Silent Mutations Research Articles

Identification of the novel KIR3DL1*00703 allele in a Chinese Han individual.

The novel KIR3DL1*00703 allele differs from the closest allele KIR3DL1*00701 by a single silent mutation.

Single Base Substitution Mutation Profiling in Non-Matched and Matched Metastases across Various Tumor Histologies and Anatomic Sites

<h3>Purpose/Objective(s)</h3> As the landscape of oligometastatic care continues to evolve, it has become clear that not all histologies (or anatomic sites) respond equivalently to systemic therapy or radiation. This study aims to gain more insight into metastatic tumor biology by evaluating single base substitution mutations across various metastatic sites and within matched primary-metastatic patient samples. <h3>Materials/Methods</h3> We evaluated our prospectively collected institutional biorepository for metastatic (Met) tumors which underwent targeted exome sequencing for 1,327 cancer-related genes. The patient cohorts included: 1) non-matched Mets (N-Met; n=794 [707 tumors]) and 2) a subset of matched primary (Pri)-Met (M-Pri-Met; n=207 [390 tumors]). Exome data were enriched for somatic non-silent mutations by filtering out artifacts and known germline and silent mutations. Mutation signatures based on 6 nucleotide base alterations (96 subtypes) were generated and compared across samples followed by hierarchical clustering analysis to assess mutation signature correlation to the COSMIC mutation database. The mutation signatures were assessed across unique histology groups from Prim (n=17) and Met (n=16) sites and compared by Kruskal-Wallis test. Also, counts of individual mutations were related to given mutation signatures. A generalized linear mixed model was used to infer the odds ratio (OR) of mutation incidence in Pri vs Met tumors. <h3>Results</h3> Across all Mets, we found that C>T and G>A alterations were identified in 23% and 21% of tumors, respectively, whereas T>A was <2%. Notable differences were seen in the proportions of mutation signatures (n=9) across Pri histology (range p < 0.03 to 1.0e⁻¹⁶) and among different Met sites within a given histology. For example, our 6^th signature was associated with AID/APOBEC family activity and varied across Pri histology (p = 1.0e⁻¹³) and within Met sites (p = 4.3e⁻⁰⁹). In contrast, our 4^th signature was related to <i>ERCC2</i> mutation, but only differed across Pri histology and not Met site. The median number of mutations related with mutation signatures was 27 (range: 1-1404) with the highest counts in melanoma. Within our M-Pri-Met analysis, several genes were identified to be either private or shared among tumors. For example, within the M-Pri-Met breast (n=93) group, <i>EPHB4</i> mutation was concordant in 58% of samples with a LogOR 1.38 (95% CI 1.2-13.2; p=0.02) in Pri vs Met. In contrast, <i>BRAF</i> mutation was 40% concordant with higher odds of being in Met vs Pri (p=0.04). No significant differences were identified in the lung M-Pri-Met cohort (n=45). Though, individual mutations did not appear to be different in lung histology, we did observe differences in the proportions of mutation signatures within individual patient tumors. <h3>Conclusion</h3> A deeper knowledge of similarities and differences among metastases may aid in biologically-informed metastasis-directed therapy. This analysis highlights diversity in mutation type and incidence among metastases.

Circadian Regulation of the Rod Contribution to Mesopic Vision in Mice.

At intermediate (mesopic) light levels, rods and cones are both active and can contribute to vision. This presents a challenge to the retina because the visual responses originating with rods and cones are distinct, yet their visual responses must be seamlessly combined. The current study aimed to establish how the circadian clock regulates rod and/or cone vision in these conditions, given the strong time-of-day change in the reliance on each photoreceptor. Visual responses were recorded in the retina and visual thalamus of anaesthetized male mice at distinct circadian time points, and the method of receptor silent substitution was used to selectively stimulate different photoreceptor types. With stimuli designed to only activate rods, responses in the mesopic range were highly rhythmic and peaked in amplitude in the subjective night. This rhythm was abolished following intravitreal injection of the gap junction blocker meclofenamic acid, consistent with a circadian variation in the strength of electrical coupling of photoreceptors. In contrast, responses to stimuli designed to only activate cones were arrhythmic within the mesopic to photopic range when adapted to the background irradiance. The outcome was that combined rod-plus-cone responses showed a stable contrast-response relationship across mesopic-photopic backgrounds in the circadian day, whereas at night, responses were significantly amplified at lower light levels. These data support the idea that the circadian clock is a key regulator of vision, in this case defining the relative amplitude of rod/cone vision across the mesopic transition according to time of day.SIGNIFICANCE STATEMENT Although the importance of circadian clocks in regulating vision has been long recognized, less is known about how the clock shapes vision in conditions where both rods and cones are active (mesopic conditions). Here, the novel approach of receptor silent substitution has been applied to trace rod and cone visual responses in mice across the circadian cycle and has identified pronounced rhythms in rod, but not cone, vision. This has the effect of boosting responses in dimmer backgrounds at night at the cost of impaired contrast-response stability across the mesopic to photopic range. Thus, the circadian clock drives anticipatory changes in the relative contribution of rods versus cones to vision, which match the prevailing visual environment.

Prime editing optimized RTT permits the correction of the c.8713C>T mutation in DMD gene

Duchenne muscular dystrophy is a severe debilitating genetic disease caused by different mutations in the DMD gene leading to the absence of dystrophin protein under the sarcolemma. We used CRISPR-Cas9 prime editing technology for correction of the c.8713C>T mutation in the DMD gene and tested different variations of reverse transcription template (RTT) sequences. We increased by 3.8-fold the editing percentage of the target nucleotide located at+13. A modification of the protospacer adjacent motif sequence (located at+6) and a silent mutation (located at+9) were also simultaneously added to the target sequence modification. We observed significant differences in editing efficiency in interconversion of different nucleotides and the distance between the target, the nicking site, and the additional mutations. We achieved 22% modifications in myoblasts of a DMD patient, which led to dystrophin expression detected by western blot in the myotubes that they formed. RTT optimization permitted us to improve the prime editing of a point mutation located at+13 nucleotides from the nick site to restore dystrophin protein.

Silent - but not neutral!

An ingenious system for generating thousands of point mutations in yeast genes and measuring their effect on fitness shows convincingly that, for the chosen subset of representative non-essential genes, silent mutations have as much effect on fitness as missense mutations. In other words, silent mutations are not neutral, at least under these conditions. This result has important implications for evolutionary biology.

P432 Detection of erg11 gene mutation in fluconazole resistant Candida albicans isolates

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectiveTo determine the profile of fluconazole-resistant Candida isolates (FRCS) and the proportion of erg11 gene mutation in fluconazole-resistant C. albicans (FRCA) isolatesMethodsAfter getting approval from ethics the study was conducted on 150 isolates of FRCS obtained from patients admitted to our tertiary care hospital from September 2019 to December 2021. All the isolates were revived onto Sabouraud dextrose agar (SDA) and identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Fluconazole-resistance was detected by VITEK 2 automated system. Deoxyribonucleic acid (DNA) of FRCA isolates were extracted by QIAamp DNA Mini kit (Qiagen-51 304, Hilden, Germany) as per manufacturer's instruction. A single primer pair targeting the nucleotide sequence from 1067 to 1576 bp region of erg11 gene was used for amplification as per Xu et al. with minor modification and 500 bp polymerase chain reaction (PCR) products were observed by gel electrophoresis. A positive clinical isolate of FRCA was used as a positive control. The PCR products were purified and subjected to Sanger sequencing with an ABI PRISM DNA analyzer (Applied Biosystems) and mutations were detected by using Mega (version 7) software by comparing with the published GenBank sequence AF153844.1 of C. albicans strain ATCC 28516. Categorical variables were expressed in percentages and continuous variables were expressed in mean (average).ResultsOut of the total of 150 FRCS, the most common fluconazole-resistant agent of candidiasis was C. auris, which was isolated from 60 (40%) cases followed by 24 (16%) isolates of C. glabrata, 19 (12.7%) isolates of C. krusei, 18 (12%) isolates of C. parapsilosis, 17 (11.3%) isolates of C. albicans, 10 (6.7%) isolates of C. tropicalis, and only two (1.3%) isolates of C. guilliermondii. Among these 17 FRCA isolates, 9 isolates were obtained from genital swabs, 5 from blood, and a single isolate each from the perianal swab, ear swab, and sputum. A total of 12 (70.6%) out of 17 FRCA isolates yielded 500 bp region in erg11 gene. Gel electrophoresis image of PCR products of erg11 gene is depicted in Figure 1. Sequencing of these products detected a single missense mutation A1309G (V437I) in one FRCA isolate. Most of the FRCA isolates had an average of 4 silent mutations (observed in 6 isolates) and a single isolate had 6 silent mutations. A total of 8 different silent mutations were observed among FRCA isolates T1296C (83.3%), C1203T (66.7%), A1440G (66.7%), C1302T (50%), T1470C (50%), T1140C (25%), T1110C (16.7%), and T1284C (8.3%).Conclusion C. auris was the most common fluconazole resistant isolate observed, followed by C. glabrata, C. krusei, C. parapsilosis, C. albicans, C. tropicalis, and C. guilliermondii. There were many silent mutations observed in erg11 gene of FRCA isolates and detected only a single missense mutation (V437I). Fluconazole-resistant in FRCA isolates may be due to mechanisms other than the studied one in our region.

Detection of Voltage-Gated Sodium Channel (VGSC) Mutation in Deltamethrin-Resistant Blattella germanica L. from Indonesia.

&lt;b&gt;Background and Objective:&lt;/b&gt; German cockroach (&lt;i&gt;Blattella germanica&lt;/i&gt; L.) is one of the most common residential pests in Indonesia. Controlling the population face obstacles due to insecticide resistance, especially to deltamethrin. This research investigated the resistance status and the possibility of a Voltage-Gated Sodium Channel (VGSC) knockdown resistance mutation (L1014F) in two field strains of German cockroaches collected from two cities in Indonesia (Bukittinggi, named RMKN-BKT and Bandung, named KRSA-BDG) with VCRU-WHO as the standard strain. &lt;b&gt;Materials and Methods:&lt;/b&gt; This study started with a bioassay test to determine the lethal dose of 50% (LD&lt;sub&gt;50&lt;/sub&gt;) for each strain and followed by a molecular test for mutation detection. &lt;b&gt;Results:&lt;/b&gt; The results showed that the RMKN-BKT and KRSA-BDG strains were highly resistant to deltamethrin with RR50 values of 80,090 times and 73,272 times, respectively. Only the RMKN-BKT strain was shown to carry L1014F kdr mutation which lead to an amino acid replacement from leucine (TTG) to phenylalanine (TTC). Two silent mutations were also found in both field strains at codons 983 (TGC/cysteine→TGT/cysteine) and 984 (GGG/ glycine→GGA/glycine) which were suggested as polymorphism phenomena. The absence of the L1014F mutation in the Bandung strain does not exclude the possibility of the presence of the VGSC mutation at other points. &lt;b&gt;Conclusion:&lt;/b&gt; It requires subsequent investigation in mutation detection at other points and the possible presence of other resistance mechanisms to get a precise solution in the population control. Bioinsecticides may stand as a breakthrough so that the strategy will no longer focus on insecticides.

A p53 transcriptional signature in primary and metastatic cancers derived using machine learning.

The tumor suppressor gene, TP53, has the highest rate of mutation among all genes in human cancer. This transcription factor plays an essential role in the regulation of many cellular processes. Mutations in TP53 result in loss of wild-type p53 function in a dominant negative manner. Although TP53 is a well-studied gene, the transcriptome modifications caused by the mutations in this gene have not yet been explored in a pan-cancer study using both primary and metastatic samples. In this work, we used a random forest model to stratify tumor samples based on TP53 mutational status and detected a p53 transcriptional signature. We hypothesize that the existence of this transcriptional signature is due to the loss of wild-type p53 function and is universal across primary and metastatic tumors as well as different tumor types. Additionally, we showed that the algorithm successfully detected this signature in samples with apparent silent mutations that affect correct mRNA splicing. Furthermore, we observed that most of the highly ranked genes contributing to the classification extracted from the random forest have known associations with p53 within the literature. We suggest that other genes found in this list including GPSM2, OR4N2, CTSL2, SPERT, and RPE65 protein coding genes have yet undiscovered linkages to p53 function. Our analysis of time on different therapies also revealed that this signature is more effective than the recorded TP53 status in detecting patients who can benefit from platinum therapies and taxanes. Our findings delineate a p53 transcriptional signature, expand the knowledge of p53 biology and further identify genes important in p53 related pathways.

Compendium of proteins containing segments that exhibit zero-tolerance to amino acid variation in humans.

Genetic missense tolerance ratio (MTR) analysis systematically evaluates all possible segments in a given protein‐encoding transcript found in the human population. This method scores each segment for the number of observed missense variants versus the number of silent mutations in that same segment. An MTR score of 0 indicates that no missense mutations are observed within a given segment. This is indicative of evolutionary purifying selection, which excludes mutations in that segment from the general human population. Here, we conducted MTR analysis on each of the roughly 20,000 protein‐encoding human genes. It was seen that there are 257 genes with at least one 31‐residue encoding segment with MTR = 0 (1.3% of all human genes). The proteins encoded by these 257 genes were tabulated along with information regarding the sequence location of each intolerant segment, the likely function of the protein, and so forth. The most functionally‐enriched family among these proteins is a collection of several dozen proteins that are directly involved in RNA splicing. Some of the other proteins with zero‐tolerance segments have thus far escaped significant characterization. Indeed, while a number of these proteins have previously been genetically linked to human disorders, many have not. We hypothesize that this compendium of human proteins with zero‐tolerance segments can be used to complement disease mutation data as a pointer to genes and proteins that are associated with interesting and underexplored human biology.

Genetic characterization of diphtheria tox B to evaluate vaccine efficacy in Indonesia.

Blocking the attachment of diphtheria toxins to host cells through the intact receptor binding site (tox B) was the initial mechanism of action of the diphtheria vaccine. Diphtheria outbreaks in populations with good vaccination coverage can be caused by mutations or changes in the genetic structure of the tox B protein. The aim of this study was to characterize the Tox B protein produced by Corynebacterium diphtheriae isolated from 2018 to 2019 in patients in Jakarta who had already received the diphtheria vaccine. Of the 89 throat swab specimens of patients with a clinical diagnosis of diphtheria, 10 were positive for diphtheria and toxin. PCR was used to amplify the tox B DNA fragment in the 10 positive isolates. DNA sequencing was conducted with overlapping primers and the DNA sequences were analysed by using SeqScape V2.7. Of the 10 isolates, nine isolate showed a DNA mutation (G30A), but the mutation did not change the amino acid encoding arginin (silent mutation). Our findings indicate that the efficacy of the diphtheria vaccine used in Indonesia has not decreased because of mutations in the tox B genes not change the amino acid. Overall, there are no amino acid changes in the tox B protein, indicating that the outbreaks are not affected by mutation in tox B. Another possible mechanism - overexpression of the toxin - is likely responsible for causing diphtheria in patients who have a complete history of immunization in Indonesia.

Identification of an ERN1 target site within EGFP mRNA.

EGFP (enhanced green fluorescent protein) is one of the most common tools used in life sciences, including research focusing on proteostasis. Here we report that ERN1 (endoplasmic reticulum to nucleus signaling 1), which is upregulated by UPR (unfolded protein response), targets an RNA hairpin loop motif in EGFP mRNA. A silent mutation introduced into EGFP mRNA abolished the ERN1‐dependent mRNA decay. Therefore, experiments that employ EGFP as a reporter gene in studies that involve upregulation of the UPR pathway should be interpreted carefully, and a mutant devoid of the ERN1 target motif may be more suitable for such studies.

Electroretinographic responses to luminance and cone-isolating white noise stimuli in macaques.

Electroretinograms (ERGs) are mass potentials with a retinal origin that can be measured non-invasively. They can provide information about the physiology of the retina. Often, ERGs are measured to flashes that are highly unnatural stimuli. To obtain more information about the physiology of the retina, we measured ERGs with temporal white noise (TWN) stimuli that are more natural and keep the retina in a normal range of operation. The stimuli can be combined with the silent substitution stimulation technique with which the responses of single photoreceptor types can be isolated. We characterized electroretinogram (ERG) responses driven by luminance activity or by the L- or the M-cones. The ERGs were measured from five anesthetized macaques (two females) to luminance, to L-cone isolating and to M-cone isolating stimuli in which luminance or cone excitation were modulated with a TWN profile. The responses from different recordings were correlated with each other to study reproducibility and inter-individual variability. Impulse response functions (IRFs) were derived by cross-correlating the response with the stimulus. Modulation transfer functions (MTFs) were the IRFs in the frequency domain. The responses to luminance and L-cone isolating stimuli showed the largest reproducibility. The M-cone driven responses showed the smallest inter-individual variability. The IRFs and MTFs showed early (high frequency) components that were dominated by L-cone driven signals. A late component was equally driven by L- and M-cone activity. The IRFs showed characteristic similarities and differences relative to flash ERGs. The responses to TWN stimuli can be used to characterize the involvement of retinal cells and pathways to the ERG response. It can also be used to identify linear and non-linear processes.

Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9.

The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, which is poorly efficient. Recently, the use of CRISPR/Cas9 technology was shown to greatly improve the speed and efficiency of the production of recombinant VACV expressing a heterologous gene. However, the ability to rapidly recover viruses bearing single nucleotide substitutions is still challenging. Notwithstanding, ongoing studies on the VACV and its interaction with the host cell could benefit from viral gene targeted mutagenesis. Here, we present a modified version of the CRISPR/Cas9 system for the rapid selection of mutant VACV carrying point mutations. For this purpose, we introduced a silent mutation into the donor gene (which will replace the wildtype gene) that serves a double function: it is located in the PAM (NGG) sequence, which is essential for Cas9 cleavage, and it alters a restriction site. This silent mutation, once introduced into the VACV genome, allows for rapid selection and screening of mutant viruses carrying a mutation of interest in the targeted gene. As a proof of concept, we produced several recombinant VACVs, with mutations in the E9L gene, upon which, phenotypic analysis was performed.

Highly Effective Markerless Genetic Manipulation of Streptococcus suis Using a Mutated PheS-Based Counterselectable Marker.

Streptococcus suis is an important zoonotic pathogen, however, an efficient markerless genetic manipulation system is still lacking for further physiological and pathological studies on this bacterium. Several techniques have been developed for markerless genetic manipulation of S. suis utilizing either a temperature-sensitive vector or a counterselectable markers (CSMs), however, at present, the efficiency of these techniques is not very satisfactory. In this study, we developed a strategy for markerless genetic manipulation of S. suis employing a CSM based on a conditionally lethal mutant allele of pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase (PheS). This mutant pheS, mPheS, was constructed by introducing site-directed mutations for a T261S/A315G double-substitution and a number of silent mutations to decrease its similarity with the endogenous wild type pheS gene (wtPheS). Additionally, five potentially strong promoters from S. suis were screened for their ability to drive high-level expression of mPheS, thus endowing the carrier strain with sufficient sensitivity to the phenylalanine analog p-chloro-phenylalanine (p-Cl-phe). Insertion of these P-mPheS cassettes into a vector or into the chromosomal locus via a linked erythromycin resistance gene revealed that mPheS allele driven by promoters P0530 and P1503 renders S. suis sensitive to as low as 0.01% (or 0.5 mM) of p-Cl-phe. This offers two potential CSMs for S. suis with p-Cl-phe as a counterselective agent. P1503-mPheS was revealed to be 100% efficient for counter-selection in S. suis by application in a precise gene deletion. Using P1503-mPheS as a CSM, a two-step insertion and excision strategy for markerless genetic manipulation of S. suis were developed, supplying a powerful tool for markerless genetic manipulation of S. suis.

Assessment of the genetic changes of the attenuated Hanvet1.vn strain compared with original virulent 02HY strain of the porcine reproductive and respiratory syndrome virus

The attenuated porcine reproductive and respiratory syndrome virus (PRRSV) strain Hanvet1.vn was developed by Hanvet Pharmaceutical Co., Ltd. by inoculating the virulent strain 02HYon Marc-145 cells for 80 generations and used to produce PRRS vaccine. In this study, we published the results of sequencing, analyzing and comparing the genome of the attenuated PRRSV strain Hanvet1.vn compared with the original pathogenic strain 02HY. The genomes of strains Hanvet1.vn and 02HY have 8 reading frames, coding for 8 non-structural and structural proteins: NSP1a, NSP1b, GP2, GP3, GP4, GP5, MP, NP. After sequencing and translating into proteins, the gene sequence of each open reading frame (ORF) of strain Hanvet1.vn was compared with the sequence of pathogenic strain 02HY to find nucleotide and amino acid changes. The results showed that the Hanvet1.vn pathogenic strain genome (Genbank Accession KU842720) when compared with the pathogenic strain 02HY genome (Submission2490633) had89 nucleotide mutations that changed 51 amino acids in 7 ORFs and 7 proteins, respectively. Particularly, ORF6 encoding for the M protein is completely unchanged. The size of each reading frame is also exactly the same. It showed that there were no insertion and deletion (Indel) mutations in the ORFs of the attenuated strain after 80 generations of inoculation. There was a change in the genome that made the strain Hanvet1.vn become attenuated, but the gene encoding for the GP5 protein that induces the production of neutralizing antibodies only changed two nucleotides at position 471 (A-&gt;G), causing the TCA codon to turn into a TCG codon. This is a silent mutation and both codons code for the amino acid Serine (S). The second mutation at position 587 (A-&gt;T) causes Glutamine (Q) to transform into Leucine (L). However, this modification does not belong to the GP5 antigenic epitopes. In clonclusion, after 80 passages, despite changes occurred in genes of Hanvet1.vn strain for becoming an attenuated strain, the GP5 protein of the attenuated strain did not change its antigenic amino acids.

Polymorphisms of dtxR Gene of Corynebacterium diphtheriae Isolated from Diphtheria Outbreak in Indonesia

Background: In Corynebacterium diphtheriae, the dtxR gene plays a role in regulating diphtheria toxin synthesis. The dtxR gene is often used as a marker for identifying C. diphtheriae by the polymerase chain reaction (PCR) method because it is present in all strains of this bacterium. Mutations in the dtxR gene can cause the over-synthesis of diphtheria toxin and reduce PCR assays' sensitivity. Objectives: This study aimed to describe the polymorphisms in the dtxR gene of C. diphtheriae isolated from a diphtheria outbreak in Indonesia. Methods: Forty-eight isolates of C. diphtheriae were obtained from clinical samples (throat/nasopharyngeal swabs) of diphtheria cases and close contacts. The isolates were revived on a blood agar plate (BAP), bacterial colonies were harvested, and deoxyribonucleic acid (DNA) was extracted. The DNA sequencing was carried out using a Whole-genome Sequencing (WGS) approach. The data were converted and analyzed with U-gene software. The dtxR gene analysis was performed with C. diphtheriae PW8 as references. Results: There were 59-point mutation locations in 48 isolates examined. None of these single nucleotide polymorphisms (SNPs) coded for amino acid changes. Based on the mutation pattern, seven clades/groups of the dtxR gene of 48 C. diphtheriae isolates were examined. Conclusions: At least seven types of DNA sequences and more than 50 SNPs of the dtxR gene were identified in 48 C. diphtheriae isolates from a diphtheria outbreak in Indonesia. Although all of them are silent mutations, they must be considered in the design of PCR examination in diphtheria laboratories.

Translated Mutant DSPP mRNA Expression Level Impacts the Severity of Dentin Defects.

Hereditary dentin defects are conventionally classified into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia (DD). Mutations in the dentin sialophosphoprotein (DSPP) gene have been identified to cause DGI type II and III and DD type II; therefore, these are not three different conditions, but rather allelic disorders. In this study, we recruited three families with varying clinical phenotypes from DGI-III to DD-II and performed mutational analysis by candidate gene analysis or whole-exome sequencing. Three novel mutations including a silent mutation (NM_014208.3: c.52-2del, c.135+1G>C, and c.135G>A; p.(Gln45=)) were identified, all of which affected pre-mRNA splicing. Comparison of the splicing assay results revealed that the expression level of the DSPP exon 3 deletion transcript correlated with the severity of the dentin defects. This study did not only expand the mutational spectrum of DSPP gene, but also advanced our understanding of the molecular pathogenesis impacting the severity of hereditary dentin defects.

Genomic landscape of pathogenic mutation of APC, KRAS, TP53, PIK3CA, and MLH1 in Indonesian colorectal cancer.

Colorectal cancer (CRC) needs several mutations to occur in various genes, and can vary widely in different individuals; hence it is essential to be discovered in a specific population. Until recently, there has been no known study describing APC, TP53, PIK3CA, KRAS, and MLH1 of CRC in Indonesian population. This study describes the nature and location of mutation in CRC patients treated at three different hospitals in Jakarta. This descriptive study was conducted on CRC patients who underwent neoadjuvant, surgical, and adjuvant therapy at RSCM, RSKJ, and MRCCC in 2017-2018. DNA analysis was performed using next-generation sequencing and aligned against GRCh38. The pathogenic variant was identified using ACMG classification and FATHMM score. Data related to behavior and survival were collected from medical records. Twenty-two subjects in which APC, TP53, and PIKCA were mutated. KRAS mutation occurred in 64%, while MLH1 in 45%. There were five mutation types: nonsense, missense, frameshift, splice-site, and silent mutation. There are four groups of co-occurring mutations: APC, TP53, PIK3CA (triple mutation/TM) alone; TM+KRAS; TM+MLH1; and TM+KRAS+MLH1, presenting different nature and survival. Indonesia has a distinct profile of pathogenic mutation, mainly presenting with locally-advanced stage with various outcomes and survival rate.

Abstract 3061: Elevated bile acid metabolism is associated with breast cancer microbiome, less cell proliferation, and better survival

Abstract BACKGROUND: Bile acids are generated and secreted by hepatocytes and are involved in intestinal circulation. Recently it has been reported that specific bile acids are accumulated in the breast cancer tumor microenvironment (TME), leading to a favorable prognosis for patients and reducing the aggressiveness and metastatic potential of the cancer. We hypothesized that breast cancers with increased bile acid metabolism may have a favorable clinical outcome due to the suppression of cancer cell proliferation. MATERIALS AND METHODS: We analyzed total of 6285 patients from three independent breast cancer cohorts with TCGA as an exploring cohort and METABRIC, GSE96058 as validation cohorts. We divided each cohort into "high" and "low" groups by the top and bottom quartiles of the score following the previous reports. RESULTS: High bile acid metabolism breast cancer enriched multiple metabolic pathway gene sets such as fatty acid and xenobiotic metabolism, as well as adipogenesis and protein secretion by gene set enrichment analysis (GSEA). Adipocytes and preadipocytes were significantly infiltrated in the high score group. Considering the possibility that activation of bile acid metabolism is due to accumulation of microbiome-derived bile acids in breast cancer, we identified that four microorganisms (Anaerococcus, Collimonas, Gammaretrovirus and Hymenobacter) significantly correlated with the bile acid metabolism using TCGA 16s RNA seq data. Surprisingly, protein secretion gene set was significantly enriched to tumors that are abundant in three out of four of these bacterial species. This may suggest that bile acid metabolism are elevated in response to bile acids secreted by the microbiome present in breast cancer TME. The bile acid metabolism was highest in ER+HER2- and lowest in triple-negative, the most aggressive form of breast cancer. The score was negatively correlated with Ki67 gene expression and histological grade, consistent with previous reports that breast cancers with high bile acid accumulation were less proliferative. In agreement, breast cancers with low bile acid metabolism was associated with significantly high silent and non-silent mutation rates, Intratumor Heterogeneity, Proliferation score and Homologous Recombination Defects, and enriched G2M checkpoint gene set. Finally, survival analysis showed that high bile acid metabolism group had significantly better disease free, disease specific and overall survival. CONCULUSIONS: Bile acid metabolism was associated with involvement of microbiome in TME, the suppression of cell proliferation, and better prognosis in breast cancer. Citation Format: Rongrong Wu, Quratulain Sabih, Masanori Oshi, Takashi Ishikawa, Li Yan, Kazuaki Takabe. Elevated bile acid metabolism is associated with breast cancer microbiome, less cell proliferation, and better survival [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 3061.

Frequency of Cap+1 Mutation in Beta Thalassemia and its Associated Haematological Features

Objective: To study the frequency of Cap+1 mutation and associated hematological parameters in suspected beta thalassemia patients.&#x0D; Study Design: Analytical cross-sectional study.&#x0D; Place and Duration of Study: Department of Hematology, Armed Forces Institute of Pathology (AFIP) Rawalpindi from Aug 2017 to Aug 2018.&#x0D; Methodology: 960 patients suspected to have beta thalassemia were inducted into the study. After detailed history and examination basic hematological parameters (Hemoglobin, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin) were analyzed using automated analyzer (Sysmex XE-5000). Molecular genetic analysis by conventional PCR was carried out for CAP+1 mutation. Genomic DNA purification kit (Gentra system USA) was used for extracting DNA from whole blood in order to study the molecular genetics for Cap+1 mutation. Primers were designed for detection and analysis of normal and mutant DNA.&#x0D; Results: The frequency of Cap+1 mutation was observed in 3.2 ± 1.7% (31/960) in all suspected cases of beta thalassemia with a normal range of Hemoglobin (12.4 ± 1.1 g/dl), Mean Corpuscular Volume (86.4 ± 2.1fl/red cell) and Mean Corpuscular Hemoglobin (29 ± 1.7pg/cell).&#x0D; Conclusion: Cap+1 mutation is a silent mutation and its diagnosis remains a challenge because of its normal clinical presentation and normal to mildly deranged basic hematological parameters. Detection of CAP+1 at molecular level has revolutionized the thalassemia prevention program in Pakistan.