PPP2R5E: New gene potentially involved in specific learning disorders and myopathy.

Protein phosphatase 2A (PP2A) is a family of multifunctional enzymatic complexes crucial for cellular signalling, playing a pivotal role in brain function and development. Mutations in specific genes encoding PP2A complexes have been associated with neurodevelopmental disorders with hypotonia and high risk of seizures. In the current work, we present an individual with specific learning problems, motor coordination disorders, hypotonia and behavioural issues. Although whole exome sequencing (WES) did not unveil pathogenic variants in known genes related to these symptoms, a de novo heterozygous variant Glu191Lys was identified within PPP2R5E, encoding the PP2A regulatory subunit B56ε. The novel variant was not observed in the four healthy brothers and was not detected as parental somatic mosaicism. The mutation predicted a change of charge of the mutated amino acid within a conserved LFDSEDPRER motif common to all PPP2R5 B-subunits. Biochemical assays demonstrated a decreased interaction with the PP2A A and C subunits, leading to disturbances in holoenzyme formation, and thus likely, function. For the first time, we report a potential causal link between the observed variant within the PPP2R5E gene and the symptoms manifested in the subject, spanning specific learning problems and motor coordination disorders potentially associated with myopathy.

Clinical characteristics, longitudinal adaptive functioning, and association with electroencephalogram activity in PPP2R5D-related neurodevelopmental disorder.

Protein phosphatase 2 regulatory subunit B56δ related neurodevelopmental disorder (PPP2R5D-related NDD) is largely caused by de novo heterozygous missense PPP2R5D variants. We report medical characteristics, longitudinal adaptive functioning, and in-person neurological, motor, cognitive, and electroencephalogram (EEG) activity for PPP2R5D-related NDD. Forty-two individuals (median age 6 years, range = 0.8-25.3) with pathogenic/likely pathogenic PPP2R5D variants were assessed, and almost all variants were missense (97.6%) and de novo (85.7%). Common clinical symptoms were developmental delay, hypotonia, macrocephaly, seizures, autism, behavioral challenges, and sleep problems. The mean Gross motor functional measure-66 was 60.2 ± 17.3% and the mean Revised upper limb module score was 25.9 ± 8.8. The Vineland-3 adaptive behavior composite score (VABS-3 ABC) at baseline was low (M = 61.7 ± 16.8). VABS-3 growth scale value scores increased from baseline in all subdomains (range = 0.6-5.9) after a mean follow-up of 1.3 ± 0.3 years. EEG beta and gamma power were negatively correlated with VABS-3 score; p < 0.05. Individuals had a mean Quality-of-life inventory-disability score of 74.7 ± 11.4. Twenty caregivers (80%) had a risk of burnout based on the Caregiver burden inventory. Overall, the most common clinical manifestations of PPP2R5D-related NDD were impaired cognitive, adaptive function, and motor skills; and EEG activity was associated with adaptive functioning. This clinical characterization describes the natural history in preparation for clinical trials.

Unmasking Protein Phosphatase 2A Regulatory Subunit B as a Crucial Factor in the Progression of Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is one of the major causes of heart failure. Although significant progress has been made in elucidating the underlying mechanisms, further investigation is required for clarifying molecular diagnostic and therapeutic targets. In this study, we found that the mRNA level of protein phosphatase 2 regulatory subunit B' delta (Ppp2r5d) was altered in the peripheral blood plasma of DCM patients. Knockdown of Ppp2r5d in murine cardiomyocytes increased the intracellular levels of reactive oxygen species (ROS) and inhibited adenosine triphosphate (ATP) synthesis. In vivo knockdown of Ppp2r5d in an isoproterenol (ISO)-induced DCM mouse model aggravated the pathogenesis and ultimately led to heart failure. Mechanistically, Ppp2r5d-deficient cardiomyocytes showed an increase in phosphorylation of STAT3 at Y705 and a decrease in phosphorylation of STAT3 at S727. The elevated levels of phosphorylation at Y705 in STAT3 triggered the upregulation of interleukin 6 (IL6) expression. Moreover, the decreased phosphorylation at S727 in STAT3 disrupted mitochondrial electron transport chain function and dysregulated ATP synthesis and ROS levels. These results hereby reveal a novel role for Ppp2r5d in modulating STAT3 pathway in DCM, suggesting it as a potential target for the therapy of the disease.

Loss of protein phosphatase 2A regulatory subunit PPP2R5A is associated with increased incidence of stress-induced proarrhythmia.

Background: Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme that controls Ca2+ homeostasis and contractility of the heart via dephosphorylation of regulatory proteins. In some genetically modified mouse models with increased arrhythmogenicity, a reduced expression of the regulatory subunit B56α of PP2A was found as a concomitant effect. Whether there is a general correlation between the abundance of B56α and the promotion of cardiac arrhythmogenesis remains unclear. Methods: The aim of this study was therefore to investigate the role of PP2A-B56α in the propensity for arrhythmic activity in the heart. The experimental analysis of this question has been addressed by using a mouse model with deletion of the PP2A-B56α gene, PPP2R5A (KO), in comparison to wild-type animals (WT). Evidence for arrhythmogenicity was investigated in whole animal, isolated heart and cardiomyocytes by ECG, recording of monophasic action potential (MAP) induced by programmed electrical stimulation (PES), measurement of Ca2+ transients under increased pacing frequencies and determination of total K+ channel currents (I K). Results: ECG measurements showed a prolongation of QT time in KO vs. WT. KO mice exhibited a higher rate of premature ventricular contractions in the ECG. MAP measurements in Langendorff-perfused KO hearts showed increased episodes of ventricular tachyarrhythmia induced by PES. However, the KO hearts showed values for MAP duration that were similar to those in WT hearts. In contrast, KO showed more myocardial cells with spontaneous arrhythmogenic Ca2+ transient events compared to WT. The whole-cell patch-clamp technique applied to ventricular cardiomyocytes revealed comparable peak potassium channel current densities between KO and WT. Conclusion: These findings support the assumption that a decrease or even the loss of PP2A-B56α leads to an increased propensity of triggered arrhythmias. This could be based on the increased spontaneous Ca2+ tansients observed.

PPP2R5D heterozygous pathogenic variant causes early-onset parkinsonism and treatment implications: A case report.

Neurodevelopmental disorders with early-onset parkinsonism have diverse genetic aetiologies and can mimic Parkinson's disease. We report the clinical evaluation and neuroimaging studies of a woman with intellectual disability and levodopa-responsive akinetic rigid parkinsonism. Whole-genome sequencing of family trio identified a de novo missense variant in PPP2R5D in the proband.

Characterizing ambulatory function in children with PPP2R5D-related neurodevelopmental disorder.

BACKGROUND: Individuals with PPP2R5D-related neurodevelopmental disorder have an atypical gait pattern characterized by ataxia and incoordination. Structured, quantitative assessments are needed to further understand the impact of these impairments on function. RESEARCH QUESTION: How do gait parameters and ambulatory function of individuals with PPP2R5D-related neurodevelopmental disorder compare to age and sex matched healthy norms? METHODS: Twenty-six individuals with PPP2R5D pathogenic genetic variants participated in this observational, single visit study. Participants completed at least one of the following gait assessments: quantitative gait analysis at three different speeds (preferred pace walking (PPW), fast paced walking (FPW) and running, six-minute walk test (6MWT), 10-meter walk run (10MWR), and timed up and go (TUG). Descriptive statistics were used to summarize gait variables. Percent of predicted values were calculated using published norms. Paired t-tests and regression analyses were used to compare gait variables. RESULTS: The median age of the participants was 8 years (range 4-27) and eighteen (69.2 %) were female. Individuals with PPP2R5D-related neurodevelopmental disorder walked slower and with a wider base of support than predicted for their age and sex. Stride velocity ranged from 48.9 % to 70.1 % and stride distance from 58.5 % to 81.9 % of predicted during PPW. Percent of predicted distance walked on the 6MWT ranged from 30.6 % to 71.1 % representing varied walking impairment. Increases in stride distance, not cadence, were associated with changes in stride velocity in FPW (R2 = 0.675, p =< 0.001) and running conditions (R2 = 0.918, p =< 0.001). SIGNIFICANCE: We quantitatively assessed the abnormal gait in individuals with PPP2R5D-related neurodevelopmental disorder. These impairments may affect ability to adapt to environmental changes and participation in daily life. Rehabilitative interventions targeting gait speed and balance may improve function and safety for individuals with PPP2R5D-related neurodevelopmental disorder.

Validation of a modified version of the gross motor function measure in PPPR5D related neurodevelopmental disorder.

BACKGROUND: Protein phosphatase 2 regulatory subunit B' Delta (PPP2R5D)-related neurodevelopmental disorder is a rare genetic condition caused by pathogenic variants in the PPP2R5D gene. Clinical signs include hypotonia, gross motor delay, intellectual disability (ID), epilepsy, speech delays, and abnormal gait among other impairments. As this disorder was recognized within the last decade, there are only 103 people published diagnoses to date. A thorough understanding of the motor manifestations of this disorder has not yet been established. Knowledge of the natural history of PPP2R5D related neurodevelopmental disorder will lead to improved standard of care treatments as well as serve as a baseline foundation for future clinical trials. Appropriate outcome measures are necessary for use in clinical trials to uniformly measure function and monitor potential for change. The aim of this study was to validate the gross motor function measure (GMFM) in children and adults with PPP2R5D-related neurodevelopmental disorder in order to better characterize the disorder. RESULTS: Thirty-eight individuals with PPP2R5D pathogenic variants, median age 8.0 years (range 1-27) were evaluated. Gross motor, upper limb and ambulatory function were assessed using the GMFM-66, six-minute walk test (6MWT), 10-meter walk run (10MWR), timed up and go (TUG), and revised upper limb module (RULM). The pediatric disability inventory computer adapted test (PEDI-CAT) captured caregiver reported assessment. Median GMFM-66 score was 60.6 (SD = 17.3, range 21.1-96.0). There were strong associations between the GMFM-66 and related mobility measures, 10MWR (rs = -0.733; p < 0.001), TUG (rs= -0.747; p = 0.003), 6MWT (r = 0.633; p = 0.006), RULM (r = 0.763; p < 0.001), PEDICAT-mobility (r = 0.855; p < 0.001), and daily activities (r = 0.822; p < 0.001) domains. CONCLUSIONS: The GMFM is a valid measure for characterizing motor function in individuals with PPP2R5D related neurodevelopmental disorder. The GMFM-66 had strong associations with the RULM and timed function tests which characterized gross motor, upper limb and ambulatory function demonstrating concurrent validity. The GMFM-66 was also able to differentiate between functional levels in PPP2R5D related neurodevelopmental disorder demonstrating discriminant validity. Future studies should examine its sensitivity to change over time, ability to identify sub-phenotypes, and suitability as an outcome measure in future clinical trials in individuals with PPP2R5D variants.

Novel Variants of PPP2R1A in Catalytic Subunit Binding Domain and Genotype-Phenotype Analysis in Neurodevelopmentally Delayed Patients.

Neurodevelopmental disorders (NDDs) are a group of high-incidence rare diseases with genetic heterogeneity. PPP2R1A, the regulatory subunit of protein phosphatase 2A, is a recently discovered gene associated with NDDs. Whole/clinical exome sequencing was performed in five patients with a family with NDDs. In vitro experiments were performed to evaluate the mutants' expression and interactions with the complex. The genotype-phenotype correlations of reported cases as well as our patients with PPP2R1A variants were reviewed. We reported five unrelated individuals with PPP2R1A variants, including two novel missense variants and one frameshift variant. The protein expression of the Arg498Leu variant was less than that of the wild-type protein, the frameshift variant Asn282Argfs*14 was not decreased but truncated, and these two variants impaired the interactions with endogenous PPP25RD and PPP2CA. Furthermore, we found that pathogenic variants clustered in HEAT repeats V, VI and VII, and patients with the Met180Val/Thr variants had macrocephaly, severe ID and hypotonia, but no epilepsy, whereas those with Arg258 amino acid changes had microcephaly, while a few had epilepsy or feeding problems. In this study, we reported five NDD patients with PPP2R1A gene variants and expanded PPP2R1A pathogenic variant spectrum. The genotype and phenotype association findings provide reminders regarding the prognostication and evidence for genetic counseling.

A Novel Mouse Model of Combined Hepatocellular-Cholangiocarcinoma Induced by Diethylnitrosamine and Loss of Ppp2r5d.

Primary liver cancer (PLC) can be classified in hepatocellular (HCC), cholangiocarcinoma (CCA), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). The molecular mechanisms involved in PLC development and phenotype decision are still not well understood. Complete deletion of Ppp2r5d, encoding the B56δ subunit of Protein Phosphatase 2A (PP2A), results in spontaneous HCC development in mice via a c-MYC-dependent mechanism. In the present study, we aimed to examine the role of Ppp2r5d in an independent mouse model of diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Ppp2r5d deletion (heterozygous and homozygous) accelerated HCC development, corroborating its tumor-suppressive function in liver and suggesting Ppp2r5d may be haploinsufficient. Ppp2r5d-deficient HCCs stained positively for c-MYC, consistent with increased AKT activation in pre-malignant and tumor tissues of Ppp2r5d-deficient mice. We also found increased YAP activation in Ppp2r5d-deficient tumors. Remarkably, in older mice, Ppp2r5d deletion resulted in cHCC-CCA development in this model, with the CCA component showing increased expression of progenitor markers (SOX9 and EpCAM). Finally, we observed an upregulation of Ppp2r5d in tumors from wildtype and heterozygous mice, revealing a tumor-specific control mechanism of Ppp2r5d expression, and suggestive of the involvement of Ppp2r5d in a negative feedback regulation restricting tumor growth. Our study highlights the tumor-suppressive role of mouse PP2A-B56δ in both HCC and cHCC-CCA, which may have important implications for human PLC development and targeted treatment.

Quantitative proteomics and phosphoproteomics of PP2A-PPP2R5D variants reveal deregulation of RPS6 phosphorylation via converging signaling cascades.

Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.

Quantum-based modeling implies that bidentate Arg89-substrate binding enhances serine/threonine protein phosphatase-2A(PPP2R5D/PPP2R1A/PPP2CA)-mediated dephosphorylation.

PP2A-serine/threonine protein phosphatases function as heterotrimeric holoenzymes, composed of a common scaffold (A-subunit encoded by PPP2R1A/PPP2R1B), a common catalytic (C-subunit encoded by PPP2CA/PPP2CB), and one of many variable regulatory (B) subunits. The site of phosphoprotein phosphatase (PPP) hydrolysis features a bimetal system (M1/M2), an associated bridge hydroxide [W1(OH-)], and a highly-conserved core sequence. In the presumptive common mechanism, the phosphoprotein's seryl/threonyl phosphate coordinates the M1/M2 system, W1(OH-) attacks the central P atom, rupturing the antipodal bond, and simultaneously, a histidine/aspartate tandem protonates the exiting seryl/threonyl alkoxide. Based on studies of PPP5C, a conserved arginine proximal to M1 is also expected to bind the substrate's phosphate group in a bidentate fashion. However, in PP2A isozymes, the role of the arginine (Arg89) in hydrolysis is not clear because two independent structures for PP2A(PPP2R5C) and PP2A(PPP2R5D) show that Arg89 engages in a weak salt bridge at the B:C interface. These observations raise the question of whether hydrolysis proceeds with or without direct involvement of Arg89. The interaction of Arg89 with B:Glu198 in PP2A(PPP2R5D) is significant because the pathogenic E198K variant of B56δ is associated with irregular protein phosphorylation levels and consequent developmental disorders (Jordan's Syndrome; OMIM #616355). In this study, we perform quantum-based hybrid [ONIOM(UB3LYP/6-31G(d):UPM7)] calculations on 39-residue models of the PP2A(PPP2R5D)/pSer (phosphoserine) system to estimate activation barriers for hydrolysis in the presence of bidentate Arg89-substrate binding and when Arg89 is otherwise engaged in the salt-bridge interaction. Our solvation-corrected results yield ΔH‡ ≈ ΔE‡ = +15.5 kcal/mol for the former case, versus +18.8 kcal/mol for the latter, indicating that bidentate Arg89-substrate binding is critical for optimal catalytic function of the enzyme. We speculate that PP2A(PPP2R5D) activity is suppressed by B:Glu198 sequestration of C:Arg89 under native conditions, whereas the PP2A(PPP2R5D)-holoenzyme containing the E198K variant has a positively-charged lysine in this position that alters normal function.

An Exploration of Osteosarcoma Metastasis Diagnostic Markers Based on Tumor-Associated Neutrophils.

BACKGROUND: The high rate of the recurrence and metastasis of osteosarcoma (OS) is the major cause of its poor prognosis. There is a strong correlation between tumor-associated neutrophils (TANs) and tumor progression, progression, and metastasis. This study aimed to identify potential markers that could predict OS metastasis based on analysis of TANs in the tissues of OS patients. METHODS: A single-cell sequencing dataset (GSE152048), containing seven primary OS lesions, two recurrent OS lesions, and two lung metastatic OS lesions was used for TANs subset identification using the R software (version 4.1.0, R Project for Statistical Computing, Vienna, Austria; https://www.r-project.org/). Immune cell infiltration and immune score were analyzed using CIBERSORT algorithm and ESTIMATE database, respectively. The differentially expressed genes (DEGs) of TANs were used for weighted gene co-expression network analysis (WGCNA) to screen key genes associated with OS metastasis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis were used to analyze the functions and signaling pathways involved in key genes. The mRNA levels of protein phosphatase 2 regulatory subunit B'gamma (PPP2R5C) were validated in human osteosarcoma cell lines U2-OS and MG63, human normal cervical endometrial cell line HUCEC, and human foreskin fibroblast (HFF-1) cell line by real-time qPCR (RT-qPCR). PPP2R5C-siRNA991 was transfected into U2-OS and MG63 for 48 h, then the expression levels of PPP2R5C, AKT serine/threonine kinase (AKT), and phospho-AKT (p-AKT) were determined by RT-qPCR and Western blotting. Cell proliferation, migration, and apoptosis were measured by cell counting kit-8 (CCK-8), Transwell, and flow cytometry, respectively. RESULTS: We identified TANs subsets in primary, metastatic, and recurrent OS. Immune infiltration analysis showed that TANs were expressed in OS. Compared with non-metastatic OS, metastatic OS had lower stromal score, immune score, ESTIMATE score, and higher tumor purity. WGCNA classified DEGs into five clusters, according to their function and identified PPP2R5C, protein phosphatase 2 regulatory subunit B'epsilon (PPP2R5E), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) and CREB binding protein (CREBBP), as potential markers that may affect TANs-induced OS metastasis via hypoxia inducible factor 1 (HIF-1), phosphatidylinositol 3-kinases (PI3K)-AKT and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathways. In vitro experiments demonstrated that the mRNA and protein expressions of PPP2R5C, PPP2R5E, YWHAG, and CREBBP were highly expressed in U2-OS and MG63 cells (p < 0.01). Furthermore, PPP2R5C reduced proliferation and migration (p < 0.01) and increased apoptosis and p-AKT protein levels in U2-OS and MG6 cells (p < 0.01). CONCLUSIONS: PPP2R5C affects OS metastasis via PI3K/AKT pathway, which may be a potential marker for OS metastasis and recurrence.

Combined detection of SDC2/ADHFE1/PPP2R5C methylation in stool DNA for colorectal cancer screening.

BACKGROUND: Colorectal cancer (CRC) is a disease of global concern, and its increasing incidence suggests the need for early and accurate diagnosis. The aim of this study was to investigate the value of combined detection of SDC2, ADHFE1 and PPP2R5C gene methylation in stool samples for early CRC screening. METHODS: Stool samples from patients with CRC (n = 105), advanced adenoma (AA) (n = 54), non-advanced adenoma (NA) (n = 57), hyperplastic or other polyps (HOP) (n = 47) or no evidence of disease (NED) (n = 100) were collected from September 2021 to September 2022. The methylation levels of SDC2, ADHFE1 and PPP2R5C were quantified by quantitative methylation-specific polymerase chain reaction (qMSP), and faecal immunochemical testing (FIT) was performed. The diagnostic value was assessed using reporter operating characteristic (ROC) curve analysis. RESULTS: The sensitivity of combined detection of SDC2/ADHFE1/PPP2R5C methylation in predicting CRC (0-IV) was 84.8%, the specificity was 98.0%, and the AUC was 0.930 (95% CI 0.889-0.970). Compared to FIT and serum tumour biomarkers, it showed better diagnostic performance for different stages of CRC. CONCLUSION: The results of this study verified that the methylation levels of SDC2, ADHFE1 and PPP2R5C in stool DNA were significantly increased in CRC patients. Combined detection of SDC2/ADHFE1/PPP2R5C methylation is a potential non-invasive diagnostic method for CRC and precancerous lesion screening. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trials Registry, ChiCTR2100046662, registered on 26 May 2021, prospective registration.

Deregulation of the miR-19b/PPP2R5E Signaling Axis Shows High Functional Impact in Colorectal Cancer Cells.

MicroRNA (miR)-19b is deregulated in colorectal cancer (CRC) and locally advanced rectal cancer (LARC), predicting worse outcome and disease progression in CRC patients, and acting as a promising prognostic marker of patient recurrence and pathological response to 5-fluorouracil (5-FU)-based neoadjuvant chemoradiotherapy in LARC. Moreover, there is a strong inverse correlation between miR-19b and PPP2R5E in LARC, and both predict the response to neoadjuvant therapy in LARC patients. However, the functional role of the miR-19b/PPP2R5E axis in CRC cells remains to be experimentally evaluated. Here, we confirm with luciferase assays that miR-19b is a direct negative regulator of PPP2R5E in CRC, which is concordant with the observed decreased PP2A activity levels after miR-19b overexpression. Furthermore, PPP2R5E downregulation plays a key role mediating miR-19b-induced oncogenic effects, increasing cell viability, colonosphere formation ability, and the migration of CRC cells. Lastly, we also confirm the role of miR-19b mediating 5-FU sensitivity of CRC cells through negative PPP2R5E regulation. Altogether, our findings demonstrate the functional relevance of the miR-19b/PPP2R5E signaling pathway in disease progression, and its potential therapeutic value determining the 5-FU response of CRC cells.

An in-frame deletion affecting the critical acid loop of PPP2R5D is associated with a neonatal lethal form of PPP2R5D-related neurodevelopmental disorder.

Heterozygous pathogenic variants in PPP2R5D gene are associated with PPP2R5D-related neurodevelopmental disorder, a rare autosomal dominant condition, characterized by neurodevelopmental impairment in childhood, macrocephaly/megalencephaly, hypotonia, epilepsy, and dysmorphic features. Up-to-date, only approximately 100 cases have been published in the literature and the full phenotypic and genotypic spectrum have not yet been fully described. PPP2R5D gene encodes the B56δ subunit of the PP2A enzyme complex. We describe a neonatal form of PPP2R5D-related disorder with early infantile death, caused by a novel in-frame deletion causing loss of 8 amino acids and insertion of serine at position 201 (p.Phe194_Pro201delinsSer) of the B56δ subunit. This deletion is predicted to disrupt a critical acidic loop of amino acids important for binding other subunits of the PP2A enzyme complex, and harbors many of the residues previously reported to cause a mild-moderate form of this condition. This report describes a neonatal lethal presentation of the PPP2R5D-related neurodevelopmental disorder and provides additional evidence that disruption of the acidic loop is an important pathomechanism underlying PPP2R5D-related disorder.

A novel nonsense mutation in PPP2R5D is associated with neurodevelopmental disorders and shows incomplete penetrance in a Chinese pedigree.

BACKGROUND AND PURPOSE: PPP2R5D mutations are known to cause neurodevelopmental disorders in an autosomal-dominant manner. To date, the vast majority of the reported cases in the literature have been sporadic with de novo mutations. There are no data regarding PPP2R5D mutation penetrance. We aimed to unravel the underlying genetic defects in 3 generations of a family affected by intellectual disability, neurodevelopmental delay, and facial dysmorphology. METHODS: We performed detailed clinical examinations and whole-exome sequencing in the family. RESULTS: We identified a novel mutation, c.1321 C>T (p.Arg441*), in PPP2R5D. The mutation cosegregated with affected family members I-2 and II-7 but not II-3, who bears the mutation but is phenotypically healthy. Our whole-exome sequencing also excluded the involvement of pathogenic mutations in other genes known to be related to neurodevelopmental disorders. The mutation was predicted to introduce a premature stop codon at position 441, thereby truncating the 162 amino acids at the C-terminus of the encoded protein. CONCLUSIONS: We report a familial transmitted PPP2R5D-related neurodevelopmental disorder as well as a novel nonsense pathogenic mutation and its incomplete penetrance. Our study expands the mutational and phenotypic spectra of PPP2R5D-related neurodevelopmental disorders, broadens our understanding of these disorders, and will thus be valuable for mutation-based pre- and postnatal screening and genetic diagnosis of neurodevelopmental disorders.

Analysis of the Association of Two SNPs in the Promoter Regions of the PPP2R5C and SLC39A5 Genes with Litter Size in Yunshang Black Goats.

Screening for candidate genes and genetic variants associated with litter size is important for goat breeding. The aim of this study was to analyze the relationship between single nucleotide polymorphisms (SNPs) in PPP2R5C and SLC39A5 and litter size in Yunshang black goats. KASP genotyping was used to detect the SNP genetic markers in the PPP2R5C and SLC39A5 in a population of 569 Yunshang black goats. The results show that there were two SNPs in the PPP2R5C and SLC39A5 promoter regions. Association analysis revealed that the polymorphisms PPP2R5C g.65977743C>T and SLC39A5 g.50676693T>C were significantly associated with the litter size of the third parity of Yunshang black goats (p < 0.05). To further explore the regulatory mechanism of the two genes, the expression of different genotypes of PPP2R5C and SLC39A5 was validated by RT-qPCR and Western blotting. The expression of PPP2R5C was significantly higher in individuals with the TT genotype than in those with the TC and CC genotypes (p < 0.05). The expression of SLC39A5 was also significantly higher in individuals with the TT genotype than in TC and CC genotypes (p < 0.05). Dual luciferase reporter analysis showed that the luciferase activity of PPP2R5C-C variant was significantly higher than that of PPP2R5C-T variant (p < 0.05). The luciferase activity of SLC39A5-T variant was significantly higher than that of SLC39A5-C variant (p < 0.05). Software was used to predict the binding of transcription factors to the polymorphic sites, and the results show that SOX18, ZNF418, and ZNF667 and NKX2-4 and TBX6 might bind to PPP2R5C g.65977743C>T and SLC39A5 g.50676693T>C, respectively. These results provide new insights into the identification of candidate genes for marker-assisted selection (MAS) in goats.

Evolutionary Conservation of PP2A Antagonism and G2/M Cell Cycle Arrest in Maedi-Visna Virus Vif.

The canonical function of lentiviral Vif proteins is to counteract the mutagenic potential of APOBEC3 antiviral restriction factors. However, recent studies have discovered that Vif proteins from diverse HIV-1 and simian immunodeficiency virus (SIV) isolates degrade cellular B56 phosphoregulators to remodel the host phosphoproteome and induce G2/M cell cycle arrest. Here, we evaluate the conservation of this activity among non-primate lentiviral Vif proteins using fluorescence-based degradation assays and demonstrate that maedi-visna virus (MVV) Vif efficiently degrades all five B56 family members. Testing an extensive panel of single amino acid substitution mutants revealed that MVV Vif recognizes B56 proteins through a conserved network of electrostatic interactions. Furthermore, experiments using genetic and pharmacologic approaches demonstrate that degradation of B56 proteins requires the cellular cofactor cyclophilin A. Lastly, MVV Vif-mediated depletion of B56 proteins induces a potent G2/M cell cycle arrest phenotype. Therefore, remodeling of the cellular phosphoproteome and induction of G2/M cell cycle arrest are ancient and conserved functions of lentiviral Vif proteins, which suggests that they are advantageous for lentiviral pathogenesis.