Pathogenic Missense Mutations Research Articles

Clinicopathologic, Genomic, and Immunophenotypic Landscape of ATM Mutations in Non-Small Cell Lung Cancer.

ATM is the most commonly mutated DNA damage and repair gene in non-small cell lung cancer (NSCLC); however, limited characterization has been pursued. Clinicopathologic, genomic, and treatment data were collected for 5,172 patients with NSCLC tumors which underwent genomic profiling. ATM IHC was performed on 182 NSCLCs with ATM mutations. Multiplexed immunofluorescence was performed on a subset of 535 samples to examine tumor-infiltrating immune cell subsets. A total of 562 deleterious ATM mutations were identified in 9.7% of NSCLC samples. ATM-mutant (ATMMUT) NSCLC was significantly associated with female sex (P = 0.02), ever smoking status (P < 0.001), non-squamous histology (P = 0.004), and higher tumor mutational burden (DFCI, P < 0.0001; MSK, P < 0.0001) compared with ATM-wild-type (ATMWT) cases. Among 3,687 NSCLCs with comprehensive genomic profiling, co-occurring KRAS, STK11, and ARID2 oncogenic mutations were significantly enriched among ATMMUT NSCLCs (Q < 0.05), while TP53 and EGFR mutations were enriched in ATMWT NSCLCs. Among 182 ATMMUT samples with ATM IHC, tumors with nonsense, insertions/deletions, or splice site mutations were significantly more likely to display ATM loss by IHC (71.4% vs. 28.6%; P < 0.0001) compared with tumors with only predicted pathogenic missense mutations. Clinical outcomes to PD-(L)1 monotherapy (N = 1,522) and chemo-immunotherapy (N = 951) were similar between ATMMUT and ATMWT NSCLCs. Patients with concurrent ATM/TP53 mutations had significantly improved response rate and progression-free survival with PD-(L)1 monotherapy. Deleterious ATM mutations defined a subset of NSCLC with unique clinicopathologic, genomic, and immunophenotypic features. Our data may serve as resource to guide interpretation of specific ATM mutations in NSCLC.

Analysis of concordance between microsatellite instability by next generation sequencing (NGS-MSI) and mismatch repair deficiency by immunohistochemistry (IHC-MMR) in &gt;28,000 colorectal tumors.

30 Background: The use of immune checkpoint inhibitors (ICI) in MSI-H/MMRd CRC patients has profoundly changed the treatment landscape in the metastatic setting; the most recent NICHE-2 trial showed 100% response rate in MSI-H/MMRd locally advanced CRC patients treated with neoadjuvant ICI, further highlighting the importance of identifying these patients accurately to enhance patient care. Here we report the concordance of NGS-MSI and IHC-MMR from a very large cohort of CRC tumors and study the molecular characteristics and clinical outcomes of these patients. Methods: A total of 28,105 CRC tumors were analyzed by NGS (592 genes, NextSeq or WES, NovaSeq) and IHC. MMRd was defined as complete loss of ≥1 IHC stains (MLH1, MSH2, MSH6, or PMS2) and proficient (MMRp) as any positive staining for all four proteins. MSI status was determined from over 7,000 target microsatellite loci covered by NGS. Central pathology review (CPR) of MMR IHC was done on 73 cases with discordant MMR/MSI results. Real-world overall survival was obtained from insurance claims and calculated from either tissue collection or treatment start to last contact; Kaplan-Meier estimates were calculated for molecularly defined patients. Results: For CRC tumors with NGS and IHC, 28031/28105 were concordant (99.74%), 0.09% were MMRd/MSS, and 0.2% were MMRp/MSI-H. After CPR, 23/24 (96%) of MMRd/MSS cases and 46/49 (94%) of MMRp/MSI-H were confirmed. Of the 46 post-CPR MMRp/MSI-H CRC samples, 52% had ≥1 pathogenic missense mutation in including MLH1/PMS2/MSH2/6 or MLH3, MSH3, PMS1 or POLE. Of the 23 post-CPR MMRd/MSS CRC samples, 38% had MLH1 loss, 91% PMS2 loss, no MSH2 loss and 9% MSH6 loss. When comparing clinical outcomes with concordant MSS/MMRp tumors, concordant MSI-H/MMRd patients had significantly longer OS (HR=1.131 [95% CI: 1.02-1.254], p&lt;0.001) and post-ICI survival (HR= 2.695 [95% CI: 1.932-3.76], p&lt;0.001). Compared to MSS/MMRd tumors (N=21), MSI-H/MMRp (N=29) trends to have longer OS (HR=2.163, [95% CI: 0.939-4.983], p=0.064) and insufficient ICI-treated patients were available for analysis. Conclusions: Here we report from &gt;28,000 CRC tumors that the concordance of IHC-MMR/NGS-MSI is 99.74%. Clinical outcome from a large cohort of patients show NGS is not inferior compared to IHC in identifying patients with MSI-H/MMRd. The additional lens that NGS-MSI offers is of value in identifying CRC patients who may benefit from ICI therapy. [Table: see text]

Biophysical characterization of pathogenic missense mutation in the ARID domain of ARID1A.

The ARID1A, AT- rich interactive domain containing protein 1A, is frequently mutated in cancer (e.g., 6% of every cancer and 45% of all ovarian cancer). ARID1A is a member of the Switch/Sucrose non-Fermentable (SWI/SNF) complex responsible for chromatin remodeling and gene expression. Within ARID1A lies an ancient and highly conserved DNA-binding domain (ARID domain) of ∼100 amino acids with a helix-turn-helix motif. The Cancer Genome Atlas (TCGA) reports on mutations in the ARID domain for which 37% of the mutations are frameshift, another 37% are missense and remaining are stop gain. Here we use experimental and computational approaches to identify how missense mutation impaired the ARID-to-DNA interactions by impacting protein stability, binding affinity, and structural dynamics potentially explaining their pathogenicity. Specifically, we identified the most mutated variants in the TCGA (i.e., R1020S, M1022K, K1047Q, G1063V, Q1098H). Next, we used web server bioinformatic pathogenic predictors tools (SAMPDI, CUPSAT, SAMBA-3D, SAAMBE-SEQ, SAAFEC, SAAFEC-SEQ, nMCS) to compute biophysical properties such as the protein stability and DNA binding energy and their likelihood for pathogenicity. We used denaturation assays to experimentally determine the stability of selected variants and compare it with the wildtype. To determine the binding affinity, we run electrophoresis mobility shift assays, intrinsic fluorescence, fluorescence anisotropy, and microscale thermophoresis approaches. We compare experimental and bioinformatic tools to decipher the subtle impacts of missense mutation in the ARID domain. Our findings will help us understand the impact of missense mutations in the structure-function relationship.

GSN gene frameshift mutations in Alzheimer’s disease

BackgroundThe pathogenic missense mutations of the gelsolin (GSN) gene lead to familial amyloidosis of the Finnish type (FAF); however, our previous study identified GSN frameshift mutations existed in patients with...

POST COVID RECURRENT FEVER IN A CHILD WITH GENETIC SUSCEPTIBILITY TO FAMILIAL MEDITERRANEAN FEVER

<h3>Introduction</h3> Children and adults with post-acute sequelae of COVID (PASC) sometimes report recurrent fevers. We present the case of 13-year-old male with PASC recurrent fever who was found to have a heterozygous mutation in the gene associated with Familial Mediterranean fever (FMF). <h3>Case Description</h3> Our patient presented with recurrent fever 6 months after acute SARs-CoV-2 infection in January 2022. He endorsed prior episodes of unexplained fever and flushing prior but his recurrent fevers (39 C) post COVID had become more frequent occurring every few days. Laboratory analysis when he was afebrile demonstrated normal serum ESR, CRP, and CMP and normal cytokine levels. Genetic (In-Vitae) panel testing for autoinflammatory syndromes revealed a pathogenic heterozygous missense mutation in MEFV (p.A744S), the gene that controls pyrin production. A trial of colchicine was recommended, and he was referred to pediatric rheumatology. <h3>Discussion</h3> FMF is a genetic disorder that causes recurrent episodes of fever and almost always presents before age 20. Clinically significant FMF gene mutations lead to poorly functional pyrin; a protein that controls inflammation. Most children look and feel well between episodes but some children have such frequent episodes that they do not fully recover. In such cases, daily use of Colchicine should be considered as a safe and effective medication to control fever episodes. This report highlights the utility of genetic testing to identify potential causes of recurrent fever after COVID. Children with PASC related recurrent fevers should have genetic testing for susceptibility to FEF as this is a potentially treatable condition.

The use of pharmacological chaperones in rare diseases caused by reduced protein stability.

Rare diseases are most often caused by inherited genetic disorders that, after translation, will result in a protein with altered function. Decreased protein stability is the most frequent mechanism associated with a congenital pathogenic missense mutation and it implies the destabilization of the folded conformation in favour of unfolded or misfolded states. In the cellular context and when experimental data is available, a mutant protein with altered thermodynamic stability often also results in impaired homeostasis, with the deleterious accumulation of protein aggregates, metabolites and/or metabolic by-products. In the last decades, a significant effort has enabled the characterization of rare diseases associated to protein stability defects and triggered the development of innovative therapeutic intervention lines, say, the use of pharmacological chaperones to correct the intracellular impaired homeostasis. Here, we review the current knowledge on rare diseases caused by reduced protein stability, paying special attention to the thermodynamic aspects of the protein destabilization, also focusing on some examples where pharmacological chaperones are being tested.

MOLECULAR CYTOGENETIC AND NEXT-GENERATION SEQUENCING STUDIES OF AN ADOLESCENT WITH ACUTE MYELOID LEUKEMIA AND INV(16) ASSOCIATED WITH A KIT PATHOGENIC MUTATION

Inv(16)(p13q22)/CBFb-MYH11 in Acute Myeloid Leukemia (AML) is hard to characterize and comprises about 5%‒7% of all cases and 5%‒9% in children. Inv(16) is associated with a good prognosis both in adults and children. However, approximately 30%‒40% of affected patients experience relapse. In this context, additional type I and II mutations have been related to inv(16). KIT mutations associated with inv(16) have been related to a worse prognosis. Here we describe the clinical and molecular data of a pediatric patient with AML, presenting inv(16) associated with a KIT pathogenic mutation. A 12-year-old boy with bleeding was referred to Pedro Ernesto University Hospital (HUPE), Rio de Janeiro, Brazil. The patient presented with suspected hyperleukocyte acute leukemia. Laboratory tests revealed a WBC count of 80 × 10 3 , a platelet count of 9 × 10 3 , and a hemoglobin level of 5.1 g/dL. Flow cytometry results demonstrated an immunophenotypic profile characteristic of LMA-M4. We conducted G-banding, FISH, and NGS studies. Cytogenetic analysis was non-informative. Based on the clinical and immunophenotypic data, we conducted an experimental design using PML-RARA and CBFB-MYH11 FISH probes. The patient was negative for the PML-RARA fusion, but CBFb-MYH11 was confirmed. The NGS Assay revealed a pathogenic missense mutation in KIT. The results were negative for FLT3/ITD mutations. AML patients presenting inv(16)/CBFb-MYH11 usually have a good prognosis with better remission and overall survival rates compared to those with normal karyotypes. However, due to the high rates of disease relapse, the prognostic value of alterations associated with inv(16)/CBFb-MYH11 has been widely discussed in the literature. Our patient presented with excessive bleeding and heavy hemorrhage, which initially raised the suspicion of acute promyelocytic leukemia. Due to the heterogeneity of the CBFb-MYH11 fusion, featuring distinct clinical and genetic profiles depending on its molecular size molecular and the association of these profiles with specific genomic alterations, a precise genomic characterization is necessary. KIT mutations, such as the one observed in our patient, are present as alterations associated with inv(16) in approximately 9%‒53% of AML cases. Previous studies have demonstrated the potential prognostic impact of KIT mutations in the affected patients. In this context, missense mutations may lead KIT to an overactive state. This type of mutation has been shown to play a pivotal role in mediating clinical response and disease refractoriness in patients treated with targeted therapeutics. Therefore, the information provided through the NGS can be crucial for treatment adaptation. In this work, we contribute with the literature showing the clinical, molecular, and genomic data of an adolescent with AML and inv(16) associated with a KIT pathogenic mutation. Besides, we highlight the importance of NGS studies in characterizing the genomic profile of patients affected by this neoplasm. Although, the prognostic implications of these findings remain to be investigated, especially in the context of new generation tyrosine kinase inhibitors targeting KIT in future clinical trials for inv(16)/CBFb-MYH11 pediatric patients.

Severe Phenotype in Patients with X-linked Hydrocephalus Caused by a Missense Mutation in L1CAM.

Objective:The study aimed to show the clinical characteristics and prognosis of the L1 syndrome in patients with L1CAM mutations in the extracellular region.Materials and Methods:Three affected boys and their siblings and parents from a large family were included in this study. Genetic etiology was investigated by whole-exome sequencing in the index patient. The pathogenic variant was detected by whole-exome sequencing and was validated by Sanger sequencing in 3 patients and other family members.Results:We present 2 brothers and their cousin with prenatal onset hydrocephalus, severe developmental and speech delay, corpus callosum agenesis/hypogenesis, epilepsia, and adducted thumbs. A hemizygous missense mutation NM_024003 (c.A2351G:p.Y784C) on exon 18 of L1CAM gene was found in the 3 patients and their carrier mother. This missense mutation in the conserved region of the second fibronectin type III-like repeats located in the extracellular region of L1CAM resulted in the severe phenotype of X-linked inherited L1 syndrome in the patients.Conclusion:L1 syndrome should be considered in the differential diagnosis of male children with intellectual disability, hydrocephalus, and adducted thumbs. While truncating mutations of L1CAM may cause a more severe phenotype, missense mutations cause milder forms. However, pathogenic missense mutations affecting key amino acid residues lead to severe phenotype likely.

Loss-of-function, gain-of-function and dominant-negative mutations have profoundly different effects on protein structure

Most known pathogenic mutations occur in protein-coding regions of DNA and change the way proteins are made. Taking protein structure into account has therefore provided great insight into the molecular mechanisms underlying human genetic disease. While there has been much focus on how mutations can disrupt protein structure and thus cause a loss of function (LOF), alternative mechanisms, specifically dominant-negative (DN) and gain-of-function (GOF) effects, are less understood. Here, we investigate the protein-level effects of pathogenic missense mutations associated with different molecular mechanisms. We observe striking differences between recessive vs dominant, and LOF vs non-LOF mutations, with dominant, non-LOF disease mutations having much milder effects on protein structure, and DN mutations being highly enriched at protein interfaces. We also find that nearly all computational variant effect predictors, even those based solely on sequence conservation, underperform on non-LOF mutations. However, we do show that non-LOF mutations could potentially be identified by their tendency to cluster in three-dimensional space. Overall, our work suggests that many pathogenic mutations that act via DN and GOF mechanisms are likely being missed by current variant prioritisation strategies, but that there is considerable scope to improve computational predictions through consideration of molecular disease mechanisms.

Functional Restoration of BRCA1 Nonsense Mutations by Aminoglycoside-Induced Readthrough.

BRCA1 is a major tumor suppressor that functions in the accurate repair of DNA double-strand breaks via homologous recombination (HR). Nonsense mutations in BRCA1 lead to inactive truncated protein products and are associated with high risk of breast and ovarian cancer. These mutations generate premature termination codons (PTCs). Different studies have shown that aminoglycosides can induce PTC suppression by promoting stop codon readthrough and restoring full-length (FL) protein expression. The use of these compounds has been studied in clinical trials for genetic diseases such as cystic fibrosis and Duchenne muscular dystrophy, with encouraging results. Here we show proof-of-concept data demonstrating that the aminoglycoside G418 can induce BRCA1 PTC readthrough and restore FL protein synthesis and function. We first demonstrate that G418 treatment restores BRCA1 FL protein synthesis in HCC1395, a human breast tumor cell line carrying the R1751X mutation. HCC1395 cells treated with G418 also recover HR DNA repair and restore cell cycle checkpoint activation. A set of naturally occurring BRCA1 nonsense variants encoding different PTCs was evaluated in a GFP C-terminal BRCA1 construct model and BRCA1 PTC readthrough levels vary depending on the stop codon context. Because PTC readthrough could generate FL protein carrying pathogenic missense mutations, variants representing the most probable acquired amino acid substitutions in consequence of readthrough were functionally assessed by a validated transcription activation assay. Overall, this is the first study that evaluates the readthrough of PTC variants with clinical relevance in the breast and ovarian cancer-predisposing gene BRCA1.

Abstract 57: Patients with germline ATM mutations develop clonal hematopoiesis characterized by co-occurrence of multiple somatic ATM alterations

Abstract BACKGROUND The DNA repair gene ATM is remarkable for its roles across the spectrum of neoplastic biology, including inherited risk of multiple malignancies, somatic tumor biology, and clonal hematopoiesis (CH). We hypothesized that comparison of liquid biopsy (LB) versus tumor tissue (TT) from germline ATM pathogenic mutation carriers (gATM+) might offer additional insights into the role of ATM in CH. METHODS 34,825 LB samples and 384,847 TT specimens were sequenced for up to 324 genes during routine clinical care and analyzed for all classes of genomic alterations (Frampton 2013, Woodhouse 2020). Individual variants were assessed for germline versus somatic origin using the validated somatic-germline zygosity algorithm (Sun 2018). RESULTS Germline ATM mutations were observed in 0.9% (113/12,217) of LB and 0.8% (3,029/384,847) of TT (P&amp;lt;0.001). Up to 27 ATM mutations were identified per gATM+ sample, with a range of 1-27 in LB and 1-8 in TT. Co-occurrence of 3+ ATM mutations in a single sample was 18-fold higher among gATM+ subjects undergoing LB versus TT (23.9% vs. 1.2%, P&amp;lt;0.001). In LB, the incidence of 3+ ATM mutations increased with age for all groups, but the frequency in gATM+ subjects diverged as patient age advanced. In the age 80+ subgroup, 57.1% (28/49) of gATM+ subjects had 3+ ATM mutations, compared to 3.3% (133/4,055) of gATM− subjects (P&amp;lt;0.001). A significant difference was maintained after correcting for the contribution of the germline ATM mutation to the total number of ATM mutations in gATM+ subjects. We hypothesized that multiple ATM variants per sample could be caused by high TMB or positive selection for multiple subclonal hits in the ATM gene. In TT, observation of 3+ ATM mutations was associated with high TMB in 80.5% (513/637) of cases. In contrast, 88.5% (555/627) of LB samples with 3+ ATM mutations had low TMB (Pdiff&amp;lt;0.001), a pattern consistent with positive selection in LB. LB from gATM+ cases with 3+ ATM mutations all had one variant with ~50% variant allele fraction (VAF), while 80.0% (546/682) of the co-mutations had VAF &amp;lt;1%. Co-mutations included 32.0% (793/2,485) truncating alterations and 68.1% (1,692/2,485) missense alterations. Low VAF missense co-mutations were clustered in the FAT and PIKKc domains, which are known pathogenic missense mutation hotspots. This distribution suggests that some missense alterations within these hotspots that are currently classified as VUS are under positive selection and therefore warrant consideration for reclassification. CONCLUSION The finding of numerous ATM mutations under positive selection in gATM+ subjects was specific to LB, increased in incidence with age, and co-mutations tended to have low VAF. All these features are consistent with polyclonal hematopoiesis (polyCH) involving ATM co-mutations in gATM+ subjects. Additional investigation is needed to extend our understanding of this phenomenon. Citation Format: Brennan Decker, Angela A. Kou, Tyler Janovitz, Douglas A. Mata, Ethan S. Sokol, Dexter X. Jin, Hanna Tukachinsky, Jo-Anne Vergilio, Julia A. Elvin, Geoffrey R. Oxnard. Patients with germline ATM mutations develop clonal hematopoiesis characterized by co-occurrence of multiple somatic ATM alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 57.

Insilico identification of the rare-coding pathogenic mutations and structural modeling of human NNAT gene associated with anorexia nervosa.

Increased susceptibility towards anorexia nervosa (AN) was reported with reduced levels of neuronatin (NNAT) gene. We sought to investigate the most pathogenic rare-coding missense mutations, non-synonymous single-nucleotide polymorphisms (nsSNPs) of NNAT and their potential damaging impact on protein function through transcript level sequence and structure based in silico approaches. Gene sequence, single nucleotide polymorphisms (SNPs) of NNAT was retrieved from public databases and the putative post-translational modification (PTM) sites were analyzed. Distinctive in silico algorithms were recruited for transcript level SNPs analyses and to characterized high-risk rare-coding nsSNPs along with their impact on protein stability function. Ab initio 3D-modeling of wild-type, alternate model prediction for most deleterious nsSNP, validation and recognition of druggable binding pockets were also performed. AN 3D therapeutic compounds that followed rule of drug-likeness were docked with most pathogenic variant of NNAT to estimate the drugs' binding free energies. Conclusively, 10 transcript (201-205)-based nsSNPs from 3 rare-coding missense variants, i.e., rs539681368, rs542858994, rs560845323 out of 840 exonic SNPs were identified. Transcript-based functional impact analyses predicted rs539681368 (C30Y) from NNAT-204 as the high-risk rare-coding pathogenic nsSNP,deviating protein functions. The 3D-modeling analysis of AN drugs' binding energies indicated lowest binding free energy (ΔG) and significant inhibition constant (Ki) with mutant models C30Y. Mutant model (C30Y) exhibiting significant drug binding affinity and the commonest interaction observed at the acetylation site K59. Thus, based on these findings, we concluded that the identified nsSNP may serve as potential targets for various studies, diagnosis and therapeutic interventions. No level of evidence-open access bioinformatics research.

Cancer genomic profiling identified dihydropyrimidine dehydrogenase deficiency in bladder cancer promotes sensitivity to gemcitabine

Chemotherapy is a standard therapy for muscle-invasive bladder cancer (MIBC). However, genomic alterations associated with chemotherapy sensitivity in MIBC have not been fully explored. This study aimed to investigate the genomic landscape of MIBC in association with the response to chemotherapy and to explore the biological role of genomic alterations. Genomic alterations in MIBC were sequenced by targeted exome sequencing of 409 genes. Gene expression in MIBC tissues was analyzed by western blotting, immunohistochemistry, and RNA microarray. Cellular sensitivity to gemcitabine and gemcitabine metabolite was examined in bladder cancer cells after modulation of candidate gene. Targeted exome sequencing in 20 cases with MIBC revealed various genomic alterations including pathogenic missense mutation of DPYD gene encoding dihydropyrimidine dehydrogenase (DPD). Conversely, high DPYD and DPD expression were associated with poor response to gemcitabine-containing chemotherapy among patients with MIBC, as well as gemcitabine resistance in bladder cancer cells. DPD suppression rendered cells sensitive to gemcitabine, while DPD overexpression made cells gemcitabine-resistant through reduced activity of the cytotoxic gemcitabine metabolite difluorodeoxycytidine diphosphate. This study revealed the novel role of DPD in gemcitabine metabolism. It has been suggested that DPYD genomic alterations and DPD expression are potential predictive biomarkers in gemcitabine treatment.

A Budding Yeast Model System to Define Biological Pathways Altered by Pathogenic Missense Mutations in Histone Genes Identifies a Link between Histone H3K36 and the TOS4 Gene

Histones are critical for both packaging DNA into chromatin within the cell nucleus and for regulating all aspects of gene expression. DNA is packaged into nucleosomes, which consist of a hetero‐octamer of histones H2A, H2B, H3, and H4. Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. These mutations, termed oncohistone mutations, have been linked to multiple types of cancer, including pediatric gliomas and head and neck cancers. In humans, a histone H3 lysine 36 to methionine missense mutation (H3K36M) causes chondroblastomas. Our lab has employed a budding yeast model system to explore how missense mutations such as H3K36M alter histone function. These studies, as well as those from previous groups, reveal that changes at H3K36 confer sensitivity to growth on plates containing caffeine, suggesting that H3K36 mutants show changes in stress response pathways. To further define these pathways, the laboratory employed a high copy suppressor screen to identify genes that when overexpressed could suppress the caffeine‐sensitive growth phenotype of H3K3M cells. This screen identified several genes linked to epigenetic regulation, including TOS4, which encodes a forkhead‐associated (FHA) domain protein that interacts with Rpd3L and Set3 histone deacetylase complexes. To begin to define the function of Tos4 required for this suppression, two amino acid substitutions within the Tos4 FHA domain that disrupt interaction with histone deacetylase complexes (R122A, N161A) were generated. This Tos4 variant still suppresses the caffeine‐sensitive growth of H3K36M cells, suggesting these interactions are not required for suppression. My studies focus on defining the function of Tos4 required for this suppression. Results from such studies may provide insight for novel therapeutic targets in humans diagnosed with these distinct oncohistone‐driven cancer types.

Sodium Phenylbutyrate Rescues Thyroid Hormone Transport in Brain Endothelial-Like Cells.

Background: Monocarboxylate transporter 8 (MCT8) deficiency is a rare genetic disease leading to a severe developmental delay due to a lack of thyroid hormones (THs) during critical stages of human brain development. Some MCT8-deficient patients are not as severely affected as others. Previously, we hypothesized that these patients' mutations do not affect the functionality but destabilize the MCT8 protein, leading to a diminished number of functional MCT8 molecules at the cell surface. Methods: We have already demonstrated that the chemical chaperone sodium phenylbutyrate (NaPB) rescues the function of these mutants by stabilizing their protein expression in an overexpressing cell system. Here, we expanded our previous work and used iPSC (induced pluripotent stem cell)-derived brain microvascular endothelial-like cells (iBMECs) as a physiologically relevant cell model of human origin to test for NaPB responsiveness. The effects on mutant MCT8 expression and function were tested by Western blotting and radioactive uptake assays. Results: We found that NaPB rescues decreased mutant MCT8 expression and restores transport function in iBMECs carrying patient's mutation MCT8-P321L. Further, we identified MCT10 as an alternative TH transporter in iBMECs that contributes to triiodothyronine uptake, the biological active TH. Our results indicate an upregulation of MCT10 after NaPB treatment. In addition, we detected an increase in thyroxine (T4) uptake after NaPB treatment that was not mediated by rescued MCT8 but an unidentified T4 transporter. Conclusions: We demonstrate that NaPB is suitable to stabilize a pathogenic missense mutation in a human-derived cell model. Further, it activates TH transport independent of MCT8. Both options fuel future studies to investigate repurposing the Food and Drug Administration-approved drug NaPB in selected cases of MCT8 deficiency.

Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants.

Many pathogenic missense mutations are found in protein positions that are neither well-conserved nor fall in any known functional domains. Consequently, we lack any mechanistic underpinning of dysfunction caused by such mutations. We explored the disruption of allosteric dynamic coupling between these positions and the known functional sites as a possible mechanism for pathogenesis. In this study, we present an analysis of 591 pathogenic missense variants in 144 human enzymes that suggests that allosteric dynamic coupling of mutated positions with known active sites is a plausible biophysical mechanism and evidence of their functional importance. We illustrate this mechanism in a case study of β-Glucocerebrosidase (GCase) in which a vast majority of 94 sites harboring Gaucher disease-associated missense variants are located some distance away from the active site. An analysis of the conformational dynamics of GCase suggests that mutations on these distal sites cause changes in the flexibility of active site residues despite their distance, indicating a dynamic communication network throughout the protein. The disruption of the long-distance dynamic coupling caused by missense mutations may provide a plausible general mechanistic explanation for biological dysfunction and disease.

Shedding light on the phenotypic\u2013genotypic correlation of rare treatable and potentially treatable pediatric movement disorders

BackgroundAdvances in genetic science have led to the identification of many rare treatable pediatric movements disorders (MDs). We explored the phenotypic–genotypic spectrum of pediatric patients presenting with MDs. By this, we aimed at raising awareness about such rare disorders, especially in our region. Over the past 3 years, we reviewed the demographic data, clinical profile, molecular genetics and other diagnostic workups of pediatric patients presenting with MDs.ResultsTwelve patients were identified; however, only six patients were genetically confirmed. The phenomenology of MDs ranged from paroxysmal kinesigenic choreoathetosis (1 patient), exercise-induced dyskinesia (2 patients), ataxia (2 patients) and dystonia (2 patients). Whole-exome sequencing in addition to the functional studies for some patients revealed a specific genetic diagnosis being responsible for their MDs. The genetic diagnosis of our patients included infantile convulsions and paroxysmal choreoathetosis syndrome and episodic ataxia due to “pathogenic homozygous mutation of PRRT2 gene,” glucose transporter type 1 deficiency-exercise induced dyskinesia due to “De Novo pathogenic heterozygous missense mutation of exon 4 of SLC2A1 gene,” aromatic L amino acid decarboxylase deficiency due to “pathogenic homozygous mutation of the DDC gene,” myopathy with extrapyramidal signs due to “likely pathogenic homozygous mutations of the MICU1 gene,” mitochondrial trifunctional protein deficiency due to “homozygous variant of uncertain significance (VUS) of HADHB gene” and glutaric aciduria II with serine deficiency due to “homozygous VUS for both ETFDH and PHGDH genes.” After receiving the treatment as per recognized treatment protocols, two patients showed complete resolution of symptoms and the rest showed variable responses.ConclusionIdentifying the genetic etiology of our patients guided us to provide either disease-specific treatment or redirected our management plan. Hence, highlighting the value of molecular genetic analysis to avoid the diagnostic odyssey and identify treatable MDs.

Generation of an induced pluripotent stem cell line (EURACi014-A) from a Parkinson’s disease patient with an A53T mutation in the SNCA gene by an integration-free reprogramming method

The SNCA gene encodes the presynaptic α-synuclein (aSyn) protein, and its mutations are associated with autosomal dominant Parkinson’s disease (PD). We describe the generation of an induced pluripotent stem cell (iPSC) line of a patient carrying a pathogenic Ala53Thr missense mutation in the SNCA gene. Human dermal fibroblasts were reprogrammed using a non-integrating episomal method. The generated iPSC line (EURACi014-A; iPS-1.1) shows expression of pluripotency markers, the potential to differentiate into all three germ layers, and a stable karyotype. Hence, this line represents a valuable resource for the study and modeling of the processes directly controlled by aSyn.

Three patients of transthyretin amyloidosis in a Japanese family with amyloidogenic transthyretin Thr49Ser (p.Thr69Ser) variant

Transthyretin (TTR)-related hereditary amyloidosis (ATTRv) is a rare autosomal dominant disorder that is caused by pathogenic missense mutation of the TTR gene. As of today, more than 150 TTR gene variants have been reported to occur as causal mutations. Herein, we present three familial patients of ATTRv caused by the Thr49Ser (p.Thr69Ser) variant, including their phenotypes and penetrance.The first patient was a 68-year-old woman with a history of carpal tunnel syndrome, who was referred to our department with heart failure symptoms. Echocardiography, 99mTechnetium (Tc)-pyrophosphate scintigraphy, and myocardial biopsy confirmed her diagnosis as TTR-related amyloidosis. Genetic testing for the TTR gene was performed, which confirmed the presence of a Thr49Ser (p.Thr69Ser) variant. The second patient, a 45-year-old woman, who was the niece of the first patient, presented with dyspnea on exertion. Her clinical manifestations included cardiac symptoms in addition to polyneuropathy. Similarly, myocardial biopsy showed TTR amyloid deposition within cardiac tissues, and TTR gene sequencing detected the presence of a Thr49Ser (p.Thr69Ser) variant. The final patient was a 42-year-old man, who was the nephew of the first patient, presented with numbness in his hands. Abdominal wall fat pad biopsy showed TTR amyloid deposition, and TTR gene sequencing was performed considering the familial history to confirm the presence of Thr49Ser (p.Thr69Ser) variant. No cardiac symptoms or dysfunctions have been observed yet, but imaging has detected TTR amyloid deposition in the heart.The present three patients with Thr49Ser (p.Thr69Ser) variant showed variation in phenotypes including cardiac and neurological manifestations at a fairly young age. In addition, the familial relationship in this report suggested that this variant is highly penetrant. Early genetic diagnosis due to collecting the genetic information from family medical history may be beneficial to improve patient prognosis via early therapeutic intervention.

Analysis of missense variants in the human genome reveals widespread gene-specific clustering and improves prediction of pathogenicity

SummaryWe used a machine learning approach to analyze the within-gene distribution of missense variants observed in hereditary conditions and cancer. When applied to 840 genes from the ClinVar database, this approach detected a significant non-random distribution of pathogenic and benign variants in 387 (46%) and 172 (20%) genes, respectively, revealing that variant clustering is widespread across the human exome. This clustering likely occurs as a consequence of mechanisms shaping pathogenicity at the protein level, as illustrated by the overlap of some clusters with known functional domains. We then took advantage of these findings to develop a pathogenicity predictor, MutScore, that integrates qualitative features of DNA substitutions with the new additional information derived from this positional clustering. Using a random forest approach, MutScore was able to identify pathogenic missense mutations with very high accuracy, outperforming existing predictive tools, especially for variants associated with autosomal-dominant disease and cancer. Thus, the within-gene clustering of pathogenic and benign DNA changes is an important and previously underappreciated feature of the human exome, which can be harnessed to improve the prediction of pathogenicity and disambiguation of DNA variants of uncertain significance.