Loss Of Protein Function Research Articles

The Implications of Insufficient Zinc on the Generation of Oxidative Stress Leading to Decreased Oocyte Quality.

Zinc is a transition metal that displays wide physiological implications ranging from participation in hundreds of enzymes and proteins to normal growth and development. In the reproductive tract of both sexes, zinc maintains a functional role in spermatogenesis, ovulation, fertilization, normal pregnancy, fetal development, and parturition. In this work, we review evidence to date regarding the importance of zinc in oocyte maturation and development, with emphasis on the role of key zinc-binding proteins, as well as examine the effects of zinc and reactive oxygen species (ROS) on oocyte quality and female fertility. We summarize our current knowledge about the participation of zinc in the developing oocyte bound to zinc finger proteins as well as loosely bound zinc ion in the intracellular and extracellular environments. These include aspects related to (1) the impact of zinc deficiency and overwhelming production of ROS under inflammatory conditions on the offset of the physiological antioxidant machinery disturbing biomolecules, proteins, and cellular processes, and their role in contributing to further oxidative stress; (2) the role of ROS in modulating damage to proteins containing zinc, such as zinc finger proteins and nitric oxide synthases (NOS), and expelling the zinc resulting in loss of protein function; and (3) clarify the different role of oxidative stress and zinc deficiency in the pathophysiology of infertility diseases with special emphasis on endometriosis-associated infertility.

Genome-wide identification, evolution, and expression analysis of MLO gene family in melon (Cucumis melo L.).

Powdery mildew (PM) is one of the main fungal diseases that appear during the cultivation of the melon fruit crop. Mildew Resistance Locus "O" (MLO) is known as a gene family and has seven conserved transmembrane domains. An induced functional loss of a specific MLO gene could mainly confer PM resistance to melons. However, the genomic structure of MLO genes and its main role in PM resistance still remain unclear in melon. In this study, bioinformatic analysis identified a total of 14 MLO gene family members in the melon genome sequence, and these genes were distributed in an uneven manner on eight chromosomes. The phylogenetic analysis divided the CmMLO genes into five different clades, and gene structural analysis showed that genes in the same clade had similar intron and exon distribution patterns. In addition, by cloning the CmMLO gene sequence in four melon lines, analyzing the CmMLO gene expression pattern after infection, and making microscopic observations of the infection pattern of PM, we concluded that the CmMLO5 (MELO3C012438) gene plays a negative role in regulating PM-resistance in the susceptible melon line (Topmark), and the critical time point for gene function was noticed at 24 and 72 hours after PM infection. The mutational analysis exhibited a single base mutation at 572 bp, which further results in loss of protein function, thus conferring PM resistance in melon. In summary, our research evidence provides a thorough understanding of the CmMLO gene family and demonstrates their potential role in disease resistance, as well as a theoretical foundation for melon disease resistance breeding.

Multi‐Dimensional Structure and Dynamics Landscape of Proteins in Mammalian Cells Revealed by In‐Cell NMR

AbstractGoverning function, half‐life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in‐cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co‐existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.

Multi-Dimensional Structure and Dynamics Landscape of Proteins in Mammalian Cells Revealed by In-Cell NMR.

Governing function, half-life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in-cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co-existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.

Comprehensive proteome and lysine acetylome analysis after artificial aging reveals the key acetylated proteins involved in wheat seed oxidative stress response and energy production.

Lysine acetylation is a common post-translational modification of proteins within all organisms. However, quantitative acetylome characterization in wheat seed during aging in storage has not been reported. This study reports the first large-scale acetylome analysis of wheat seeds after artificial aging treatment, using the quantitative proteomic approach. In total, 11,002 acetylation sites, corresponding to 4262 acetylated proteins were identified, of which 1207 acetylated sites, representing 783 acetylated proteins, were significantly more or less acetylated after artificial aging. Functional analysis demonstrated that the majority of the acetylated proteins are closely involved with cellular and metabolic functions. In particular, key enzymes in the oxidative stress response and energy metabolism were significantly differentially acetylated and appear to be heavily involved in wheat seed aging. The acetylome analysis was verified by quantitative real-time PCR and enzyme activity determination. Lysine-acetylation results in a weaker oxidative stress response and lower energy production efficiency, resulting in the apoptosis of wheat seed cells, insufficient energy supply at the germination stage, and consequently, marked loss of seed vigor. PRACTICAL APPLICATIONS: It is known that the loss of protein function is an important reason for the decrease of seed vigor. Therefore, the change of protein function in the process of wheat seed aging was studied by proteome and lysine acetylome analysis technology. The results showed that the oxidation-reduction imbalance and the decrease of energy production efficiency of seeds were the important reasons for the decrease of their vigor. This will provide a new idea for green and safe storage of grain.

The location of protein oxidation in dystrophic skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

Creating De Novo Overlapped Genes.

Future applications of synthetic biology will rely on deploying engineered cells outside of lab environments for long periods of time. Currently, a significant roadblock to this application is the potential for deactivating mutations in engineered genes. A recently developed method to protect engineered coding sequences from mutation is called Constraining Adaptive Mutations using Engineered Overlapping Sequences (CAMEOS). In this chapter we provide a workflow for utilizing CAMEOS to create synthetic overlaps between two genes, one essential (infA) and one non-essential (aroB), to protect the non-essential gene from mutation and loss of protein function. In this workflow we detail the methods to collect large numbers of related protein sequences, produce multiple sequence alignments (MSAs), use the MSAs to generate hidden Markov models and Markov random field models, and finally generate a library of overlapping coding sequences through CAMEOS scripts. To assist practitioners with basic coding skills to try out the CAMEOS method, we have created a virtual machine containing all the required packages already installed that can be downloaded and run locally.

The effects of missense OPN3 mutations in melanocytic lesions on protein structure and light-sensitive function.

Opsin 3 (OPN3), a member of the light-sensitive, retinal-dependent opsin family, is widely expressed in a variety of human tissues and plays a multitude of light-dependent and light-independent roles. We recently identified five missense variants of OPN3, including p. I51T, p. V134A, p. V183I, p. M256I and p. C331Y, in human melanocytic tumours. However, it remains unclear how these OPN3 variants affect OPN3 protein structure and function. Herein, we conducted structural and functional studies of these variant proteins in OPN3 by molecular docking and molecular dynamics simulations. Moreover, we performed in vitro fluorescence calcium imaging to assess the functional properties of five single-nucleotide variant (SNV) proteins using a site-directed mutagenesis method. Notably, the p. I51T variant was not able to effectively dock with 11-cis-retinal. Additionally, in vitro, the p. I51T SNVs failed to induce any detectable changes in intracellular Ca2+ concentration at room temperature. Taken together, these results reveal that five SNVs in the OPN3 gene have deleterious effects on protein structure and function, suggesting that these mutations, especially the p. I51T variant, significantly disrupt the canonical function of the OPN3 protein. Our findings provide new insight into the role of OPN3 variants in the loss of protein function.

Loss of Protein Function Causing Severe Phenotypes of Female-Restricted Wieacker Wolff Syndrome due to a Novel Nonsense Mutation in the ZC4H2 Gene.

Pathogenic variants of zinc finger C4H2-type containing (ZC4H2) on the X chromosome cause a group of genetic diseases termed ZC4H2-associated rare disorders (ZARD), including Wieacker-Wolff Syndrome (WRWF) and Female-restricted Wieacker-Wolff Syndrome (WRWFFR). In the current study, a de novo c.352C>T (p.Gln118*) mutation in ZC4H2 (NM_018684.4) was identified in a female neonate born with severe arthrogryposis multiplex congenita (AMC) and Pierre-Robin sequence (cleft palate and micrognathia). Plasmids containing the wild-type (WT), mutant-type (MT) ZC4H2, or GFP report gene (N) were transfected in 293T cell lines, respectively. RT-qPCR and western blot analysis showed that ZC4H2 protein could not be detected in the 293T cells transfected with MT ZC4H2. The RNA seq results revealed that the expression profile of the MT group was similar to that of the N group but differed significantly from the WT group, indicating that the c.352C>T mutation resulted in the loss of function of ZC4H2. Differentially expressed genes (DEGs) enrichment analysis showed that c.352C>T mutation inhibited the expression levels of a series of genes involved in the oxidative phosphorylation pathway. Subsequently, expression levels of ZC4H2 were knocked down in neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) by lentiviral-expressed small hairpin RNAs (shRNAs) against ZC4H2. The results also demonstrated that decreasing the expression of ZC4H2 significantly reduced the growth of NSCs by affecting the expression of genes related to the oxidative phosphorylation signaling pathway. Taken together, our results strongly suggest that ZC4H2 c.352C>T (p.Gln118*) mutation resulted in the loss of protein function and caused WRWFFR.

A novel missense variant in the CASK gene causes intellectual developmental disorder and microcephaly with pontine and cerebellar hypoplasia

BackgroundVariants in the CASK gene result in a wide range of observed phenotypes in humans, such as FG Syndrome 4 and intellectual disabilities. Intellectual developmental disorder with microcephaly and pontine and cerebellar hypoplasia (MICPCH) is an X-linked disorder that affects females and is characterized by severely impaired intellectual development and variable degrees of pontocerebellar hypoplasia. Variants in CASK are the main genetic cause of MICPCH. Variants in CASK can explain most patients with MICPCH, but there are still some patients whose disease aetiology cannot be explained.Case presentationAn 11-month-old female diagnosed with MICPCH exhibited general developmental delays, microcephaly, and cerebellar hypoplasia. Whole-exome sequencing (WES) was used to find a novel heterozygous missense variant (NM_003688.3: c.638T>G) of CASK in this patient. Strikingly, this variant reduced the expression of CASK at the protein level but not at the mRNA level. By using protein structure prediction analysis, this study found that the amino acid change caused by the variant resulted in further changes in the stability of the protein structure, and these changes caused the downregulation of protein expression and loss of protein function.ConclusionIn this study, we first reported a novel heterozygous pathogenic variant and a causative mechanism of MICPCH. The amino acid change cause by this variant led to changes in the protein structure and a decrease in its stability, which caused a loss of protein function. This study could be helpful to the genetic diagnosis of this disease.

Population-Based Penetrance of Deleterious Clinical Variants

Population-based assessment of disease risk associated with gene variants informs clinical decisions and risk stratification approaches. To evaluate the population-based disease risk of clinical variants in known disease predisposition genes. This cohort study included 72 434 individuals with 37 780 clinical variants who were enrolled in the BioMe Biobank from 2007 onwards with follow-up until December 2020 and the UK Biobank from 2006 to 2010 with follow-up until June 2020. Participants had linked exome and electronic health record data, were older than 20 years, and were of diverse ancestral backgrounds. Variants previously reported as pathogenic or predicted to cause a loss of protein function by bioinformatic algorithms (pathogenic/loss-of-function variants). The primary outcome was the disease risk associated with clinical variants. The risk difference (RD) between the prevalence of disease in individuals with a variant allele (penetrance) vs in individuals with a normal allele was measured. Among 72 434 study participants, 43 395 were from the UK Biobank (mean [SD] age, 57 [8.0] years; 24 065 [55%] women; 2948 [7%] non-European) and 29 039 were from the BioMe Biobank (mean [SD] age, 56 [16] years; 17 355 [60%] women; 19 663 [68%] non-European). Of 5360 pathogenic/loss-of-function variants, 4795 (89%) were associated with an RD less than or equal to 0.05. Mean penetrance was 6.9% (95% CI, 6.0%-7.8%) for pathogenic variants and 0.85% (95% CI, 0.76%-0.95%) for benign variants reported in ClinVar (difference, 6.0 [95% CI, 5.6-6.4] percentage points), with a median of 0% for both groups due to large numbers of nonpenetrant variants. Penetrance of pathogenic/loss-of-function variants for late-onset diseases was modified by age: mean penetrance was 10.3% (95% CI, 9.0%-11.6%) in individuals 70 years or older and 8.5% (95% CI, 7.9%-9.1%) in individuals 20 years or older (difference, 1.8 [95% CI, 0.40-3.3] percentage points). Penetrance of pathogenic/loss-of-function variants was heterogeneous even in known disease predisposition genes, including BRCA1 (mean [range], 38% [0%-100%]), BRCA2 (mean [range], 38% [0%-100%]), and PALB2 (mean [range], 26% [0%-100%]). In 2 large biobank cohorts, the estimated penetrance of pathogenic/loss-of-function variants was variable but generally low. Further research of population-based penetrance is needed to refine variant interpretation and clinical evaluation of individuals with these variant alleles.

The Periplasmic Oxidoreductase DsbA Is Required for Virulence of the Phytopathogen Dickeya solani.

In bacteria, the DsbA oxidoreductase is a crucial factor responsible for the introduction of disulfide bonds to extracytoplasmic proteins, which include important virulence factors. A lack of proper disulfide bonds frequently leads to instability and/or loss of protein function; therefore, improper disulfide bonding may lead to avirulent phenotypes. The importance of the DsbA function in phytopathogens has not been extensively studied yet. Dickeya solani is a bacterium from the Soft Rot Pectobacteriaceae family which is responsible for very high economic losses mainly in potato. In this work, we constructed a D. solani dsbA mutant and demonstrated that a lack of DsbA caused a loss of virulence. The mutant bacteria showed lower activities of secreted virulence determinants and were unable to develop disease symptoms in a potato plant. The SWATH-MS-based proteomic analysis revealed that the dsbA mutation led to multifaceted effects in the D. solani cells, including not only lower levels of secreted virulence factors, but also the induction of stress responses. Finally, the outer membrane barrier seemed to be disturbed by the mutation. Our results clearly demonstrate that the function played by the DsbA oxidoreductase is crucial for D. solani virulence, and a lack of DsbA significantly disturbs cellular physiology.

Proteomic Analysis of Subchronic Furan Exposure in the Liver of Male Fischer F344 Rats.

Furan is a volatile compound formed during the thermal processing of foods. Chronic exposure has been shown to cause cholangiocarcinoma and hepatocellular tumors in rodent models. We conducted a 90 day subchronic study in Fisher 344 rats exposed to various doses by gavage to determine the NOAEL. Previous reports have outlined changes in the liver using gross necropsy examination, histopathology, clinical biochemistry, hematology, immunohistochemistry, and toxicogenomics. The data revealed that males were more sensitive than females. The focus of this study was to evaluate the toxicoproteomic changes by 2-dimensional differential in gel electrophoresis followed by mass spectrometry analysis. To compliment previous studies, protein expression changes were evaluated of male animals after 90 days of exposure to doses of 0, 0.03, 0.5, and 8.0 mg/kg bw/d. Significant statistical treatment-related changes compared to the controls identified 45 protein spots containing 38 unique proteins. Proteins identified are implicated in metabolism, redox regulation, protein folding/proteolysis as well as structural and transport proteins. At lower doses, multiple cytoprotective pathways are activated to maintain a homeostasis but ultimately the loss of protein function and impairment of several pathways could lead to adverse health effects at higher doses of furan administration.

The expanding clinical and genetic spectrum of alsin-related disorders: the first cohort of Brazilian patients

There are three types of autosomal recessive disorders involving pathogenic variants in the ALS2 gene (OMIM*606352), infantile ascending hereditary spastic paraplegia (IAHSP), juvenile primary lateral sclerosis (JPLS) and juvenile amyotrophic lateral sclerosis (JALS), which are rare and related to retrograde degeneration of motor neurons. ALS2 pathogenic variants are distributed widely across the entire coding sequence and mostly result in a loss of protein function. Rarely, patients with JALS have been reported with lower motor neuron involvement. Here, we report the first Brazilian cohort (six patients) of JPLS with novel ALS2 pathogenic variants, and we propose an expanding clinical and genetic spectrum of alsin-related disorders. A review of the literature in PubMed from 2001 to September 2020 allowed us to identify 26 publications about the three different phenotypes caused by ALS2 variants (only case reports or families), encompassing 35 nonrelated families. We compiled data (sex, age, age at onset, first symptoms, atypical clinical features, molecular data, and clinical evolution (improvement or death)) from these studies and analyzed them in a general context on the basis of demographic features.

Clinical and genetic analysis of six Chinese children with Poirier-Bienvenu neurodevelopmental syndrome caused by CSNK2B mutation.

Mutations in CSNK2B lead to Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), a rare neurodevelopmental disorder. Only 14 cases of POBINDS have been reported worldwide. The main manifestations are seizures, often tonic-clonic, with or without intellectual disability, growth retardation, and developmental language retardation. We conducted a comprehensive phenotypic mining and trio-whole exome sequencing on six children with POBINDS for gene diagnosis and analyzed the different variants using bioinformatics analysis software and related experiments. This paper reviews previous literature and discusses two common missense variants that lead to structural changes. Among the six patients, four, one, and one had tonic-clonic, myoclonic, and febrile seizures, respectively. Language development disorder, motor development disorder, and developmental delay/intellectual disability (DD/ID) are the main clinical features. All children had de novo mutations in CSNK2B, including three missense variants (c.410G > T/p.(Cys137Phe), c.494A > G/p.(His165Arg), and c.3G > A/p.(Met1Ile)), two splice variants (c.292-2A > T, c.558-3T > G), and one frameshift variant (c.499delC/p.(Leu167Serfs*60)). Three missense variants were predicted to be harmful by various software programs, and two splicing variants were found to produce new exonic splicing enhancers by the minigene assay. Western blot analysis showed that the frameshift variant resulted in decreased protein expression. According to a literature review, c.3G > A/p.(Met1Ile), c.292-2A > T, c.558-3T > G, and c.499delC/p.(Leu167Serfs*60) are novel variants of CSNK2B. The decrease or loss of protein function caused by CSNK2B mutations may be a pathogenic factor in this cohort. The severity of the POBINDS phenotype differs, and refractory epilepsy may be accompanied by a more serious DD/ID, language disorder, and motor retardation. At present, there is no specific treatment, and antiepileptic therapy usually requires the combination of two or more anti-epileptic drugs.

Hypercholesterolemia risk associated Abca6 does not regulate lipoprotein metabolism in mice or hamster

Hypercholesterolemia has strong heritability and about 40–60% of hypercholesterolemia is caused by genetic risk factors. A number of monogenic genes have been identified so far for familial hypercholesterolemia (FH). However, in the general population, more than 90% of individuals with LDL cholesterol over 190 mg/dL do not carry known FH mutations. Large scale whole-exome sequencing has identified thousands of variants that are predicted to be loss-of-function (LoF) and each individual has a median of about twenty rare LoF variants and several hundreds more common LoF variants. However, majority of those variants have not been characterized and their functional consequence remains largely unknown. Rs77542162 is a common missense variant in ABCA6 and is strongly associated with hypercholesterolemia in different populations. ABCA6 is a cholesterol responsive gene and has been suggested to play a role in lipid metabolism. However, whether and how rs77542162 and ABCA6 regulate lipoprotein metabolism remain unknown. In current study, we systemically characterized the function of rs77542162 and ABCA6 in cultured cells and in vivo of rodents. We found that Abca6 is specifically expressed on the basolateral surface of hepatocytes in mouse liver. The rs77542162 variant disrupts ABCA6 protein stability and results in loss of functional protein. However, we found no evidence that Abca6 plays a role in lipoprotein metabolism in either normal mice or hypercholesterolemia mice or hamsters. Thus, our results suggest that Abca6 does not regulate lipoprotein metabolism in rodents and highlight the challenge and importance of functional characterization of disease-associated variants in animal models.

Protein haploinsufficiency drivers identify MYBPC3 variants that cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Variants in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are the leading cause of HCM. However, the pathogenicity status of hundreds of MYBPC3 variants found in patients remains unknown, as a consequence of our incomplete understanding of the pathomechanisms triggered by HCM-causing variants. Here, we examined 44 nontruncating MYBPC3 variants that we classified as HCM-linked or nonpathogenic according to cosegregation and population genetics criteria. We found that around half of the HCM-linked variants showed alterations in RNA splicing or protein stability, both of which can lead to cMyBP-C haploinsufficiency. These protein haploinsufficiency drivers associated with HCM pathogenicity with 100% and 94% specificity, respectively. Furthermore, we uncovered that 11% of nontruncating MYBPC3 variants currently classified as of uncertain significance in ClinVar induced one of these molecular phenotypes. Our strategy, which can be applied to other conditions induced by protein loss of function, supports the idea that cMyBP-C haploinsufficiency is a fundamental pathomechanism in HCM.

Fine Mapping and Candidate Gene Identification of a White Flower Gene BrWF3 in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Flower color is an important trait in plants. However, genes responsible for the white flower trait in Chinese cabbage are rarely reported. In this study, we constructed an F2 population derived from the Y640-288 (white flower) and Y641-87 (yellow flower) lines for the fine mapping of the white flower gene BrWF3 in Chinese cabbage. Genetic analysis indicated that BrWF3 was controlled by a single recessive gene. Using BSA-seq and KASP assays, BrWF3 was fine-mapped to an interval of 105.6 kb. Functional annotation, expression profiling, and sequence variation analyses confirmed that the AtPES2 homolog, Bra032957, was the most likely candidate gene for BrWF3. Carotenoid profiles and transmission electron microscopy analysis suggested that BrWF3 might participate in the production of xanthophyll esters (particularly violaxanthin esters), which in turn disrupt chromoplast development and the formation of plastoglobules (PGs). A SNP deletion in the third exon of BrWF3 caused the loss of protein function, and interfered with the normal assembly of PGs, which was associated with reduced expression levels of genes involved in carotenoid metabolism. Furthermore, we developed and validated the functional marker TXBH83 for BrWF3. Our results provide insight into the molecular mechanism underlying flower color pigmentation and reveal valuable information for marker-assisted selection (MAS) breeding in Chinese cabbage.

Abstract PR006: Correlation of immunohistochemistry with TP53 sequence and clinical outcomes in GOG-086P

Abstract Introduction: GOG-086P was one of the first attempts to combine molecular inhibitors such as bevacizumab or temsirolimus with chemotherapy in patients with advanced endometrial cancer. An exploratory analysis of this study indicates that bevacizumab combined with chemotherapy is superior to temsirolimus plus chemotherapy in patients with TP53 mutated tumors as determined by next generation sequencing. The functional status of p53 in a tumor can be inferred by sequence analysis and/or by immunohistochemistry (IHC). In this next study of the GOG-086P cohort, we sought to determine whether p53 IHC alone or integrated with TP53 sequencing was similarly predictive of outcome and whether IHC was predictive of the sequence analysis. Methods: A total of 349 patients were enrolled on GOG-086P. Two hundred and forty-three patients have TP53 sequence data, and 213 have p53 protein expression data measured by IHC. Tumor histology and sequence data were correlated with p53 IHC categorizing each case as p53 negative or null, wild type (WT) or over-expressed (OE), and these variables were further correlated with progression-free survival (PFS) and overall survival (OS) in the chemotherapy + bevacizumab arms versus the chemotherapy + temsirolimus arm. Results: The majority of patients (118) had tumors with WT p53 protein expression by IHC and non-mutated or WT TP53 sequence, with expected concordance between IHC and sequence analysis. However, there were five discordant cases with OE p53 protein in the absence of a TP53 mutation. Fourteen patients had null IHC protein expression and a concordant TP53 sequence mutation expected to result in the total loss of protein. Four cases that were p53 null by IHC had a discordant WT TP53 sequence. Seventeen patients had tumors with a missense TP53 mutation but had WT p53 protein expression by IHC, reflecting a potential discordance between sequencing and IHC versus a missense mutation that did not result in loss of protein function. Six of these 17 discordant cases may be explained by co-mutations in TP53 and POLE or microsatellite instability where p53 proteins, though mutated, function more like WT. Reassuringly and as expected, high concordance was found in the 55 cases that had OE protein by IHC and a missense mutation in TP53. On integrated analysis, a TP53 mutation with p53 protein OE was associated with significantly lower hazard ratio for PFS (HR=0.41; 95% CI 0.22-0.83) and OS (HR=0.28; 95% CI 0.14-0.59), i.e., longer PFS and OS, for patients receiving bevacizumab + chemotherapy relative to temsirolimus + chemotherapy. Conclusions: Mutations in TP53, which are common in patients with advanced endometrial cancer, represent a developing platform upon which to design personalized treatment regimens. We demonstrate that IHC is often but not always predictive of the TP53 sequence. However, most cases with over-expressed p53 protein by IHC had a mutation in TP53, and bevacizumab + chemotherapy prolonged PFS and OS in such patients. Citation Format: Kristina W. Thiel, Eric J. Devor, Virginia Filiaci, Douglas A. Levine, Fanny Dao, Narciso Olvera, David Mutch, Carol Aghajanian, Kimberly K. Leslie. Correlation of immunohistochemistry with TP53 sequence and clinical outcomes in GOG-086P [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR006.

GLYT1 encephalopathy: Further delineation of disease phenotype and discussion of pathophysiological mechanisms.

GLYT1 encephalopathy is a form of glycine encephalopathy caused by disturbance of glycine transport. The phenotypic spectrum of the disease has not yet been completely described, as only four unrelated families with the disorder have been reported to date. Common features of affected patients include neonatal hypotonia, respiratory failure, encephalopathy, myoclonic jerks, dysmorphic features, and musculoeskeletal anomalies. All reported affected patients harbor biallelic genetic variants in SLC6A9. SNP array together with Sanger sequencing were performed in a newborn with arthrogryposis and severe neurological impairment. The novel genetic variant c.997delC in SLC6A9 was detected in homozygous state in the patient. At protein level, the predicted change is p.(Arg333Alafs*3), which most probably results in a loss of protein function. The variant cosegregated with the disease in the family. A subsequent pregnancy with ultrasound anomalies was also affected. The proband presented the core phenotypic features of GLYT1 encephalopathy, but also a burst suppression pattern on the electroencephalogram, a clinical feature not previously associated with the disorder. Our results suggest that the appearance of this pattern correlates with higher cerebrospinal fluid glycine levels and cerebrospinal fluid/plasma glycine ratios. A detailed discussion on the possible pathophysiological mechanisms of the disorder is also provided.