propose that the lysine methyltransferase gene, KMT5B (OMIM: 610881), may fit into a category of epigenetic modifier genes that are critical for typical neurodevelopment, such as EHMT1 and ARID1B, which are associated with Kleefstra syndrome (OMIM: 610253) and Coffin-Siris syndrome (OMIM: 135900), respectively. Based on our current knowledge of the H4K20 methyl modification, we discuss emerging themes and interesting questions on how this histone modification, and particularly KMT5B expression, might impact neurodevelopment along with current challenges and potential avenues for future research.

Neurodevelopmental disease genes implicated by de novo mutation and copy number variation morbidity.

We combined de novo mutation (DNM) data from 10,927 individuals with developmental delay and autism to identify 253 candidate neurodevelopmental disease genes with an excess of missense and/or likely gene-disruptive (LGD) mutations. Of these genes, 124 reach exome-wide significance (P < 5 × 10-7) for DNM. Intersecting these results with copy number variation (CNV) morbidity data shows an enrichment for genomic disorder regions (30/253, likelihood ratio (LR) +1.85, P = 0.0017). We identify genes with an excess of missense DNMs overlapping deletion syndromes (for example, KIF1A and the 2q37 deletion) as well as duplication syndromes, such as recurrent MAPK3 missense mutations within the chromosome 16p11.2 duplication, recurrent CHD4 missense DNMs in the 12p13 duplication region, and recurrent WDFY4 missense DNMs in the 10q11.23 duplication region. Network analyses of genes showing an excess of DNMs highlights functional networks, including cell-specific enrichments in the D1+ and D2+ spiny neurons of the striatum.

Associations between Familial Rates of Psychiatric Disorders and De Novo Genetic Mutations in Autism.

The purpose of this study was to examine the confluence of genetic and familial risk factors in children with Autism Spectrum Disorder (ASD) with distinct de novo genetic events. We hypothesized that gene-disrupting mutations would be associated with reduced rates of familial psychiatric disorders relative to structural mutations. Participants included families of children with ASD in four groups: de novo duplication copy number variations (DUP, n = 62), de novo deletion copy number variations (DEL, n = 74), de novo likely gene-disrupting mutations (LGDM, n = 267), and children without a known genetic etiology (NON, n = 2111). Familial rates of psychiatric disorders were calculated from semistructured interviews. Results indicated overall increased rates of psychiatric disorders in DUP families compared to DEL and LGDM families, specific to paternal psychiatric histories, and particularly evident for depressive disorders. Higher rates of depressive disorders in maternal psychiatric histories were observed overall compared to paternal histories and higher rates of anxiety disorders were observed in paternal histories for LGDM families compared to DUP families. These findings support the notion of an additive contribution of genetic etiology and familial factors are associated with ASD risk and highlight critical need for continued work targeting these relationships.

De Novo Mutations in Protein Kinase Genes CAMK2A and CAMK2B Cause Intellectual Disability.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

The evolution and population diversity of human-specific segmental duplications.

Segmental duplications contribute to human evolution, adaptation and genomic instability but are often poorly characterized. We investigate the evolution, genetic variation and coding potential of human-specific segmental duplications (HSDs). We identify 218 HSDs based on analysis of 322 deeply sequenced archaic and contemporary hominid genomes. We sequence 550 human and nonhuman primate genomic clones to reconstruct the evolution of the largest, most complex regions with protein-coding potential (n=80 genes/33 gene families). We show that HSDs are non-randomly organized, associate preferentially with ancestral ape duplications termed "core duplicons", and evolved primarily in an interspersed inverted orientation. In addition to Homo sapiens-specific gene expansions (e.g., TCAF1/2), we highlight ten gene families (e.g., ARHGAP11B and SRGAP2C) where copy number never returns to the ancestral state, there is evidence of mRNA splicing, and no common gene-disruptive mutations are observed in the general population. Such duplicates are candidates for the evolution of human-specific adaptive traits.