Pathogenic Missense Mutations Research Articles

Prevalence of germline TP53 variants among early-onset breast cancer patients from Polish population

BackgroundThe objective of this study was to determine spectrum and prevalence of germline mutations in TP53 gene among Polish women with early-onset breast cancer (BC), which has not been determined until now.MethodsA cohort of 100 females with BC diagnosed ≤ 30 years of age and with a positive family history of cancer was used as a discovery cohort. 1880 women with BC ≤ 45 years old and a control group of 2000 healthy women were genotyped as a replication phase of this study.ResultsFour heterozygous pathogenic missense mutations were detected in a group of 100 patients with early-onset breast cancer. On the basis of software prediction and available literature data, all these variants were defined as pathogenic. None of these TP53 variants were detected among 1880 breast cancer patients and 2000 healthy controls. No large mutations were found among early-onset cases using MLPA reaction.ConclusionGermline pathogenic TP53 variants were found in 4% early-onset Polish BC patients. No founder mutations were identified in Polish population. To improve the treatment and surveillance screening, the search for germline TP53 pathogenic variants is recommended for all female BC cases diagnosed ≤ 30 years old.

Unexpected expression of mismatch repair protein is more commonly seen with pathogenic missense than with other mutations in Lynch syndrome

It has been observed that some patients with colorectal cancer due to germline or double somatic pathogenic variants in the mismatch repair (MMR) genes may have intact protein expression in their tumors as assessed by immunohistochemistry (IHC). This has been speculated to occur more frequently in Lynch syndrome (LS) cases due to pathogenic missense mutations, leading to expression of a full-length but nonfunctional protein with retained antigenicity. Our goals were to study the frequency of unexpected MMR expression in colorectal cancers among LS cases with missense mutations, LS cases with truncating mutations, as well as cases with double somatic MMR mutationsand evaluate if the unexpected MMR expression is more common in certain categories. IHC slides were available for 82 patients with MMR deficiency without methylation, which included 56 LS cases and 26 double somatic MMR mutation cases. Sixteen of 82 MMR-defective cases showed unexpected MMR expression, with 10 cases showing tumor staining weaker than the control and 6 cases (7%) showing intact staining. Unexpected MMR expression was most commonly seen with LS cases with missense mutations (4 of 9, 44%), followed by MMR double somatic mutation cases (7 of 26, 27%), and finally by LS cases with truncating mutations (5 of 47, 11%). Cautious interpretation of MMR IHC is advised when dealing with tumor staining that is weaker than the control regardless of the percentage of tumor stainingas these cases may harbor pathogenic MMR gene mutations. Missense mutations may account for some LS cases that may be missed by IHC alone. Strict adherence to proper interpretation of IHC with attention to staining intensity and the status of heterodimer partner protein will prevent many potential misses.

Molecular profiling of lamellar ichthyosis pathogenic missense mutations on the structural and stability aspects of TGM1 protein

Lamellar ichthyosis (LI) is a rare inherited disease where affected infants present a extensive skin scaling characterized by hyperkeratosis. Inherited mutations in the Transglutaminase 1 (TGM1) protein is one of the known causative genetic factor for the LI. The main objective of this study is to explore the impact of LI causative missense mutations on the structural and stability aspects of TGM1 protein using structural modeling, molecular docking and molecular dynamics approaches. By testing all LI causative TMG1 mutations against multiple stability prediction methods, we found that L362R and L388P mutations positioned in the Transglut_core domain were most destabilizing to the stability of TGM1 protein. These 2 mutations were 3D protein modeled and further analyzed by molecular docking and dynamic simulation methods. Molecular docking of these TGM1 mutant structures with chitosan, a natural polyphenolic compound and known inducer for transglutaminase enzyme, has shown stable molecular interactions between the native TGM1-chitosan and TGM1(L388P)-chitosan complex, when compared to the TGM1(L362R)-chitosan complex. Interestingly, molecular dynamics analysis have also yielded similar findings, where L388P-chitosan complex is shown to develop B-sheets and attain better stability, whereas TGM1-L362R complex possessed coils over the simulation period, pointing its highly destabilizing behavior on the protein structure. This study concludes that missense mutations in Transglut_core domain of the TGM1 are deleterious to the stability and structural changes of TGM1 protein and also suggest that chitosan molecule could act as a natural activator against few pathogenic TGM1 mutations. Communicated by Ramaswamy H. Sarma

Computational approach towards identification of pathogenic missense mutations in AMELX gene and their possible association with amelogenesis imperfecta.

Amelogenin gene (AMEL-X) encodes an enamel protein called amelogenin, which plays a vital role in tooth development. Any mutations in this gene or the associated pathway lead to developmental abnormalities of the tooth. The present study aims to analyze functional missense mutations in AMEL-X genes and derive an association with amelogenesis imperfecta. The information on missense mutations of human AMEL-X gene was collected from Ensembl database (https://asia.ensembl.org). Three different computational tools viz., SIFT, PolyPhen and PROVEAN were used to identify the deleterious or pathogenic forms of mutations in the gene studied. I-Mutant Suit was used to identify the stability of the proteins identified as deleterious by the three tools. Further, MutPred analysis revealed the pathogenicity of these mutations. Among 96 missense variants reported in AMEL-X gene, 18 were found to be deleterious using the three prediction tools (SIFT, PolyPhen and PROVEAN). When these variants were subjected to protein stability analysis, about 14 missense variants showed decreased stability whereas the other 8 variants showed increased stability. Further, these variants were analyzed using MutPred which identified 9 variants to be highly pathogenic. ExAC database revealed that all the pathogenic mutations had a minor allele frequency less than 0.01. The in silico analysis revealed highly pathogenic mutations in amelogenin gene which could have a putative association with amelogenesis imperfecta. These mutations should be screened in patients for early diagnosis of susceptibility to AI.

Identification of SDHA germline mutations in sporadic SDHA mutant gastrointestinal stromal tumors (GIST): The need of a genetic counselling.

11537 Background: SDH- deficient GIST, as defined by the loss of expression of SDHB, account up to about 10% of all gastric GIST and generally affect younger population. Germline mutations in SDHB, SDHC, and SDHD occur in about 20–30% of SDH- deficient, that may be referred to a hereditary condition known as hereditary GIST-paraganglioma syndrome (Carney-Stratakis Syndrome), whereas germline SDHA mutations have been rarely described in apparently sporadic cases. Currently, even germline testing is recommended for SDH- deficient GIST, there are no clear guidelines for genetic counselling and follow-up of SDH x mutation carriers and relatives, especially for SDHA mutant GIST not yet linked to well-defined hereditary syndrome. The aim of this work was to study the SDHA gene in the normal DNA of patients with SDHA mutant GIST. Methods: Thirteen patients carrying SDHA-mutant GIST were studied (8F/5M). Median age of diagnosis was 45,9 years (range 25-74). All GIST were located in the stomach and 3 patients out 13 presented a metastatic disease. In all cases except one, the GIST was the only cancer presentation and no personal or familial history of cancer was revealed. All cases were negative for SDHB immunohistochemistry. Germline mutations were identified through Sanger sequencing of SDHA in the normal counterpart. Results: Germline mutations were identified in all patients for which normal counterpart was available: 4 cases harboured truncating mutations (S384X, R31X and W119X); 5 cases carried pathogenic missense mutations (G233V, R171H, R589Q, G257A and R600Q) and 2 cases had splice site alterations (c.457-3_457-1 delCAG and c.456+9 C > T). In 8 cases the tumor DNA showed the loss of the corresponding wild-type allele, while in the other 3 cases compound heterozygosity for an additional somatic mutation was detected (R589W, R451C,and R171C). In 2 patients, unfortunately, normal DNA was not available, however both tumours carried two mutational hits on SDHA (one with heterozygous G419R and E564K, and one with homozygous R585Q). Of note, 5 patients presented un-usaul SDHA related clinical characteristics as were not young adult ( > 50 years-old) or no multifocal GIST. Conclusions: We demonstrated that germline SDHA mutations are highly frequent in SDHA- deficient GIST. Therefore, although a clear syndrome has not been defined, genetic counselling and follow-up of SDHA mutation carriers and relatives should be clarified.

SUN-LB13 Idiopathic Infantile Hypercalcemia Secondary to CYP24A1 Mutation: A Rare Case Without Exogenous Vitamin D Supplementation

Background: Mutations in CYP24A1, which encodes 24-hydroxylase, the key enzyme for Vitamin D breakdown, cause symptomatic hypercalcemia and nephrocalcinosis in infants on Vitamin D supplementation. New, symptomatic diagnoses of idiopathic infantile hypercalcemia without exogenous supplementation are rare. Previous case reports describe a seasonal effect with worsening hypercalcemia and hypercalciuria during summertime, attributed to increased sun exposure and endogenous Vitamin D production. Clinical Case: A 10-month-old female presented to endocrine care with hypercalcemia and nephrocalcinosis, detected on renal ultrasound (US) due to history of UTI. Her first renal US and serum calcium (Ca) at 3mo of age were normal. Subsequent renal US at 6mo and 9mo of age demonstrated nephrocalcinosis, prompting nephrology and endocrine evaluation. History was significant for failure to thrive. She was born in the fall, with worsening hypercalcemia and nephrocalcinosis during the summer. Diet consisted of standard infant formula and age appropriate solid foods with no added Vitamin D supplementation (~300 IU/day in her formula). She had no family history of nephrocalcinosis, nephrolithiasis, bone disease, or disorders of Ca regulation. Initial labs were notable for Ca corrected for albumin 11.5 (7.8-11.1 mg/dL), PTH <4 (8.7-77.1 pg/mL), 25-OH-Vitamin D 81 (30-96 ng/mL), 1,25-OH-Vitamin D 23.1 (26.1-95 pg/mL), Urine Ca/creatinine ratio of 0.9 mg/mg (<0.81), normal chromosomal microarray, and normal thyroid function tests. She was started on reduced mineral formula PM 60/40. One week later, repeat Ca level increased to Ca corrected 14.2 (7.8-11.1 mg/dL). She was admitted for IV fluids and pamidronate, and was transitioned to a low Ca and Vitamin D formula (Calcilo), with improvement in Ca levels. Testing revealed an increased ratio of 25-OH-Vitamin D to 24,25-OH-Vitamin D of 192 (normal <25), and genetic testing showed 2 pathogenic missense mutations in CYP24A1 genes: c.1226T>C p.(Leu409Ser) and c.1186C>T p.(Arg396Trp). The Leu409Ser mutation has shown a small amount of 24-hydroxylase activity in previous in vitro analysis. She has continued a low Ca diet with stable Ca corrected of 10.7-10.8 (8.7-9.8 mg/dL) and significantly improved weight gain. Conclusion: This is one of the few documented cases of symptomatic idiopathic infantile hypercalcemia secondary to CYP24A1 mutation in an infant without exogeneous Vitamin D supplementation. Her nephrocalcinosis and hypercalcemia worsened over the summer, suggesting increased sun exposure may have been a contributing factor. This case demonstrates that 1,25-OH-Vitamin D levels may be normal or low in this condition, particularly for individuals with the Leu409Ser mutation who may retain partial 24-hydroxylase function.

Molecular dynamics simulations to decipher the structural and functional consequences of pathogenic missense mutations in the galactosylceramidase (GALC) protein causing Krabbe’s disease

Krabbe disease (KD), also known as globoid cell leukodystrophy disease, is an autosomal recessive lysosomal storage genetic disorder, which is caused by the deficiency of galactocerebrosidase (GALC) coding gene (GALC). This study aimed to use extensive computational pipelines in understanding the missense mutations in GALC. We retrieved 176 mutations from the public databases and subjected them to pathogenicity, stability, and conservation analyses. The PredictSNP, iStable, and ConSurf prediction tools predicted 45, 95, and 47 mutations to be deleterious, destabilizing, and highly conserved, respectively. The R396L and R396W were the most deleterious and destabilizing to GALC, and were therefore prioritized for further analysis. Systematic validation on the impact of the R396L and R396W mutations to the chaperone alpha lobeline was performed using the molecular docking approach. The docking analysis revealed that the mutant R396W interacted with minimal binding affinity compared with both the R396L mutant and native GALC. Furthermore, the repetitive molecular dynamics simulation analysis showed that the mutant R396W demonstrated less compactness and reduced number of intramolecular hydrogen bonds compared with the mutant R396L and the native GALC. Overall, we observed higher structural and functional modifications in R396W positioned in the substrate-binding site. This was highly supported by the MMPBSA and DSSP analysis of the GROMACS. DSSP showed the transformation of turns to bends, indicating a loss of stability due to the R396W mutation. This study is expected to serve as a platform for prioritizing mutant proteins that could be a platform for both drug and target therapeutics Communicated by Ramaswamy H. Sarma

Oral-Facial-Digital Syndrome Type 1: Further Clinical and Molecular Delineation in 2 New Families.

Oral-facial-digital syndrome type 1 (OFD1) [OMIM 311200] is a rare genetic disorder associated with congenital anomalies of the oral cavity, face, and digits. This condition is associated with mutations in the OFD1 gene. Our objective was to recruit patients with the OFD1 clinical phenotype without genetic confirmation, aiming to identify genetic variants in the OFD1 gene. Three patients from 2 unrelated families were recruited into our study. We employed a variety of genomic techniques on these patients, including candidate gene analysis, array comparative genomic hybridization, whole-exome sequencing, and whole-genome sequencing. We investigated 3 affected patients from 2 unrelated families with a clinical diagnosis of OFD1. We discovered a novel pathogenic dominant missense mutation c.635G>C (p.Arg212Pro) in the OFD1 gene in one family. A novel frameshift, loss-of-function mutation c.306delA (p.Glu103LysfsTer42) was detected in the affected patient in the second family. These new genetic variants will add to the spectrum of known OFD1 mutations associated with the OFD1 disorder. Our study also confirms the variable phenotypic presentation of OFD1 and its well-recognized association with central nervous system malformations and renal anomalies. Molecular diagnostic confirmation achieved in these families will have positive implications for their medical management.

Map of Genetically Constrained Regions in Human Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) and regions (IDRs) are pivotal for biological processes like signaling, recognition, and regulation, and have been found to be implicated in many diseases. Evolutionary selection of IDPs thus has been extensively investigated through multiple sequence alignment (MSA) methods. Recent large-scale catalogues of human genome sequences have enabled the detection of constrained regions depleted of genetic variation. Here, we aim to identify the prevalence, characteristics, and functions of genetically constrained IDRs. We mapped the missense tolerance ratio (MTR), quantifying the depletion of amino acid substitutions in genome aggregation database (a sample of 138,632 individuals) on 210 human IDPs from DisProt7.0 database. We identified 63 IDRs (length<100 residues) having 25th percentile MTR, indicating these IDRs are >75% constrained. For further verification, we annotated IDRs using MPC (Missense badness, PolyPhen-2, and Constraint) score to identify regions in genes that are depleted of substitutions. We found 70 more IDRs with MPC < 1.0 (lower observed versus expected substitutions). We then investigated the functions and interactions of the 133 missense constrained IDRs (mcIDRs). We spotted 25 mcIDRs carrying molecular recognition features (assemblers or effectors), 14 acting as flexible linkers/spacers, 51 to have disordered state and 45 involved in protein-protein interactions. Looking further into the cross-species MSA (BLAST search, 50% identity cut-off), we found 53 mcIDRs with >90% and 26 mcIDRs with <30% conserved residues. We further examined the enrichment of disease-associated loci in mcIDRs, and found 645 rare singletons (new mutations in evolution) and 635 pathogenic missense mutations. We exploited two genetic constraint metrics to identify IDRs that are restricted against substitutions. The observed functional diversity of the constrained IDRs, their contrast with evolutionary conservations, and their accumulation of disease-associated loci will advance our understanding of IDP-based regulations in human pathology.

Interpretation of somatic POLE mutations in endometrial carcinoma.

Pathogenic somatic missense mutations within the DNA polymerase epsilon (POLE) exonuclease domain define the important subtype of ultramutated tumours (‘POLE‐ultramutated’) within the novel molecular classification of endometrial carcinoma (EC). However, clinical implementation of this classifier requires systematic evaluation of the pathogenicity of POLE mutations. To address this, we examined base changes, tumour mutational burden (TMB), DNA microsatellite instability (MSI) status, POLE variant frequency, and the results from six in silico tools on 82 ECs with whole‐exome sequencing from The Cancer Genome Atlas (TCGA). Of these, 41 had one of five known pathogenic POLE exonuclease domain mutations (EDM) and showed characteristic genomic alterations: C>A substitution > 20%, T>G substitutions > 4%, C>G substitutions < 0.6%, indels < 5%, TMB > 100 mut/Mb. A scoring system to assess these alterations (POLE‐score) was developed; based on their scores, 7/18 (39%) additional tumours with EDM were classified as POLE‐ultramutated ECs, and the six POLE mutations present in these tumours were considered pathogenic. Only 1/23 (4%) tumours with non‐EDM showed these genomic alterations, indicating that a large majority of mutations outside the exonuclease domain are not pathogenic. The infrequent combination of MSI‐H with POLE EDM led us to investigate the clinical significance of this association. Tumours with pathogenic POLE EDM co‐existent with MSI‐H showed genomic alterations characteristic of POLE‐ultramutated ECs. In a pooled analysis of 3361 ECs, 13 ECs with DNA mismatch repair deficiency (MMRd)/MSI‐H and a pathogenic POLE EDM had a 5‐year recurrence‐free survival (RFS) of 92.3%, comparable to previously reported POLE‐ultramutated ECs. Additionally, 14 cases with non‐pathogenic POLE EDM and MMRd/MSI‐H had a 5‐year RFS of 76.2%, similar to MMRd/MSI‐H, POLE wild‐type ECs, suggesting that these should be categorised as MMRd, rather than POLE‐ultramutated ECs for prognostication. This work provides guidance on classification of ECs with POLE mutations, facilitating implementation of POLE testing in routine clinical care. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

Identification of potential inhibitors against pathogenic missense mutations of PMM2 using a structure-based virtual screening approach

The autosomal recessive phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is characterized by defective functioning of the PMM2 enzyme, which is necessary for the conversion of mannose-6-phosphate into mannose-1-phosphate. Here, a computational pipeline was drawn to identify the most significant mutations, and further, we used a virtual screening approach to identify a new lead compound to treat the identified significant mutations. We searched for missense mutation data related to PMM2-CDG in HGMD®, UniProt, and ClinVar. Our search yielded a total of 103 mutations, of which 91 are missense mutations. The D65Y, I132N, I132T, and F183S mutations were classified as deleterious, destabilizing, and altering the biophysical properties using the PredictSNP, iStable, and Align GVGD in silico prediction tools. Additionally, we applied a multistep protocol to screen for an alternative lead compound to the existing CID2876053 (1-(3-chlorophenyl)-3,3-bis(pyridine-2-yl)urea) with affinity to these identified significant mutants. Two compounds, CHEMBL1491007 (6-chloro-4-phenyl-3-(4-pyridin-2-ylpiperazin-1-yl)-1H-quinolin-2-one) and CHEMBL3653029 (5-chloro-4-[6-[(3-fluorophenyl)methylamino]pyridin-2-yl]-N-(piperidin-4-ylmethyl)pyridin-2-amine), exhibited the highest binding affinity with the selected mutants and were chosen for further analysis. Through molecular docking, molecular dynamics simulation, and MMPBSA analysis, we found that the known compound, i.e. CID2876053, has stronger interaction with the D65Y mutant. The newly identified lead compound CHEMBL1491007 showed stronger interaction with the I132N and I132T mutants, whereas the most deleterious mutant, F183S, showed stronger interaction with CHEMBL3653029. This study is expected to aid in the field of precision medicine, and further to in vivo and in vitro analysis of these lead compounds might shed light on the treatment of PMM2-CDG. Communicated by Ramaswamy H. Sarma

SCN1A mutation spectrum in a cohort of Bulgarian patients with GEFS+ phenotype.

Dravet syndrome (DS) is the most severe form of Generalized Epilepsy with Febrile Seizures plus (GEFS+) syndrome with a clear genetic component in 85% of the cases. It is characterized by fever-provoked seizure onset around six months of age and subsequent developmental deterioration later in life. In the current study, 60 patients with fever-provoked seizures and suspicion either of GEFS+ (50 patients) or of DS (10 patients) were referred for SCN1A gene sequence analysis. SCN1A gene sequencing revealed clinically significant variants in 11 patients (18.3%); seven pathogenic (11.7%) and four likely pathogenic (6.7%). Five of these variants have not been reported previously. Among the preselected group of ten DS patients, five had pathogenic SCN1A variants which confirmed diagnosis of DS. In four patients with preliminary diagnosis GEFS+, the detected SCN1A variant enabled us to specify the diagnosis of DS in these patients. Thus, SCN1A sequencing led to confirmation of the genetic diagnosis in 50% (5/10) of DS patients, as well as clarification of the diagnosis of DS in 8% of GEFS+ patients (4/50). In this study, four patients with truncating mutations had refractory seizures and additional psychomotor abnormalities. Additionally, pathogenic missense mutations were detected in three children with comparable phenotypes, which support the observations that missense mutations in critical channel function regions can cause a devastating epileptic condition. This is the first systematic screening of SCN1A gene in our country, which expands the spectrum of SCN1A variants with five novel variants from Bulgaria and demonstrates the clinical utility of confirmatory SCN1A testing, which helps clinicians make early and precise diagnoses. It is important for a better followup, choice of proper treatment, avoidance of development of refractory seizures and neuropsychological complications. Identification of pathogenic variants in SCN1A in the milder GEFS+ and severe DS cases, will help to offer adequate prenatal diagnosis and improve the genetic counselling provided to affected families.

Association of Pathogenic Missense and Nonsense Mutations in Mitochondrial COII Gene with Familial Adenomatous Polyposis (FAP).

Nuclear genetic mutations have been extensively investigated in solid tumors. However, the role of the mitochondrial genome remains uncertain. Since the metabolism of solid tumors is associated with aerobic glycolysis and high lactate production, tumors may have mitochondrial dysfunctions. Familial adenomatous polyposis (FAP) is a rare form‌ of colorectal cancer and an autosomal dominant inherited condition that is characterized by the progress of numerous adenomatous polyps in the rectum and colon. The present study aimed at understanding the nature and effect of mitochondrial cytochrome c oxidase subunit 2 (COII) gene mutations in FAP tumorigenesis. Fifty-six (26 familial and 30 sporadic) FAP patients and 60 normal controls were enrolled in this study. COII point mutations were evaluated by PCR and direct sequencing methods, and a total of 7 mtDNA mutations were detected (3 missense, 1 nonsense, and 3 synonymous variations). Novel non-synonymous COII gene mutations were mostly in heteroplasmic state. These mutations change amino acid residues in the N-terminal and C-terminal regions of COXII. Bioinformatics analysis and three-dimensional structural modeling predicted that these missense and nonsense mutations have functional importance, and mainly affected on cytochrome c oxidase (complex IV). Also, FAP patients carried a meaningfully higher prevalence of mutations in the COII gene in comparison with healthy controls (P <0.001). Analysis of cancer-associated mtDNA mutation could be an invaluable tool for molecular assessment of FAP so that these findings can be helpful for the development of potential new biomarkers in the diagnosis of cancer for future clinical assessments.

Genetic study of a family with affected members with Waardenburg syndrome type 4 without Hirschsprung disease

Introduction. Waardenburg syndrome (WS) is an autosomally inherited disorder with the most common state compounding pigmentary abnormality and sensorineural deafness. The rarest type of the disease is WS4 with the general characteristic discriminated from other types by the attendance of Hirschsprung disease (HD). Among the several genes, one of the causative genes in WS4 is endothelin 3 (EDN3) with both autosomal recessive and dominant inheritance. Aim. The intention of the present study is to report a pathogenic mutation as the genetic cause of WS in an Iranian family with four patients without any segregation criteria for the type of the disease. Material and methods. In order to detect of causing gene or genes related to the disease, Whole exome sequencing (WES) technique in proband’s sample was done. To confirm the detected mutation in proband and some family members with or without the disease direct sequencing of END3 gene was performed using Sanger method. Results. Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. WES analysis suggested a gene (EDN3) related to WS type 4B. DNA sequencing confirmed a pathogenic missense mutation c.293C&gt;T, p.T98M in EDN3 gene in all of the four patients. Conclusion. Determination of WS can usually be missed owing to the lack of some attributes in every sufferer and also conventional clinical variance, in spite of several affected members in a single family. So, Genetic counseling is pivotal for families with multiple members influenced. We detected c.293C&gt;T, p.T98K mutation in EDN3 gene as a pathogenic variant which has been known as a likely pathogenic state in the American College of Medical Genetics and Genomics (ACMG) guidelines, despite one prior report. It will be helpful in genetic diagnosis of affected persons and increases the mutation spectrum of EDN3 gene.

Retained mismatch repair protein expression occurs in approximately 6% of microsatellite instability-high cancers and is associated with missense mutations in mismatch repair genes.

Immunohistochemistry for mismatch repair protein expression is widely used as a surrogate for microsatellite instability status- an important signature for immunotherapy and germline testing. There are no systematic analyses examining the sensitivity of immunohistochemistry for microsatellite instability-high status. Mismatch repair immunohistochemistry and microsatellite instability testing were performed routinely as clinically validated assays. We classified germline/somatic mutation types as truncating (nonsense, frameshift, in/del) versus missense and predicted pathogenicity of the latter. Discordant cases were compared to concordant groups: microsatellite instability-high/ mismatch repair-deficient for mutation comparison and microsatellite stable/ mismatch repair-proficient for immunohistochemical comparison. 32 of 443 (7%) microsatellite instability-high cases had immunohistochemistry. Four additional microsatellite instability-high research cases had discordant immunohistochemistry. Of 36 microsatellite instability-high cases with discordant immnohistochemistry, 30 were mismatch repair-proficient while 6 (5 MLH1 and 1 MSH2) retained expression of the defective mismatch repair protein and lost its partner. In microsatellite instability-high tumors with discordant immunohistochemistry, we observed an enrichment in deleterious missense mutations over truncating mutations, with nearly 70% (25/36) of cases having pathogenic germline or somatic missense mutations, as opposed to only 17% (6/36) in a matched microsatellite instability-high group with concordant immunohistochemistry (p=0.0007). In microsatellite instability-high cases with discordant immunohistochemistry and MLH1 or PMS2 abnormalities, less cells showed expression (p=0.015 and p=0.00095 respectively) compared to microsatellite stable/ mismatch repair-proficient cases. Tumor mutation burden, MSIsensor score, and truncating mismatch repair gene mutations were similar between microsatellite instability-high cases with concordant versus discordant immunohistochemical expression. Approximately 6% of microsatellite instability-high cases have retained mismatch repair protein expression and would be missed by immunohistochemistry-based testing, hindering patient access to immunotherapy. Another 1% of microsatellite instability-high cases show isolated loss of the defective gene’s dimerization partner, which may lead to germline testing of the wrong gene. These cases are enriched for pathogenic mismatch repair missense mutations.

MCF2 is linked to a complex perisylvian syndrome and affects cortical lamination

The combination of congenital bilateral perisylvian syndrome (CBPS) with lower motor neuron dysfunction remains unusual and suggests a potential common genetic insult affecting basic neurodevelopmental processes. Here we identify a putatively pathogenic missense mutation in the MCF2 gene in a boy with CBPS. Using in utero electroporation to genetically manipulate cortical neurons during corticogenesis, we demonstrate that the mouse Mcf2 gene controls the embryonic migration of cortical projection neurons. Strikingly, we find that the CBPS‐associated MCF2 mutation impairs cortical laminar positioning, supporting the hypothesis that alterations in the process of embryonic neuronal migration can lead to rare cases of CBPS.

Soft Tissue Fibroblastic Reticular Cell Tumor With Whole-Exome Sequencing Findings: An Unexpected Presentation of Lynch Syndrome

Abstract Fibroblastic reticular cell tumor (FRCT) is an exceedingly rare tumor that is histologically reminiscent of follicular dendritic cell sarcoma or interdigitating dendritic cell sarcoma but lacks the immunophenotypic features of these tumors. This tumor is classically described in lymph nodes and spleen, with only 4 cases described in soft tissues. We report a case of FRCT presenting as a right thigh mass in a 67-year-old woman with no prior malignancies. Gross examination showed a 10.6-cm tan well-circumscribed intramuscular mass. Microscopic examination revealed a discohesive population of cells with indistinct pale cytoplasm and large irregular, atypical vesicular nuclei with variably prominent nucleoli in a collagenous background infiltrated by lymphocytes. The tumor cells were positive for smooth muscle actin, cytokeratins (in a dendritic pattern), and CD163, while negative for CD21, CD35, and CD23, supporting the diagnosis of FRCT. Whole-exome sequencing revealed 631 putative somatic mutations in the tumor (&gt;10 mutations/Mb of sequence). Mutational signature analysis suggested DNA mismatch repair deficiency. Germline mutational analysis revealed a heterozygous pathogenic missense mutation of MLH1 (c.2246 T &gt; C, p.Leu749Pro). Subsequent immunohistochemical analysis showed complete loss of MLH1 and PMS2 in tumor cells. To our knowledge, this is the first case of FRCT characterized by sequencing studies and found to be associated with Lynch syndrome (LS), expanding the spectrum of LS-associated neoplasms. This case demonstrates genetic hypermutation similar to that seen in the more common epithelial lesions arising in LS, and it highlights the potential for high-throughput genetic analysis to identify mismatch repair–deficient tumors of atypical histologies, which may have significant clinical implications in the era of immunotherapy.

Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations

Recoding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3'-UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNA-binding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA recoding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2.

Pathogenic Mutations Associated with Legius Syndrome Modify the Spred1 Surface and Are Involved in Direct Binding to the Ras Inactivator Neurofibromin

Neurofibromatosis type I (NF1) and Legius syndrome are rare inherited disorders that share diagnostic symptoms including dermal abnormalities like axillary and inguinal freckling and café au lait spots. In addition, patients suffering from NF1 have a demanding risk for the development of severe tumors of the peripheral and central nervous system among other NF1-specific symptoms. NF1 and Legius syndrome are caused by alterations in the NF1 and SPRED1 genes encoding the Ras inhibitors neurofibromin and Spred1 (sprouty related EVH1 domain-containing protein), respectively. Neurofibromin functions as a Ras-specific GTPase-activating protein (Ras-GAP), and Spred1 enhances Ras inactivation by recruiting neurofibromin from the cytosol to membrane-anchored Ras. In a previous study, we mapped the Spred binding site to the GAP-related domain of neurofibromin (NF1-GAP) and identified the GAPex subdomain as critical for Spred1 binding. Here, we characterize the binding site of these proteins in more detail focusing on a mutant Spred1 variant carrying a pathogenic missense mutation (threonine 102 to arginine). Introduction of this mutation, which locates at the N-terminal EVH1 domain of Spred1, weakens the interaction with neurofibromin by about 3 orders of magnitude without perturbing the protein fold, and the binding site of NF1-GAP on the mutant Spred1(EVH1) variant can be identified by NMR spectroscopy. Taken together, our data provide structural insight into the interaction of Spred1 and neurofibromin and characterize the structural or functional consequence of selected patient-derived mutations associated with Legius syndrome.