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.

Emergence of a Homo sapiens-specific gene family and chromosome 16p11.2 CNV susceptibility.

Genetic differences that specify unique aspects of human evolution have typically been identified by comparative analyses between the genomes of humans and closely related primates, including more recently the genomes of archaic hominins. Not all regions of the genome, however, are equally amenable to such study. Recurrent copy number variation (CNV) at chromosome 16p11.2 accounts for approximately 1% of cases of autism and is mediated by a complex set of segmental duplications, many of which arose recently during human evolution. Here we reconstruct the evolutionary history of the locus and identify bolA family member 2 (BOLA2) as a gene duplicated exclusively in Homo sapiens. We estimate that a 95-kilobase-pair segment containing BOLA2 duplicated across the critical region approximately 282 thousand years ago (ka), one of the latest among a series of genomic changes that dramatically restructured the locus during hominid evolution. All humans examined carried one or more copies of the duplication, which nearly fixed early in the human lineage--a pattern unlikely to have arisen so rapidly in the absence of selection (P < 0.0097). We show that the duplication of BOLA2 led to a novel, human-specific in-frame fusion transcript and that BOLA2 copy number correlates with both RNA expression (r = 0.36) and protein level (r = 0.65), with the greatest expression difference between human and chimpanzee in experimentally derived stem cells. Analyses of 152 patients carrying a chromosome 16p11. rearrangement show that more than 96% of breakpoints occur within the H. sapiens-specific duplication. In summary, the duplicative transposition of BOLA2 at the root of the H. sapiens lineage about 282 ka simultaneously increased copy number of a gene associated with iron homeostasis and predisposed our species to recurrent rearrangements associated with disease.

De Novo Disruption of the Proteasome Regulatory Subunit PSMD12 Causes a Syndromic Neurodevelopmental Disorder.

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

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.

Disruption of POGZ Is Associated with Intellectual Disability and Autism Spectrum Disorders.

Intellectual disability (ID) and autism spectrum disorders (ASD) are genetically heterogeneous, and a significant number of genes have been associated with both conditions. A few mutations in POGZ have been reported in recent exome studies; however, these studies do not provide detailed clinical information. We collected the clinical and molecular data of 25 individuals with disruptive mutations in POGZ by diagnostic whole-exome, whole-genome, or targeted sequencing of 5,223 individuals with neurodevelopmental disorders (ID primarily) or by targeted resequencing of this locus in 12,041 individuals with ASD and/or ID. The rarity of disruptive mutations among unaffected individuals (2/49,401) highlights the significance (p = 4.19 × 10(-13); odds ratio = 35.8) and penetrance (65.9%) of this genetic subtype with respect to ASD and ID. By studying the entire cohort, we defined common phenotypic features of POGZ individuals, including variable levels of developmental delay (DD) and more severe speech and language delay in comparison to the severity of motor delay and coordination issues. We also identified significant associations with vision problems, microcephaly, hyperactivity, a tendency to obesity, and feeding difficulties. Some features might be explained by the high expression of POGZ, particularly in the cerebellum and pituitary, early in fetal brain development. We conducted parallel studies in Drosophila by inducing conditional knockdown of the POGZ ortholog row, further confirming that dosage of POGZ, specifically in neurons, is essential for normal learning in a habituation paradigm. Combined, the data underscore the pathogenicity of loss-of-function mutations in POGZ and define a POGZ-related phenotype enriched in specific features.

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).

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.

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.

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).

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 (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).

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.

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.

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.

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.

Exploring the heterogeneity of neural social indices for genetically distinct etiologies of autism.

BACKGROUND: Autism spectrum disorder (ASD) is a genetically and phenotypically heterogeneous disorder. Promising initiatives utilizing interdisciplinary characterization of ASD suggest phenotypic subtypes related to specific likely gene-disrupting mutations (LGDMs). However, the role of functionally associated LGDMs in the neural social phenotype is unknown. METHODS: In this study of 26 children with ASD (n = 13 with an LGDM) and 13 control children, we characterized patterns of mu attenuation and habituation as children watched videos containing social and nonsocial motions during electroencephalography acquisition. RESULTS: Diagnostic comparisons were consistent with prior work suggesting aberrant mu attenuation in ASD within the upper mu band (10-12 Hz), but typical patterns within the lower mu band (8-10 Hz). Preliminary exploration indicated distinct social sensitization patterns (i.e., increasing mu attenuation for social motion) for children with an LGDM that is primarily expressed during embryonic development. In contrast, children with an LGDM primarily expressed post-embryonic development exhibited stable typical patterns of lower mu attenuation. Neural social indices were associated with social responsiveness, but not cognition. CONCLUSIONS: These findings suggest unique neurophysiological profiles for certain genetic etiologies of ASD, further clarifying possible genetic functional subtypes of ASD and providing insight into mechanisms for targeted treatment approaches.