Recurrent de novo missense variants across multiple histone H4 genes underlie a neurodevelopmental syndrome.

Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.

Exome Sequencing for Prenatal Diagnosis in Nonimmune Hydrops Fetalis.

BACKGROUND: The cause of most fetal anomalies is not determined prenatally. Exome sequencing has transformed genetic diagnosis after birth, but its usefulness for prenatal diagnosis is still emerging. Nonimmune hydrops fetalis (NIHF), a fetal abnormality that is often lethal, has numerous genetic causes; the extent to which exome sequencing can aid in its diagnosis is unclear. METHODS: We evaluated a series of 127 consecutive unexplained cases of NIHF that were defined by the presence of fetal ascites, pleural or pericardial effusions, skin edema, cystic hygroma, increased nuchal translucency, or a combination of these conditions. The primary outcome was the diagnostic yield of exome sequencing for detecting genetic variants that were classified as either pathogenic or likely pathogenic according to the criteria of the American College of Medical Genetics and Genomics. Secondary outcomes were the percentage of cases associated with specific genetic disorders and the proportion of variants that were inherited. RESULTS: In 37 of the 127 cases (29%), we identified diagnostic genetic variants, including those for disorders affecting the RAS-MAPK cell-signaling pathway (known as RASopathies) (30% of the genetic diagnoses); inborn errors of metabolism and musculoskeletal disorders (11% each); lymphatic, neurodevelopmental, cardiovascular, and hematologic disorders (8% each); and others. Prognoses ranged from a relatively mild outcome to death during the perinatal period. Overall, 68% of the cases (25 of 37) with diagnostic variants were autosomal dominant (of which 12% were inherited and 88% were de novo), 27% (10 of 37) were autosomal recessive (of which 95% were inherited and 5% were de novo), 1 was inherited X-linked recessive, and 1 was of uncertain inheritance. We identified potentially diagnostic variants in an additional 12 cases. CONCLUSIONS: In this large case series of 127 fetuses with unexplained NIHF, we identified a diagnostic genetic variant in approximately one third of the cases. (Funded by the UCSF Center for Maternal-Fetal Precision Medicine and others; ClinicalTrials.gov number, NCT03412760.).

Further description of two patients with biallelic variants in NADSYN1 in association with cardiac and vertebral anomalies.

Congenital nicotinamide adenine dinucleotide (NAD) deficiency disorders are associated with pathogenic variants in the genes NADSYN1, HAAO, and KYNU. These disorders overlap with the anomalies present in vertebral, anal, cardiac, tracheoesophageal, radial and renal, and limb anomalies (VATER/VACTERL) association and often result in premature death. Children who survive typically have developmental delays or intellectual disability. Here, we describe two patients with compound heterozygous variants in NADSYN1 who presented with cardiac and vertebral defects overlapping with the VATER/VACTERL association, although the patients did not satisfy criteria for the diagnosis of VATER/VACTERL due to their lack of limb anomalies and significant renal anomalies. One patient survived into childhood with developmental delays and may represent an expansion of the survival data for NADSYN1-associated NAD deficiency disorders. Interestingly, one patient had hypoplastic left heart syndrome (HLHS) and one had an aortic coarctation and transverse hypoplasia of the aortic arch, suggesting that NADSYN1 sequencing should be performed in children presenting with congenital anomalies related to VATER/VACTERL association and with HLHS and aortic arch abnormalities.

The utility of pathologic examination and comprehensive phenotyping for accurate diagnosis with perinatal exome sequencing.

OBJECTIVE: Exome sequencing (ES) offers the ability to assess for variants in thousands of genes and is particularly useful in the setting of fetal anomalies. However, the ES pipeline relies on a thorough understanding of an individual patient's phenotype, which may be limited in the prenatal setting. Additional pathology evaluations in the pre- and postnatal settings can add phenotypic details important for clearly establishing and characterizing a diagnosis. METHODS: This is a case series of prenatal ES performed at our institution in which pathology evaluations, including autopsy, dysmorphology examination, histology, and peripheral blood smear, augmented the understanding of the fetal phenotype. ES was performed at our institution and a multidisciplinary panel reviewed and classified the variants for each case. RESULTS: We present four cases wherein pathology evaluations were beneficial for supporting a perinatal diagnosis identified with ES. In each of these cases, pathology findings provided additional data to support a more complete understanding of the relationship between the perinatal phenotype and variants identified with ES. CONCLUSION: These cases highlight challenges of perinatal ES related to incomplete prenatal phenotyping, demonstrate the utility of pathology evaluations to support diagnoses identified with ES, and further characterize the disease manifestations of specific genetic variants.

De novo variants in SNAP25 cause an early-onset developmental and epileptic encephalopathy.

PURPOSE: This study aims to provide a comprehensive description of the phenotypic and genotypic spectrum of SNAP25 developmental and epileptic encephalopathy (SNAP25-DEE) by reviewing newly identified and previously reported individuals. METHODS: Individuals harboring heterozygous missense or loss-of-function variants in SNAP25 were assembled through collaboration with international colleagues, matchmaking platforms, and literature review. For each individual, detailed phenotyping, classification, and structural modeling of the identified variant were performed. RESULTS: The cohort comprises 23 individuals with pathogenic or likely pathogenic de novo variants in SNAP25. Intellectual disability and early-onset epilepsy were identified as the core symptoms of SNAP25-DEE, with recurrent findings of movement disorders, cerebral visual impairment, and brain atrophy. Structural modeling for all variants predicted possible functional defects concerning SNAP25 or impaired interaction with other components of the SNARE complex. CONCLUSION: We provide a comprehensive description of SNAP25-DEE with intellectual disability and early-onset epilepsy mostly occurring before the age of two years. These core symptoms and additional recurrent phenotypes show an overlap to genes encoding other components or associated proteins of the SNARE complex such as STX1B, STXBP1, or VAMP2. Thus, these findings advance the concept of a group of neurodevelopmental disorders that may be termed "SNAREopathies."

Segmental overgrowth and aneurysms due to mosaic PDGFRB p.(Tyr562Cys).

Activating variants in the platelet-derived growth factor receptor ß gene (PDGFRB) have been associated with Kosaki overgrowth syndrome, infantile myofibromatosis, and Penttinen premature aging syndrome. A recently described phenotype with fusiform aneurysm has been associated with mosaic PDGFRB c.1685A > G p.(Tyr562Cys) variant. Few reports however have examined the vascular phenotypes and mosaic effects of PDGFRB variants. We describe clinical characteristics of two patients with a recurrent mosaic PDGFRB p.(Tyr562Cys) variant identified via next-generation sequencing-based genetic testing. We observed intracranial fusiform aneurysm in one patient and found an additional eight patients with aneurysms and phenotypes associated with PDGFRB-activating variants through literature search. The conditions caused by PDGFRB-activating variants share overlapping features including overgrowth, premature aged skin, and vascular malformations including aneurysms. Aneurysms are progressive and can result in morbidities and mortalities in the absence of successful intervention. Germline and/or somatic testing for PDGFRB gene should be obtained when PDGFRB activating variant-related phenotypes are present. Whole-body imaging of the arterial tree and echocardiography are recommended after diagnosis. Repeating the imaging study within a 6- to 12-month period after detection is reasonable. Finally, further evaluation for the effectiveness and safety profile of kinase inhibitors in this patient population is warranted.

Pediatric bithalamic gliomas have a distinct epigenetic signature and frequent EGFR exon 20 insertions resulting in potential sensitivity to targeted kinase inhibition.

Brain tumors are the most common solid tumors of childhood, and the genetic drivers and optimal therapeutic strategies for many of the different subtypes remain unknown. Here, we identify that bithalamic gliomas harbor frequent mutations in the EGFR oncogene, only rare histone H3 mutation (in contrast to their unilateral counterparts), and a distinct genome-wide DNA methylation profile compared to all other glioma subtypes studied to date. These EGFR mutations are either small in-frame insertions within exon 20 (intracellular tyrosine kinase domain) or missense mutations within exon 7 (extracellular ligand-binding domain) that occur in the absence of accompanying gene amplification. We find these EGFR mutations are oncogenic in primary astrocyte models and confer sensitivity to specific tyrosine kinase inhibitors dependent on location within the kinase domain or extracellular domain. We initiated treatment with targeted kinase inhibitors in four children whose tumors harbor EGFR mutations with encouraging results. This study identifies a promising genomically-tailored therapeutic strategy for bithalamic gliomas, a lethal and genetically distinct brain tumor of childhood.

Application of tail vein serial microsampling for plasma or dried plasma spots in toxicokinetic assessment in rats using acetaminophen as the model compound.

In the current study, two groups of rats (five per group) were administered a single oral dose of 500 mg/kg acetaminophen. For toxicokinetic assessment, the Group 1 animals were bled via conventional sparse (two animals/time point) sublingual vein bleeding (~0.5 ml) with anesthesia, while the Group 2 animals were bled via serial tail vein microsampling (~0.075 ml) without anesthesia. All collected blood was processed for plasma. Each Group 2 plasma sample (~30 µl) was divided into 'wet' and 'dried' (dried plasma spots). All plasma samples were analyzed by LC-MS/MS for acetaminophen and its major metabolites acetaminophen glucuronide and acetaminophen sulfate. In addition, plasma and urine samples were collected for analysis of corticosterone and creatinine to assess stress levels. Comparable plasma exposure to acetaminophen and its two metabolites was observed in the plasma obtained via conventional sparse sublingual vein bleeding and serial tail vein microsampling and between the 'wet' and 'dried' plasma obtained by the latter. Furthermore, comparable corticosterone levels or corticosterone/creatinine ratios between the two groups suggested that serial microsampling without anesthesia did not increase the levels of stress as compared with conventional sampling with anesthesia, confirming the utility of microsampling for plasma or dried plasma spots in rodent toxicokinetic assessment.

Induction of hemangiosarcoma in mice after chronic treatment with S1P-modulator siponimod and its lack of relevance to rat and human.

A high incidence of hemangiosarcoma (HSA) was observed in mice treated for 2 years with siponimod, a sphingosine-1-phosphate receptor 1 (S1P1) functional antagonist, while no such tumors were observed in rats under the same treatment conditions. In 3-month rat (90 mg/kg/day) and 9-month mouse (25 and 75 mg/kg/day) in vivo mechanistic studies, vascular endothelial cell (VEC) activation was observed in both species, but VEC proliferation and persistent increases in circulating placental growth factor 2 (PLGF2) were only seen in the mouse. In mice, these effects were sustained over the 9-month study duration, while in rats increased mitotic gene expression was present at day 3 only and PLGF2 was induced only during the first week of treatment. In the mouse, the persistent VEC activation, mitosis induction, and PLGF2 stimulation likely led to sustained neo-angiogenesis which over life-long treatment may result in HSA formation. In rats, despite sustained VEC activation, the transient mitotic and PLGF2 stimuli did not result in the formation of HSA. In vitro, the mouse and rat primary endothelial cell cultures mirrored their respective in vivo findings for cell proliferation and PLGF2 release. Human VECs, like rat cells, were unresponsive to siponimod treatment with no proliferative response and no release of PLGF2 at all tested concentrations. Hence, it is suggested that the human cells also reproduce a lack of in vivo response to siponimod. In conclusion, the molecular mechanisms leading to siponimod-induced HSA in mice are considered species specific and likely irrelevant to humans.

Correlation Between Nasal Epithelial Injury and In Vitro Cytotoxicity Using a Series of Small Molecule Protein Tyrosine Phosphatase 1B Inhibitors Investigated for Reversal of Leptin Resistance in Obesity.

This research provides a cautionary example when evaluating changes in behavioral end points with respect to postulated pharmacologic activity. Various small molecule substrate mimetic protein tyrosine phosphatase 1B (PTP1B) inhibitors were investigated as pharmacologic agents for decreasing food consumption using intranasal (IN) dosing as a means for direct nose-to-brain delivery along the olfactory/trigeminal nerve pathways. Although food consumption was decreased in diet-induced obese (DIO) mice, nasal discharge was observed. Studies were conducted to investigate local effects on the nasal airway and to develop structure-activity relationships. Intranasal administration of PTP1B inhibitors at ≥0.03 mg/d to DIO mice produced dose-dependent injury to various cell types of the nasal epithelia. Protein tyrosine phosphatase 1B inhibitors with calculated log octanol >3.0 were the most toxic. Whereas a pharmacologically inactive analog of a PTP1B inhibitor produced nasal injury, along with decreased food consumption, the marketed IN drug ketorolac produced no lesions at the same dose of 0.3 mg/d and only minor changes at 3 mg/d. Rat skin fibroblast cells were exposed in vitro to PTP1B inhibitors, ketorolac, paraquat, and the detergent sodium dodecylbenzene sulfonate (NDS) followed by measures of cytotoxicity. The most potent PTP1B inhibitors were similar to NDS, whereas ketorolac was the least toxic compound. Cytotoxic potency in vitro was similar to in vivo. In conclusion, PTP1B inhibitors injured nasal epithelium through a mechanism independent of PTP1B inhibition and likely due to nonspecific cytotoxicity such as disruption of the cell membrane. Decreased food consumption in DIO mice was due to toxicity rather than a pharmacologic mode of action.

Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity.

The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4 days before being exposed once to PM (10 or 30 µM). Following eight additional days in culture, relative to control (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P<0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60 µM) exposure destroyed all stage follicles after 4 days (P<0.05). VC from nearby wells depleted primordial follicles after 4 days (P<0.05), temporarily reduced secondary follicle number after 2 days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors.

Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries.

Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75 nM) and has not (12.5 nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12 nM; low-concentration or 75 nM; high-concentration). After four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4 days of exposure, but induced large primary follicle loss at both concentrations after 8 days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4 days after exposure. With the exception of Atg7 and cKit, DMBA increased (P < 0.05) expression of all genes investigated. Also, BECN1 and pAKT(Thr308) protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures.

Comparison of coronary CT angiography image quality with and without breast shields.

OBJECTIVE: The purpose of this study is to compare the image quality of coronary CT angiography performed with and without breast shields. MATERIALS AND METHODS: This study involved a retrospective cohort of 72 women with possible angina who underwent 64-MDCT retrospective ECG-gated coronary CT angiography at a single academic tertiary medical center. Images of 36 women scanned while wearing bismuth-coated latex breast shields and 36 control subjects scanned without shields, matched by heart rate and body mass index, were graded on a standardized Likert scale for image quality, stenosis, and plaque by two independent board-certified readers blinded to breast shields. RESULTS: Seventy-two patients (mean [± SD] age, 53 ± 9 years) were included. The pre scan heart rate, body mass index, and Agatston score did not differ between groups. The median estimated radiation dose was 13.4 versus 16.1 mSv for those with and without breast shields (p = 0.003). For shielded versus unshielded scans, 86% versus 83% of coronary segments were rated excellent or above average (p = 0.4), median image quality was 2.0 for both groups, mean signal was 474 ± 75 and 452 ± 91 HU (p = 0.27), mean noise was 33.9 ± 8.5 and 29.8 ± 8.3 HU (p = 0.04), and median signal-to-noise ratio was 14.4 and 14.7 (p = 0.56), respectively. CONCLUSION: Breast shields for women undergoing coronary CT angiography slightly increased noise but did not negatively affect signal, signal-to-noise ratio, quality, or interpretability. Breast shield use warrants further study.

TFG::ALK fusion in ALK positive large B-cell lymphoma: a case report and review of literature.

Anaplastic lymphoma kinase (ALK) positive large B-cell lymphoma (ALK+ LBCL) is an aggressive and rare subtype of B-cell lymphoma. Patients typically present with advanced clinical stage disease and do not respond to conventional chemotherapy; the median overall survival is 1.8 years. The genetic landscape of this entity remains poorly understood. Here we report a unique case of ALK+ LBCL harbouring a rare TFG::ALK fusion. Targeted next-generation sequencing showed no significant single nucleotide variants, insertions/deletions, or other structural variants beyond the TFG::ALK fusion; deep deletions of FOXO1, PRKCA, and the MYB locus were also detected. Our case report draws attention to this rare disease, highlights a need for larger genetic profiling studies, and focuses on the pathogenesis and potential therapeutic targets of this aggressive disease. To our knowledge, this is the first report of a TFG::ALK fusion in ALK+ LBCL.

HER2 IHC Expression and Gene Amplification in p53-aberrant High-grade Endometrial Endometrioid Carcinoma Suggests That This Population May Benefit From HER2 Testing and Targeted Therapy.

Among gynecologic cancers, uterine serous carcinoma (USC) has been shown to be human epidermal growth factor receptor 2 (HER2) amplified and trastuzumab has been included in the recent National Comprehensive Cancer Network (NCCN) guidelines for treatment of advanced stage or recurrent USC with HER2 overexpression/amplification. There is limited literature suggesting that a subset of high-grade endometrioid carcinomas with aberrant p53 expression may also be HER2 amplified and these patients could benefit from the addition of targeted therapy. We identified 59 p53-aberrant (mismatch repair proficient) FIGO 3 endometrioid carcinomas of the uterus. HER2 immunohistochemistry was performed in all 59 tumors and HER2 fluorescence in situ hybridization (FISH) was performed in 52 of the 59 cases. Four of the 59 cases were HER2 3+ by immunohistochemistry (6.7%), using the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2007, 2013, and 2018 criteria. HER2 FISH was performed in 3 of the 4 cases and was amplified in all 3. Nine, 8, and 7 tumors showed 2+ HER2 staining when applying 2018, 2013, and 2007 criteria, respectively, FISH was performed in 7 tumors and none were amplified. An additional 4 cases did not perfectly meet the 2018 ASCO/CAP criteria but were assigned a score of 2+, none were amplified by HER2 FISH. The remaining 42 cases showed 1+ or no staining for HER2, FISH was successfully performed in 38 tumors and none showed amplification. Approximately half of the tumors fulfilled criteria for HER2-low or HER2-very low (10 HER2-low and 20 HER2-very low). Our data shows that a subset of p53-aberrant high-grade endometrial endometrioid carcinoma express HER2 and these patients may benefit from the addition of targeted therapy. The role of targeted therapy in HER2-low gynecologic carcinoma is currently unexplored.