Aim: The transcription factor RIP140 (receptor interacting protein of 140 kDa) is involved in intestinal tumorigenesis. It plays a role in the control of microsatellite instability (MSI), through the regulation of MSH2 and MSH6 gene expression. The aim of this study was to explore its effect on the expression of POLK, the gene encoding the specialized translesion synthesis (TLS) DNA polymerase κ known to perform accurate DNA synthesis at microsatellites.Methods: Different mouse models and engineered human colorectal cancer (CRC) cell lines were used to analyze by RT-qPCR, while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression. Published DNA microarray datasets were reanalyzed. The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined. Results: RIP140 positively regulates, at the transcriptional level, the expression of the POLK gene, and this effect involves, at least partly, the p53 tumor suppressor. In different cohorts of CRC biopsies (with or without MSI), a strong positive correlation was observed between RIP140 and POLK gene expression. In connection with its effect on POLK levels and the TLS function of this polymerase, the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene. Finally, the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK, thus strengthening the functional link between the two genes in human CRC. Conclusion: The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability, and more generally to the control of genome integrity. The mismatch repair (MMR) system is one of the various cellular systems involved in the maintenance of genome integrity through the correction of mistakes that occur during DNA replication. Once impaired , it generates microsatellite instability (MSI) and a hypermutated tumor phenotype with a high frequency of point and frameshift mutations,5.Colorectal cancer (CRC) is one of the most frequent cancers worldwide and genetic instability exerts a driving role in this malignancyPOLK gene encodes Polκ, one of the Y-family TLS polymerases, which possesses unique DNA damage bypass capability. Interestingly, despite any associated proofreading exonuclease activity, Polκ was shown to display high accuracy during dinucleotide microsatellite DNA synthesis, and Polκ-/- mice display a spontaneous mutator phenotype in various tissues. This could be linked to its less open active site compared to other inaccurate specialized DNA polymerases from the Y family. In fact, Polκ possesses a unique structural feature, an extension of its N-terminal region, called N-clasp, that protrudes from the thumb domain and encircles DNA in proximity to a primer terminus. This results in a much more stable complex once Polκ binds to a mismatched DNA substrate, facilitating the extension step across various minor groove distortion or lesion. Besides its role in MSI maintenance together with MMR, Polκ seems to prevent DNA damage-induced toxic effects of methylnitrosourea, another process dependent on the MMR system. This link between MMR and Polκ is reinforced by the findings that POLK interacts with MSH2 and partially protects human cells from the MMR-dependent cytotoxicity of O6-methylguanine lesions.In addition to molecular machineries that cope with DNA repair, mammalian cells possess enzymes with translesion DNA synthesis (TLS) activity. The ,14. We and others then characterized RIP140 as a coregulator of various transcription factors, including, for instance, E2F or NFKB. The repressive activity of RIP140 involves several inhibitory domains interacting with histone deacetylases and is controlled by different post-translational modifications. Using a mouse model lacking the Rip140 gene, various physiological processes were shown to be regulated by RIP140, including female fertility and mammary gland morphogenesis, fat metabolism, pro-inflammatory cytokine response, or cognition.Our laboratory recently reported that the RIP140 (receptor interacting protein of 140 kDa) gene was involved in the normal and tumoral development of the intestinal epithelium. RIP140, also known as NRIP1 (nuclear receptor-interacting protein 1), was first identified as a transcriptional repressor of nuclear hormone receptors. In line with this result, we found that this transcription coregulator inhibits the Paneth cells lineage through the regulation of SOX9 expression and activity. In addition, RIP140 expression decreased in CRC samples compared to the adjacent healthy tissue. In sporadic CRC, RIP140 mRNA and protein levels significantly correlated with better overall survival of patients and were identified as good prognosis markers. More recently, we demonstrated that RIP140 was acting as a transcriptional regulator of MSH2 and MSH6 gene expression and was involved in the regulation of the MSI and hypermutator phenotype in CRC cells. Interestingly, a frameshift mutation in the RIP140 coding sequence identified in MSI CRC tumors exhibits a dominant negative activity and correlates with shorter overall survival of patients with advanced CRC.In the intestinal epithelium, our laboratory demonstrated that RIP140 inhibits the Wnt/β-catenin signaling pathway and, as a consequence, exerts an anti-proliferative effectPOLK gene expression at the transcriptional level, at least partly via a p53-dependent mechanism. A strong correlation was observed between the expression of the RIP140 and POLK genes in CRC biopsies. Moreover, cells knocked out for the Rip140 gene were shown to be more sensitive to methyl methane sulfonate (MMS), a drug known to induce DNA lesions activating a POLK response. Finally, our data demonstrate a significant impact of POLK gene expression on the prognosis value of RIP140 in human CRC. We propose that the modulation of POLK gene expression by RIP140 could reinforce its effect on the maintenance of genome integrity and, more particularly, on the stability of microsatellite sequences.In the present study, we explored whether RIP140 could regulate POLK as a factor involved in microsatellite stability. We demonstrated that, in both engineered mouse models and human CRC cells, RIP140 positively regulates the . pEGFP-RIP140 was a kind gift of Dr. Johanna Zilliacus, . pEF-cmyc-RIPMSI was generated by mutagenesis using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene). pEF-cmyc-RIPMSI was digested with AflII and EcoRV enzymes, and the resulting insert was cloned into pEGFP-RIP140 to create pEGFP-RIPMSI. GFP, GFP-RIP140, and GFP-RIPMSI were PCR amplified and cloned into pTRIPZ previously digested with AgeI and MluI to create pTRIPZ-GFP, pTRIPZ-RIP140 and pTRIPZ-RIPMSI, respectively. All the engineered PCR constructs were sequenced.pRL-CMV-renilla and pGL promoters were obtained from Promega . pEF-cmyc-RIP140 was previously described, and the stably transfected MEFs described by Palassin et al. were grown in DMEM-F12 medium supplemented with 10% FCS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 100 mg/mL sodium pyruvate, with 40 µg/mL puromycine for selection of stably transfected cells. HCT116, RKO, SW480, and HT29 human colon cancer cells were grown as previously described and stably transfected with the empty pEGFP vector (Clontech®) or with the same vector containing the full-length human RIP140 cDNA. The SW620 human cell line was grown identically. HCT116-GFP and HCT116-RIP140 cells were previously described and grown in McCoy medium and 750 μg/mL G418. Small interfering RNA (siRNA) transfections were performed using INTERFERin® on cells seeded the day before in a 6-well plate (3 × 105 cells per well). Each transfection was performed in triplicate, and interference efficiencies were tested by quantitative RT-PCR.Mouse embryonic fibroblasts (MEFs) derived from wild-type or RIPKO mice previously described-/- (RIPKO) mice were given by Pr MG Parker . C57BL/6/129 RIP140 transgenic (RIPTg) mice were generated using the Speedy Mouse® Technology (Nucleis) by insertion of a single copy of the human RIP140 cDNA at the HPRT locus. All animals were maintained under standard conditions, on a 12 h:12 h light/dark schedule and fed a chow diet ad libitum, according to European Union guidelines for the use of laboratory animals. In vivo experiments were performed in compliance with the French guidelines for experimental animal studies (agreement B34-172-27).C57BL/6J Rip1404 cells per well) 24 h prior to DNA transfection with Jet-PEI® . The pGL3-POLK Luc and its truncated mutant pGL3-83 vectors were previously described. The pGL3-29 vector was constructed for this work and co-transfected in HCT116 cells. The pRL-CMV-renilla plasmid (Ozyme®) was used to normalize transfection efficiency. Firefly luciferase values were measured and normalized by the Renilla luciferase activity. Values were expressed as the mean ratio of luciferase activities.HCT116 cells were plated in 96-well plates -2,5-diphenyltetrazolium bromide (MTT)  was added and incubated at 37 °C for 4 h. Formazan crystals were solubilized in DMSO and absorbance read at 560 nm on a spectrophotometer. The results were normalized to the cell density at Day 1. For cytotoxicity assays, cells were seeded in quadruplicate in a 96-well plate (2.5 × 103 cells per well) and exposed the day after to increasing concentrations of cytotoxic drugs orto vehicle alone. The cells were exposed to the drug during six days and cell proliferation was quantified each day using MTT assay. The results were normalized to the mean optical density of the control for each day. MMS and cisplatin were obtained from Sigma-Aldrich®.Cells were seeded in quadruplicate at a density of 2 × 102O, and 2 μL of the template diluted at 1:10 [see Supplementary Table 1 for primer sequences]. After pre-incubation at 95 °C, runs corresponded to 35 cycles of 15 s each at 95 °C, 5 s at 60 °C, and 15 s at 72 °C. Melting curves of the PCR products were analyzed using LightCycler® software to exclude amplification of unspecific products. The results were normalized to different housekeeping gene transcripts (mouse RS9 or human 28S). Total RNA was extracted from cells using Quick-RNA kit (Zymo Research) according to the manufacturer’s instructions. Total RNA (1 µg) was subjected to reverse-transcription using qScript cDNA SuperMix . Real-time quantitative PCR (RT-qPCR) were performed with the Roche LightCycler® 480 instrument and the PerfeCTa SYBR Green FastMix  and were carried out in a final volume of 10 μL using 0.25 µL of each primer (25 μM), 5 μL of the supplied enzyme mix, 2.5 μL of HRIPA solution was used to extract whole-cell proteins. Cell extracts were analyzed after migration of 30 µg protein extract by Western blotting using a primary polyclonal antibody against Polκ . Protein quantifications were normalized with the β-actin signal . and GSE42284, were reanalyzed for RIP140 and POLK expression using the Cancertool database. A transcriptomic dataset from the TCGA-COAD RNA-seq data was also used. Another published DNA microarray study obtained on a cohort encompassing 396 colon tumor samples with MSS and MSI CRC was also reanalyzed for RIP140 and POLK mRNA expression. Statistical significance was assessed using a Spearman correlation analysis. Correlation between RIP140 and POLK gene expression was also studied using another cohort. RNA sequencing data from the TCGA were reanalyzed using Cox proportional hazard regression. RNAseq data obtained from CRC samples from the TCGA dataset were analyzed as described previously. The Kaplan-Meier method was used to estimate overall survival (OS) calculated from the diagnosis until death. Patients lost to follow-up were censored at the time of last contact.Published DNA microarray datasets, GSE39582P-value) of 0.05 was chosen for statistical significance.All experiments were conducted independently at least three times. The results were expressed as the mean ± standard deviation (S.D.). Statistical comparisons were performed with Mann-Whitney or Spearman tests. For the Cancertool database analysis, a Pearson’s test was performed for the correlation analyses. A probability level  or overexpressed (RIPTg mice) . As shown in Polκ mRNA was observed in the intestinal epithelium of RIPKO mice, whereas an increase was noted in RIPTg mice as compared to wild-type animals (WT). These regulations appeared specific since the expression of other TLS polymerase genes from the Y subfamily such as PolI did not vary between the different genotypes  as well as the Polκ protein levels in RKO cells [To check whether this regulation can be observed in human cells, we then analyzed the effect of RIP140 on g RIP140  or transg RIP140 . We alsoKO cells .POLK gene expression by RIP140, we set up transient transfection experiments of HCT116 cells using the POLK Luc reporter construct encompassing the proximal promoter region of the POLK gene [POLK gene promoter, thus supporting a positive transcriptional regulation by RIP140. The same effects were observed in other CRC cells including RKO, SW480, and SW620 cells [To decipher the mechanisms underlying the positive regulation of OLK gene . As obse20 cells .). In particular, we described a transcriptional activation of gene expression by RIP140 through Sp1-mediated mechanisms. Since the POLK gene promoter exhibits Sp1 binding sites, we tested if the deletion of these sites affected the transcriptional response to RIP140 ectopic expression. As shown in POLK 29 Luc), suggesting that the regulation of POLK gene expression by RIP140 might be, at least in part, mediated by Sp1.Although RIP140 was first identified as a transcriptional repressor, we and others reported positive regulation of gene expression  [. This allowed us to show a significant correlation of RIP140 gene expression with that of POLK in the six different subgroups, in particular in the C2 group which corresponds to the MMR deficient subgroup. Finally, we confirmed these data in another cohort comparing the expression of “DNA replication” genes in CRC and in the adjacent normal mucosa. We confirmed that RIP140 gene expression strongly decreased in the tumor (data not shown) and that there was a strong correlation in the expression ratio  of the two genes  [To validate in human CRC biopsies the expression data obtained in mouse tissues and in CRC cells × 10-16) . This da 0.0001) .MSH2 and MSH6 genes. To compare the correlation between RIP140 and POLK gene expression in microsatellite stable (MSS) and instable (MSI) CRC tumors, we reanalyzed a transcriptomic dataset from 396 human CRC with both types of tumors. As shown in RIP140 and POLK mRNA levels in the whole cohort , thus confirming the results shown in POLK and RIP140 gene expression were significantly correlated in both MSS and MSI tumors [We recently demonstrated that RIP140 plays an important role in controlling microsatellite instability through the regulation of I tumors . . This mutation  generated a truncated protein which impaired the biological activity of the RIP140 protein. As shown in MSI protein was found to be less efficient than the WT protein to increase POLK gene expression. The same results were obtained on endogenous gene expression in HT29 CRC cells overexpressing wild-type or mutated RIP140 [MSI mutant exhibited a dominant negative effect since its ectopic expression significantly decreased POLK mRNA accumulation only in WT MEFs, which express normal levels of RIP140, and not in RIPKO MEFs, which no longer express the Rip140 gene [In MSI CRC, we identified a frameshift mutation in the RIP140 coding sequenced RIP140 . Interes140 gene .POLK gene is involved in the cellular response to cytotoxic drugs including MMS and cisplatin through a TLS replication process, we next measured the importance of RIP140 on MMS and cisplatin sensitivity by comparing IC50 ratios between RIPKO cells and their WT counterparts. As shown in RIP140 gene was knocked out . Altogether, these data suggest that the regulation of POLK gene expression by RIP140 can affect the cellular responses to various alkylating agents, including anticancer drugs.Since the POLK levels contribute to the good prognosis value of RIP140 that we previously reported in CRC patients. We reanalyzed RNAseq data obtained from 452 patients with colon adenocarcinoma that we separated into two groups of 226 patients, each with low and high POLK gene expression in the corresponding tumors using the median as a cutoff value. As shown in POLK gene expression [POLK gene expression [POLI expression [Using the Kaplan-Meier Plotter database, we then investigated whether pression  but not pression . As a copression  and D. C. We also showed that RIP140 could be involved in microsatellite stability through the regulation of MSH2/MSH6 gene expression.Colorectal cancer is a frequent neoplasm with high genomic instability including MSI due to defects in the MMR system. We previously described that the transcription factor RIP140 was a key factor in the regulation of intestinal homeostasis and tumorigenesisPOLK gene expression, in both mouse and human cells and tissues. Data obtained using luciferase reporter assays demonstrate that this regulation took place at the transcriptional level and implicated the proximal region of the POLK gene. Several transcription factors including p53, Sp1, and CREB or AhR have been shown to control the expression of the POLK gene, and some of them have their activity regulated by RIP140. The data presented herein suggest that p53 is a good candidate to mediate, at least partly, the regulation of POLK gene expression since the positive effect of RIP140 is lost in HCT116 cells no longer expressing the TP53 gene. Moreover, it has been shown that the HDAC inhibitor Trichostatin A was able to induce POLK gene expression. Since RIP140 strongly interacts with HDACs, it is possible that HDAC sequestration out of the POLK gene promoter also participates in the positive effect exerted by RIP140 on POLK gene transcription as already demonstrated for other transcription factors. Further work will be necessary to fully decipher the mechanisms used by RIP140 to transcriptionally control POLK gene expression.In the present study, we demonstrated that RIP140 also strongly regulates ; the replication checkpoint, a crucial pathway that regulates the replication stress response to a large array of insults that cause replication arrest; and the synthesis of intrinsic non-B microsatellite DNA. Consistent with these multiple and complex roles, both overexpression of POLK  and under-expression of POLK  have been documented to lead to genetic instability. Indeed, excessive Polκ can interfere with fork progression and trigger a mutator phenotype and chromosome instability, while downregulation of Polκ expression can affect fork progression and induce replicative stress. Since both situations can confer a selective growth advantage during cancer cell evolution, this underlines the importance of tight regulation of POLK gene expression at the transcriptional level for the maintenance of genome integrity. Therefore, our discovery here establishing the role of RIP140 in POLK gene regulation not only may explain why POLK is abnormally expressed in cancer, notably in CRC, but can also clarify why cancers cells respond differentially to anticancer genotoxic drugs that target DNA replication forks. This last point is illustrated in the present work by cell sensitivity to MMS treatment, which can be modulated by RIP140. Cell survival relies on a subtle equilibrium between accurate genomic DNA replication and less stringent DNA synthesis often linked to DNA damage. POLK plays an important and critical role in such equilibrium by contributing to several important DNA transaction pathways. These include translesion synthesis, which allows stalled replication forks to bypass a lesion and restartPOLK gene expression could intensify the phenotypic consequences of the regulation of MutSα gene expression by RIP140. Indeed, this TLS polymerase displays a high accuracy during dinucleotide microsatellite DNA synthesis and has been proposed to play a role in the maintenance of microsatellite stability. Moreover, Polκ interacts with MSH2 and could therefore participate in the control of MutSα activity. Defining the precise involvement of POLK gene induction in the effect of RIP140 on the maintenance of genome integrity in colon cancer cells (in particular on the stability of microsatellites) and on their resistance to anticancer drugs will obviously require further work. It would also be interesting to decipher whether, as it is suggested for the MSH2 protein, POLK could be involved in the RIP140-mediated transcriptional regulation of gene expression. In addition, it is worth mentioning that the regulation of POLK-defective cells exhibit an altered DNA replication program, thus explaining the spontaneous genetic alterations observed in POLK-deficient mice. Transcriptional deregulation of POLK gene expression may therefore participate in intestinal tumorigenesis and account, at least in part, for the tumor suppressor role of RIP140 that we previously suggested.Altogether, the present data suggest that


Genomic studies may generate massive amounts of data, bringing interpretation challenges. Efforts for the differentiation of benign and pathogenic variants gain importance. In this article, we used segregation analysis and other molecular data to reclassify to benign or likely benign several rare clinically curated variants of autosomal dominant inheritance from a cohort of 500 Brazilian patients with rare diseases. This study included only symptomatic patients who had undergone molecular investigation with exome sequencing for suspected diseases of genetic etiology. Variants clinically suspected as the causative etiology and harbored by genes associated with highly-penetrant conditions of autosomal dominant inheritance underwent Sanger confirmation in the proband and inheritance pattern determination because a “de novo” event was expected. Among all 327 variants studied, 321 variants were inherited from asymptomatic parents. Considering segregation analysis, we have reclassified 51 rare variants as benign and 211 as likely benign. In our study, the inheritance of a highly penetrant variant expected to be de novo for pathogenicity assumption was considered as a non-segregation and, therefore, a key step for benign or likely benign classification. Studies like ours may help to identify rare benign variants and improve the correct interpretation of genetic findings. Genomic studies based on next-generation sequencing (NGS) technology are cost-effective alternatives to determine the molecular diagnosis of patients with rare diseases of monogenic etiology because they allow the concomitant study of several known genes associated with genetic conditions. On the other hand, these techniques may generate massive amounts of data and unravel several rare genomic variants, which brings challenges in the interpretation of these findings.1. Among these types of evidence, segregation of a variant in an affected family may be evidence for pathogenicity; for several diseases, though, the non-inheritance  is the expected mechanism for pathogenicity. On the other hand, lack of segregation of a variant with a phenotype may provide strong evidence against pathogenicity.Variant classification is a systematic process that gathers different pieces of evidence from multiple sources, including scientific literature, control databases, in-silico predictors, among others, and aims a correct interpretation of genetic findings. The American College of Medical Genetics and Genomics in conjunction with the Association for Molecular Pathology (ACMG-AMP) created guidelines for the interpretation of sequence variants, becoming an important step toward a more uniform classification process. Among the recommendations, we highlight (1) the use of specific standard terminology: ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’, and ‘benign’ to describe variants identified in Mendelian disorders and (2) a standardized process for classification of variants based on criteria using typical types of variant evidenceAnother category recommended by ACMG-AMP is in silico analysis. Many in silico algorithms have been developed to predict the impact of missense variants and some of these tools have demonstrated a superior performance, such as REVEL [2. 3].8. Although definitive re-classification of a VUS as pathogenic or benign may eventually occur, the timeline is typically many years, and may be indefinite for rare VUS, especially if the disease is uncommon10. Therefore, efforts for the differentiation of benign and pathogenic variants gain importance.The finding of a variant of unknown significance (VUS) can be problematic for patients and clinicians working in clinical genetics settingIn this article, we used segregation analysis and other molecular data to reclassify to benign or likely benign several rare clinically curated variants of autosomal dominant inheritance from a cohort of 500 Brazilian patients with rare diseases.11. These samples were collected from 2016 to 2020 in facilities of the Fleury Group, which is one of the largest private diagnostic laboratories in Brazil.This study is focused on novel data  from molecular findings obtained from exome sequencing (ES) analysis of 500 samples of adult, pediatric and fetal patients. Full details of the clinical features of patients, molecular analysis, bioinformatics protocols, clinical data, and molecular data for primary findings, secondary findings and carrier status for recessive diseases were previously publishedThis study has been performed in accordance with the Declaration of Helsinki, followed the best standards for scientific research and strictly followed Brazilian law for research involving human subjects. Written informed consent was obtained from all the participants. This study was granted ethics committee approval from Grupo Fleury and Faculdade de Medicina da Universidade de São Paulo .12. Full details of the primary and secondary findings can be found elsewhere10. The carrier status for pathogenic or likely pathogenic variants of autosomal recessive conditions has also been published elsewhere11.Primary findings were reported when pathogenic or likely pathogenic variants were observed in a gene that was associated with the patient’s phenotype, with compatible zygosity and an adequate inheritance pattern; these cases were considered positive. Secondary findings  were reported for pathogenic and likely pathogenic variants in the ACMG gene list and exceptionally in other genes determined to be clinically relevant by our team of specialistsThe samples were collected from exclusively symptomatic patients who had undergone molecular investigation for suspected diseases of genetic etiology. The clinical data were collected through a comprehensive pretest form completed by the attending physician or family, medical reports and clinical notes provided to the laboratory.11. In summary, DNA from the proband and both parents were extracted from peripheral blood leukocytes, saliva or prenatal samples of villus biopsy or amniotic fluid after appropriate cell culture. Exome capture used Agilent Clinical Research Exome v1 according to the manufacturer’s instructions and sequencing was performed using an Illumina NextSeq platform. Exome data were aligned to the GRCh37.75/hg19 reference genome using the Burrows–Wheeler Aligner . Variants (single-nucleotide variants [SNVs] and indels) were identified following the best practices of the Broad Institute using the Genome Analysis ToolKit  software and annotated using Variant Effect Predictor . All exomes met a minimum of 95% of target bases covered at > 10 × . In-house bioinformatic pipelines were developed by a dedicated team of specialists. The mitochondrial genome and copy number variants were not studied.Molecular analysis and bioinformatics protocols followed the exact workflow as published in previous works by our group11. In summary, at least two parallel analyses were performed to preselect variants considering (1) clinical relevance and overlap of gene harboring the variant and the provand’s manifestation, (2) allele frequency < 1%, (3) relevant functional impact, (4) relevant reports from databases  and (5) previous reports from the literature. The first step of variant preselection consisted of filtering those already reported in ClinVar or HGMD as disease-causing and reclassifying them according to ACMG guidelines. Then, the same filtering-reclassifying approach would include (1) variants of predicted loss-of-function harbored by genes with evident sensitivity for haploinsufficiency and (2) rare missense variants harbored by genes with missense constraint. The last step in variant preselection included variants with allele frequency < 1% harbored by genes with clinical overlap with the proband’s phenotype.The variant analysis workflow has also been performed according to our previous worksThese preselected variants were then discussed in a board comprising three experts. After this first board meeting, selected variants would undergo Sanger confirmation in the proband and inheritance pattern determination when the parental samples were available. Paternity and maternity were not confirmed by any specific test in our cohort, although nonpaternity/nonmaternity could be inferred by genotyping multiple rare variants. Parents were considered non-affected, unless otherwise reported in the clinical notes provided to the laboratory.11: Protocol 1 , in which the samples were processed in our laboratory as described above and variants were interpreted using an in-house web interface (GTAC); Protocol 2 , in which the samples were processed in our laboratory as described above and variants were analyzed using a commercially available diagnostic decision support platform by Emedgene Technologies LTD  (www.emedgene.com); Protocol 3 , in which the samples were sent to Centogene AG  (www.centogene.com) and processed according to the protocols of this third-party laboratory, including DNA extraction, bioinformatic pipeline, CNV analysis, and variant analysis. For Protocol 3, the primary findings reported by Centogene were confirmed by in-house Sanger sequencing of the proband and genitors to determine the inheritance pattern.The samples were analyzed by three distinct protocols as followsVariants preselected as described above in the board of experts were preselected because they presented relevantly low allele frequencies (< 1%) and potential relevance to the clinical manifestation of the corresponding patient. All variants were clinically suspected as the causative etiology for all patients and were harbored by genes associated with highly penetrant conditions of autosomal dominant inheritance. As a de novo event was expected for pathogenicity assumptions, these variants underwent Sanger confirmation in the proband and inheritance pattern determination when the parental samples were available. Individual data for each case, along with the molecular data and segregation studies for all variants can be accessed in the Supplementary Material.1 with assistance of the third-party ACMG calculator Varstation ® . Internal adaptations of ACMG guidelines used in this work may be found below.All variants were reviewed and reclassified for the purpose of this article. The complete details, nomenclature and classification information are available in the Supplementary Material. Variants were classified according to ACMG guidelines1. Each benign criterion is weighted as stand-alone (BA1), strong (BS1–4), or supporting (BP1–6). Benign variants need either a stand-alone (BA1) criterion or two strong (BS1–4), while likely benign variants need one strong and one supporting criterion or at least two supporting criteria. Although variant classification followed the ACMG guidelines, some adaptations were adopted especially for benign criteria BS4, BS1, BS2, BP4 and BP5:ACMG presents specific rules to classify variantsBS4—Variant does not segregate with the disease: considering that the lack of segregation of a variant with a phenotype provides evidence against pathogenicity, this criterion was used if a variant expected to be de novo was inherited from unaffected parents in genotype-positive and phenotype-negative scenarios. If the gene harboring the variant is associated only with early-onset conditions, the non-segregation was considered a strong evidence of non-pathogenicity (BS4_strong), while if the gene is associated with later-onset manifestations the non-segregation was considered a supporting evidence (BS4_supporting). All conditions analyzed in this study were considered enough penetrant for the application of segregation criteria.BP5 —Variant found in a case with an alternate molecular cause for the disease. When a variant was observed in a case with a clear alternate genetic cause of disease  for which the reported variant is unlikely to contribute or modulate expressivity of the primary finding, this was considered supporting evidence to classify the variant as benign (BP5_supporting). Primary finding has already been defined above.BS1—Allele frequency is greater than expected for disorder: we have used a conservative approach to use this criterion: variants received a BS1_strong only when presented with a frequency greater than 0.1% for autosomal dominant diseases in the gnomAD database.BS2—Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age: since the objective of our study is to analyze the impact of variants exclusively in the autosomal dominant inheritance pattern, we have used a threshold to apply BS2_strong of 5 individuals harboring the same variant in the gnomAD database.PM2_supporting-  Absent from controls for AD in gnomAD. We adjusted all PM2 criteria to PM2_supporting in agreement with recent recommendations from the Clingen working group.13.PP3-pathogenic for REVEL score > 0.7, according to the recommendations for best practices of United Kingdom Association for Clinical Genomic Science13.BP4- REVEL score < 0.4, according to the recommendations for best practices of United Kingdom Association for Clinical Genomic Sciencehttps://research.nhgri.nih.gov/CGD/search; date of access: June, 2021) of the National Human Genome Research Institute (NHGRI), based in the United States14.Information about genes and their corresponding diseases along with inheritance mechanism and penetrance was recalled from The Clinical Genome Database website .Written informed consent was obtained from all the participants.All authors are aware, consented and approved this publication.We have analyzed a total of 334 occurrences of 327 unique heterozygous variants (seven variants recurred twice) in 210 different genes.SCNN1A and GPR98; 1%) are associated with either autosomal dominant, autosomal recessive or digenic inheritances and harbor two occurrences of two unique variants (0.6%).Regarding the 210 different genes, 171 (81.4%) are solely associated with autosomal dominant diseases and this group of genes harbors 273 occurrences of 267 unique variants (81.7%). Another 37 genes (17.6%) are associated with autosomal dominant and autosomal recessive forms and this group harbors 59 occurrences of 58 unique variants (17.7%). Two genes  were not found in both parents and in one case paternal sample was not available . Six genes (2.9%) that are associated with adult-onset conditions harbor eight occurrences of seven unique variants (2.1%), to which a BS4_supporting criterion was attributed. Finally, 114 genes (54.3%) that harbor 181 occurrences of 179 unique variants (54.7%) were not classified by NHGRI regarding the onset of the correspondent diseases: 176 variants were inherited from asymptomatic parents and were attributed the BS4_supporting criterion; two variants were not found in both parents  and in one case paternal sample was not available .Still regarding the 210 different genes, 90 genes (42.9%) are associated with exclusive pediatric-onset conditions and harbor 145 occurrences of 141 unique variants (43.1%): 138 variants were inherited from asymptomatic parents and received BS4 (strong) criterion, while two variants . Variants were inherited from mothers in 168 cases (50.3%); inherited from fathers in 158 (47.3%); were not found in both parents in four (1.2%); paternal sample was not available in 2 cases (0.6%) and in two other cases both parents were heterozygous for the same variant (0.6%).Regarding the 327 unique variants, 139 (42.5%) were not found in the gnomAD database (gnomad211) and were attributed the PM2_supporting criterion; 87 variants (26.6%) presented from one and less than five controls in gnomAD. Another 100 variants (30.6%) were present in five or more controls in gnomAD and were attributed the BS2 criterion. Among these 100 variants, 11 (3.4%) presented a frequency higher than 0.1% in gnomAD and also attributed the BS1 criterion.10.For 81 unique variants (24.8%), a clear alternate genetic cause for the patients’ conditions was unraveled by ES. For these variants, BP5 criterion was used. All alternate causes for diseases (primary findings) were published elsewhereFigure Considering all variants classified as benign or likely benign , we did not find dbSNP correspondence for 76 variants (29%) and ClinVar entrances were not found for 189 variants (72.1%). ClinVar entrances were available for 73 variants (27.9%), among which 15 were classified as benign or likely benign, 40 as VUS, one as likely pathogenic and for 17 variants ClinVar classification was conflicting among participating laboratories.The remaining 65 unique variants (19.9%) were classified as variants of unknown significance. Two thirds of these variants (n = 44) were not found in the gnomAD database.For unique variants harbored by genes associated with pediatric-onset conditions, 73% (103/141) were classified as benign or likely benign, while for variants harbored by genes associated with adult-onset conditions, 71.4% (5/7) were classified as likely benign. As for variants harbored by genes non-classified by NHGRI, 86% (154/179) were classified as benign or likely benign.We have used segregation analysis and other molecular data to reclassify rare variants to benign (n = 51) or likely benign (n = 211) in a Brazilian cohort of rare diseases. All variants are harbored by genes associated with autosomal dominant disorders. Proper interpretation of rare variants is a crucial step for adequate molecular diagnosis and, consequently, clinical management and genetic counseling. Therefore, efforts to distinguish pathogenic variants from rare benign variants are a key step in molecular diagnosis, but ascertaining which rare variants have clinical/health impact remains a major challenge.We have used a systematic approach to preselect the variants in this study. Besides a low allele frequency in control databases, reports from literature and functional impact, all variants were clinically curated: they were harbored by genes associated with autosomal dominant disorders similar to all patients’ manifestations. In other words, all variants were associated with autosomal dominant disorders and were judged highly suspicious to explain the final diagnosis that had led to exome sequencing.Segregation study was assessed to investigate the clinical relevance of the rare variants found in our cohort because a de novo event was expected for all 327 suspicious variants. Segregation is important to assess the pathogenicity of a variant. Indeed, variant segregation in patients and corresponding families are widely used for novel disease-associated genes and pathogenic/likely pathogenic variants discoveries. In this study, we have used the same approach for the opposite reason: uncover benign/likely benign variants. The inheritance of a variant expected to be de novo for pathogenicity assumption was considered in our study as a non-segregation and, therefore, a key step for benign or likely benign classification. However, this was not the sole step in this process, as discussed below.Figure Another key step in the classification is the frequency in the control database. In this study, we used only the gnomAD database, a fact that may have underestimated the “benign weight” of BS2 criterion because other databases might suggest higher frequencies especially for ethnicities not covered by gnomAD; on the other hand, the quality of variant calls and integrity of database might be an uncontrolled bias. All variants found in five or more controls in gnomAD were eventually classified as benign or likely benign because they received other benign criteria. Regarding the benign variants, 14 received exclusively the combination BS4 (variants inherited from parents and associated with conditions curated as pediatric onset) and BS2. As for the likely benign variants, nine variants received exclusively BS4_suporting (variants inherited from parents and associated with conditions not curated by NHGRI) and BS2.15. These new pieces of evidence demonstrate adequate strength of REVEL as a benignity predictor and make this tool fit adequately in our study, which aims to unravel rare neutral variants. The limited use for missense variants, though, is a limitation of REVEL approach.A fourth key step was in-silico prediction. In this study, we have used REVEL, which is an ensemble method for predicting the pathogenicity of rare missense variants, as our sole in-silico predictor. REVEL has shown better performance when compared to other methods especially for rare neutral variantsAmong the 170 that did not receive either BP5 and BS2 criteria, 106 were predicted as having benign effect by REVEL (score < 0.4) and received BP4 criterion; 104 of these variants were inherited from parents (received BS4 or BS4_supporting) and the combination of BP4 + BS4/BS4_supporting was critical for the final classification as likely benign.Finally, 65 variants did not meet minimum requirements for reclassification as benign or likely benign and were considered VUS, among which 63 were inherited from a parent (received solely BS4 or BS4_supporting).16.A total of six unique variants were not confirmed to be inherited from either parent in our study, among which four were not found in parental samples (a supposed de novo event) and for the remaining two, the paternal samples were not available, though these two variants were not found in the maternal samples. Half of these variants (n = 3) were classified as likely benign considering other molecular data. Although it is widely known that several de novo genome changes can be associated with a multitude of pathological conditions, a broad range of de novo molecular events is also characteristic for individuals obtained from a general human populationFor the majority of variants classified as benign or likely benign, we did not find a ClinVar classification . Even for variants with ClinVar entrances, we found a concordance of classification with our study only for 15 variants.ASH1L, KMT2A, DEAF1, TRPV4, CHD2, SRCAP, ANKRD11, RAI1, ZEB2, SHANK3, ARID1B, and others) and this fact may have underestimated non-segregation (unclassified genes received a BS4_supporting); (3) variants associated with adult-onset conditions were a minority (seven unique variants) and segregation analysis for these cases may be more complex to understand: we opted to apply a BS4_supporting criterion considering that parents were asymptomatic and we did not have information of other affected family members, even though we may have overestimated the non-segregation effect for these seven variants; (4) our method applies for genetic sequence variants of high penetrance and dominant inheritance: for this model, we considered all conditions sufficiently penetrant for segregation/non-segregation purposes.Our study approach focuses on variant segregation with phenotype and makes several simplifying assumptions, including (1) parents were asymptomatic: we recognize that several dominant diseases may present varying degrees of severity and it may be difficult for clinicians to identify oligosymptomatic patients; (2) non-segregation was considered a strong benign criterion for child-onset diseases and a supporting criterion for later-onset diseases or onsets non-classified solely by NHGRI: NHGRI database lacks information for several genes, including some widely known early-onset genes  that a rare variant classified as B/LB for autosomal dominant Mendelian trait may have health/disease implications for complex traits, for recessive forms or even for unknown recessive forms of a gene.It is important to note, though, that there are growing pieces of evidence that rare variants make important contributions to human phenotypic variation and disease susceptibility, though detecting the effects of rare variants in complex traits is challenging because 1) it generally requires very large sample sizes to achieve statistical power and 2) rare SNVs are population-specific, which implies difficulties for replication of disease associations across different populationsOur study presents important limitations. First, it analyzed the impact of single variants in monogenic-based models of autosomal dominant diseases and did not consider more complex interactions that might modulate phenotypes. Another limitation is that several variants were classified as likely benign/benign based solely on ACMG criteria but were not definitively proven to be benign. We took several precautions to apply some ACMG benign criteria that may have underestimated their effects: BS2 required at least five controls from gnomAD, BS1 required a frequency of at least 0.1% in gnomAD (a frequency considered high for many rare conditions). Even though we followed the ACMG criteria for variant classification, we may have falsely classified some variants as B/LB since we relied solely on presumptively assuming mechanisms without proper functional studies.Even taking extra precautions that underestimate the weight of several benign criteria  and underestimating the non-segregation for several conditions non-classified by NHGRI, the majority of variants  were reclassified as benign or likely benign. One main reason for this is that it is a lot easier for a variant to be classified as B/LB than pathogenic or likely pathogenic because only two supporting benign criteria are enough for LB classification. We used the term “easier” for two reasons: easier because it requires less criteria for B/LB classification and easier because they do not have up- or -downgrades such as several pathogenic criteria. These assumptions have made us raise concerns regarding the wide application of benign criteria. They also made us unilaterally downgrade BS4 criterion for later-onset or non-classified-onset conditions. On the other hand, the addition of extra supporting benign criteria does not influence in the final classification: for instance, a variant receiving a combination of BS2 and BP4 will have the same likely benign classification of a variant receiving BS2, BS4_supporting, BP4 and BP5, though the likelihood for this later variant to be benign is greater than the former.We did not find comprehensive studies that address segregation analysis and classification of B/LB variants in Brazil or any other Latin American countries. These countries are generally underrepresented populations in international databases and genetically heterogeneous with important genetic contributions from Amerindians, African-Americans and Western-Europeans. The more NGS and segregation studies become available in such nations, the more will be known of the rare regional benign variants.Literature presents an important limitation regarding the description of benign/likely benign variants because studies and research journals generally present a strong bias for positive results. We believe that studies like ours are valuable for identifying rare benign variants and this strategy may improve the correct interpretation of genetic findings.Supplementary Information.


IntroductionInvasive blood pressure monitoring is essential in liver transplant surgery due to expected major hemodynamic shifts. The use of central versus peripheral arterial access, however, is institution-dependent, which can affect clinical decisions regarding vasopressor therapy. Although there are studies that demonstrate inconsistencies based on arterial cannulation sites, few studies have compared femoral and radial artery blood pressures in patients undergoing liver transplant surgery. To our knowledge, there are no studies investigating the differences between continuous minute-to-minute femoral and radial artery measurements during all three phases of liver transplant surgery.ObjectiveThe main objective of this study was to evaluate for any differences between central and peripheral blood pressure measurements in liver transplant surgery and to assess for any correlation between vasopressor infusion dose and femoral-arterial pressure differences.MethodsIn this retrospective study, we reviewed and studied the data of 61 patients with American Society of Anesthesiologists (ASA) grade 4 who underwent liver transplant surgery at Loma Linda University Medical Center between January and December of 2019. All patients had both femoral and radial arterial lines placed for liver transplant surgery. Femoral and radial arterial blood pressure values were obtained continuously over 60 minutes in the pre-anhepatic phase, 45 minutes during the anhepatic phase, and 60 minutes into the neo-hepatic phase. Vasopressor infusion doses were also recorded for each patient during these time frames.ResultsThis pilot study found statistically significant differences between the mean femoral and radial systolic blood pressure , diastolic blood pressure , and mean arterial pressure  during all phases of liver transplantation. The meanSBP and MAP differences between femoral and radial arteries were highest  in the late anhepatic and early neo-hepatic phases with SBP differences of 20.8 ± 0.8 mmHg and 22.8 ± 0.8 mmHg, respectively, and MAP differences of 10.0 ± 0.4 mmHg and 9.8 ± 0.4 mmHg, respectively. Higher vasopressor infusion doses were strongly associated with greater differences in femoral-radial SBP and MAP measurements  during the anhepatic phase.ConclusionsPeripheral invasive blood pressure monitoring may result in underestimation of the central blood pressure, as was seen in all phases of liver transplantation. This may lead to excessive vasopressor use with potentially adverse effects. Although the cause for the difference between femoral and radial artery measurements is unclear, increasing vasopressor infusion dosages appears to contribute. Femoral artery blood pressure monitoring allows clinicians to interpret hemodynamic status and administer appropriate vasopressors more accurately. Invasive blood pressure monitoring is warranted in patients with significant comorbidities and in high-risk surgeries where immediate, continuous monitoring is required. Although debate persists on the ideal monitoring site, the most common cannulation site in clinical practice is the radial artery due to ease of access and low risk of complications . FemoralDespite the popularity of the radial artery for invasive blood pressure monitoring, some studies demonstrate inconsistencies between peripheral and central arterial blood pressure values in patients with septic shock, hypothermia, and after cardiopulmonary bypass -5. CompaDue to major hemodynamic shifts and the potential for significant bleeding during liver transplant surgery, continuous invasive arterial blood pressure monitoring is essential. However, the choice of peripheral and/or central arterial access can vary depending on the institution and clinician's preference. Few studies have compared femoral and radial artery blood pressures in patients undergoing liver transplant surgery ,8. To ouA retrospective study was performed after obtaining Institutional Review Board approval from Loma Linda University Medical Center in compliance with Health Insurance Portability and Accountability Act regulations . Patient demographics, femoral and radial arterial blood pressure data, and vasopressor dosages were obtained from the electronic medical record of patients who underwent liver transplant surgery at our institution between January 2019 and December 2019. Patients who experienced cardiopulmonary arrest during the surgery and patients who had missing blood pressure or clamp time data on the electronic medical record were excluded from the study.For all patients, the radial artery was cannulated with a standard 20 gauge 1.75-inch radial artery catheter , and the femoral artery was cannulated with a standard 18 Gauge 6.29-inch femoral arterial catheter (Arrow). Two different transducers, set to the level of the patient’s right atrium and zeroed to atmospheric pressure, were used to measure radial and femoral arterial blood pressures. Intraoperative clinical decisions by the anesthesiologist were based on femoral arterial blood pressure with the goal to maintain mean arterial pressure (MAP) > 65 mmHg.Minute-by-minute blood pressure data (systolic blood pressure (SBP), diastolic blood pressure (DBP), and MAP) and vasopressor infusion dosage were obtained for each patient during the pre-anhepatic, anhepatic, and neo-hepatic phases. The pre-anhepatic phase was defined as 60 minutes prior to clamping the inferior vena cava (IVC). The anhepatic phase was defined as the first 45 minutes after clamping of the IVC. Neo-hepatic phase consisted of the first 60 minutes following the removal of the IVC and portal vein clamps.For data analysis, the difference between femoral-radial blood pressures  was measured at each time-point of the pre-defined stages of liver transplantation and averaged for all patients per time-point. This was calculated with a paired t-test using data from the radial arterial blood pressure subtracted from the femoral arterial blood pressure for each blood pressure variable . For each individual vasopressor , the mean infusion dosage was recorded at each time point during the three defined phases of liver transplantation. The correlation between vasopressor infusion dose and the mean difference in blood pressure was subsequently calculated using the Spearman correlation coefficient.Patient demographics2 (range: 18.5 to 42.5 kg/m2), and the mean Model for End-Stage Liver Disease (MELD) score was 30.4 ± 8.1 (range: 15 to 40).A total of 103 patients underwent liver transplantation surgery between January 2019 and December 2019 at Loma Linda University Medical Center. However, 43 patients were excluded from the study due to missing blood pressure data, clamping times, or intraoperative cardiopulmonary arrest. Of the resulting 60 patients included in our study, 37 were male (62%). The mean age of our patients was 54.3 ± 11.3 years (range: 19 to 74 years), the mean body mass index (BMI) was 29.8 ± 5.8 kg/mCentral and peripheral arterial blood pressure comparisonsThe mean difference between femoral-radial SBP, DBP, and MAP measurements of all 60 patients is demonstrated in Table The mean difference between femoral-radial SBP, DBP, and MAP measurements at each specific time point throughout the operation is visualized in Figure The mean femoral arterial SBP, DBP, and MAP were all statistically significantly higher than the radial artery measurements during all three phases of liver transplant surgery  Table . The meaVasopressor infusion dosagesThe average doses of the vasopressor infusions for vasopressin, norepinephrine, and epinephrine were documented for the same time points as the blood pressure values for all 60 patients Figure . No otheCorrelation between vasopressor infusion doses and mean blood pressure differencesTable Pre-anhepatic PhaseDuring the pre-anhepatic phase, the correlation between individual vasopressors and the mean SBP difference was not statistically significant Table . InteresAnhepatic PhaseDuring the anhepatic phase, there was a strong positive correlation between vasopressor infusion dose and the mean SBP difference  Table . The posNeo-Hepatic PhaseFor the neo-hepatic phase, statistically significant positive correlations were found between the norepinephrine and epinephrine doses and the mean SBP difference between femoral-radial artery measurements (R = 0.47 for norepinephrine and 0.85 for epinephrine) Table . NorepinWith the prevalence of significant hemodynamic variability in orthotopic liver transplantation, invasive arterial blood pressure monitoring is essential in providing anesthesiologists with data to support clinical decision-making. Reliable and accurate methods for measuring blood pressure are vital to reducing perioperative morbidity and mortality, especially for critically ill patients undergoing high-risk surgery . While tWith the high volume of liver transplantation surgeries at our institution, our study demonstrates a similar phenomenon in which we appreciate significant blood pressure differences, with femoral artery values consistently measuring higher than the radial artery. This phenomenon is visualized in Figure Few articles compare BP measurement differences specifically in liver transplant patients and the results are conflicting Table .Arnal et al.  observedIn contrast, our study found statistically significant differences between the femoral and radial mean SBP, DBP, and MAP during all phases of liver transplantation surgery (p < 0.0001). A possible explanation for the difference in our findings is that our study examined continuous minute to minute time-points and a larger number of total time points , providing a more comprehensive picture of blood pressure measurements throughout the operation. Similar to the study by Arnal et al. , our widThe mechanisms responsible for the difference in femoral and radial artery blood pressure measurements in liver transplant surgery are not well understood. One theory is that increasing vasopressors constrict smaller caliber vessels more and reduce flow to the radial artery, contributing to the difference seen between radial and femoral artery measurements. Another theory proposes that extreme peripheral vasodilation, as in the early neo-hepatic phase of liver transplantation, can result in decreased distal pressures compared to central pressures, similar to that seen in post-cardiopulmonary bypass physiology . Other pA recent, retrospective analysis of liver transplant surgery revealed patients had an increased risk for 30-day mortality with sustained intraoperative MAPs below 60 . AlthougSmall studies in liver transplant surgery, cardiac surgery, and the intensive care unit argue that utilizing peripheral invasive blood pressure monitoring alone may result in underestimating the central blood pressure -12. Our Some studies suggest that high-dose vasopressors contribute to the discrepancy between central and peripheral blood pressure, creating a cycle that could lead to significant morbidity and mortality . Our stuLimitationsA limitation of our study was that vasopressor boluses were not included in our data, and only vasopressor infusion doses were included in the data analysis. Because all our patients were on vasopressor infusions at the time points measured, we did not compare the effect of vasopressors to no vasopressors on the blood pressure difference. Additionally, our study did not account for other confounding variables, such as the physiologic effects of liver failure or reperfusion on peripheral vs. central artery vascular changes.Though our pilot study shows statistically significant differences between femoral and radial blood pressure measurements, it is difficult to quantify these data into clinical significance. To determine clinical significance, a future study would compare two groups of patients managed separately via femoral or radial blood pressure readings to detect if differences exist in the vasopressors utilized and outcomes of morbidity and mortality.To our knowledge, our pilot study is the first to examine minute-to-minute blood pressure differences in all three phases of liver transplant surgery, with a robust dataset examining femoral and radial arterial measurements. Based on the results, central blood pressure monitoring is recommended in liver transplant surgeries and major surgeries with hemodynamic shifts, massive blood loss, and multiple vasopressor usages. Multiple vasopressors are a contributing factor to the differences between central and peripheral blood pressure measurements. Central arterial blood pressure measurements allow for a more accurate interpretation of hemodynamic status and the appropriate management of vasopressors. Further studies examining additional factors such as blood pH, temperature, patient age, MELD score, and type of operation that may cause the significant differences between central and peripheral arterial blood pressure measurements may yield further insight into how the vasculature reacts in the perioperative period.


Clinico-pathological correlation studies show that some otherwise healthy elderly individuals who never developed cognitive impairment harbor a burden of Alzheimer’s disease lesions (plaques and tangles) that would be expected to result in dementia. In the absence of comorbidities explaining such discrepancies, there is a need to identify other brain changes that meaningfully contribute to the cognitive status of an individual in the face of such burdens of plaques and tangles. Glial inflammatory responses, a universal phenomenon in symptomatic AD, show robust association with degree of cognitive impairment, but their significance in early tau pathology stages and contribution to the trajectory of cognitive decline at an individual level remain widely unexplored. We studied 55 brains from individuals at intermediate stages of tau tangle pathology (Braak III-IV) with diverging antemortem cognition , and age-matched cognitively normal controls (Braak 0-II). We conducted quantitative assessments of amyloid and tau lesions, cellular vulnerability markers, and glial phenotypes in temporal pole (Braak III-IV region) and visual cortex (Braak V-VI region) using artificial-intelligence based semiautomated quantifications. We found distinct glial responses with increased proinflammatory and decreased homeostatic markers, both in regions with tau tangles  and without overt tau deposits  in demented but not in resilient. These changes were significantly associated with markers of cortical cell damage. Similar phenotypic glial changes were detected in the white matter of demented but not resilient and were associated with higher burden of overlying cortical cellular damage in regions with and without tangles. Our data suggest that changes in glial phenotypes in cortical and subcortical regions represent an early phenomenon that precedes overt tau deposition and likely contributes to cell damage and loss of brain function predicting the cognitive status of individuals at intermediate stages of tau aggregate burden (Braak III-IV). The frequency of both amyloid plaques and neurofibrillary tangles  and dementia increases with age , 26, 72.Detailed examination of high tangle burden (Braak V-VI) demented and resilient brains by our group and others has shown that one of the most robust and significant differences is the presence of substantially increased glial responses, comprising inflammatory microglia and astrocytes , 68, aloEmerging studies are highlighting the broad heterogeneity in phenotypes of astrocytes and microglial cells in AD but the role of each of these glial phenotypes (e.g. pro-inflammatory and homeostatic) in the clinical expression of plaques and tangles and other common neurodegenerative lesions, and in the tissue injury responses  and trajectory of cognitive impairment are incompletely understood , 82, 83.In addition, recent evidence from neuroimaging and autopsy studies has suggested that white matter (WM) damage comprising axonal loss, astrogliosis, and oligodendrocyte dysfunction accounts for one of the earliest pathologic changes that may be implicated in the risk and progression of AD , 62. EarWhether an aberrant activation and/or lack of homeostatic properties of glial elements in cortical and juxtacortical WM regions substantially contribute to neuronal damage and loss of brain function in AD or reflect a mere response to neuronal injury remains controversial. The study of brains at intermediate stages of tau burden (Braak III-IV) with divergent ante-mortem cognitive status (demented vs. resilient) provides a valuable opportunity to assess early presence and interaction(s) between glial cell responses and brain changes other than Aβ and tau that occur in regions yet devoid of NFTs and that could be relevant to the earliest and most preventable phases of neurodegeneration in AD. We hypothesized that changes in cortical and subcortical glial phenotypes precede overt NFT formation and correlate with early markers of cortical cell damage predicting the cognitive fate of individuals at intermediate stages of tau burden (Braak III-IV).The study included 55 cases from the Massachusetts General Hospital ADRC Brain Bank. Cases were scored by Thal phases for Aβ deposition (0–5) , Braak sThe majority of both demented and resilient brains in these series also harbored intermediate loads of amyloid neuritic plaques (moderate CERAD scores) meeting criteria for an intermediate probability of AD at autopsy according to NIA-AA guidelines . A smallQuantitative neuropathological assessments were derived from two regions of interest, temporal pole (Brodmann’s area 38) and visual cortex (Brodmann’s area 17/18), representing regions known to be affected by NFT pathology at Braak stages III-IV and V-VI respectively. Brain tissue sections derived from formalin-fixed paraffin-embedded tissue blocks were cut at a thickness of 7 µm. Immunohistochemical staining was conducted using a Bond RX autostainer from Leica Biosystems. We used primary antibodies to label Aβ deposits , phospho-tau , phospho-TDP43 , alpha-synuclein , cellular DNA damage , constitutive and inflammatory astrocytic markers , constitutive oligodendrocyte markers , and constitutive, inflammatory and homeostatic microglial markers  followed by secondary anti-mouse and anti-rabbit horseradish peroxidase (HRP) antibody conjugation  following the Leica autostainer protocol. Immunolabeling was visualized using 3,3'-diaminobenzidine (DAB) and slides were counterstained with the nuclear marker haematoxylin using the Leica Biosystems autostainer protocol. Slides were manually dehydrated, cover-slipped using Permount mounting medium and scanned with a Nanozoomer XR  using a 20X objective.2), and lesion burdens (percentage of cortex and WM occupied by lesions) were quantified within the sum of the two cortical and subcortical ROIs  based semi-automated quantification method , which allowed us to train 2-dimensional AI models, perform human-based validation(s) and apply individual AI models for accurate cell and lesion quantification(s) in predetermined regions of interest. To this end, scanned slides were uploaded to the Aiforia cloud-based platform and stored in folders. Two regions of interest (ROIs) were selected for each brain slide in cortical and adjacent subcortical brain regions respectively. Cortical ROIs were determined based on representative and well-preserved brain regions containing the 6 cortical layers and measuring a total of 6 mm in length along the pial surface Fig. a, b. SubAiforia-based AI models and quantifications were performed in the cortex for the antibodies 4G8, AT8, γH2AX, ALDH1L1, GFAP, IBA1, CD68, HLA-DR, TMEM119 and P2RY12 and in subcortical WM for Olig2, IBA1, CD68, HLA-DR, TMEM119 and P2RY12. Models were created to quantify `objects’ (positive cells) for AT8, γH2AX, ALDH1L1, GFAP, CD68, HLA-DR, TMEM119 and P2RY12 antibodies, and `burdens’  for 4G8 and IBA1. No AI model for formal quantification of alpha synuclein or pTDP43 was performed since absence of those comorbidities was a prerequisite when selecting the brains included in the present study.To better assess colocalization of γH2AX with glial markers (IBA1 and GFAP) with increased resolution at the cellular level, we expanded human brain sections following a previously published expansion microscopy (ExM) protocol . We usedA summary of the antibodies used in this study is displayed in Table D’Agostino-Pearson normality test was applied to test for Gaussian distribution. Multiple group analyses were then performed using one-way ANOVA for parametric variables and Kruskall-Wallis for non-parametric variables. Posthoc analyses to assess for between group differences were evaluated with Holm-Šídák’s test.p < 0.05. All statistical analyses and graphs were generated using Graphpad Prism version 9.3.0 .Correlation analyses were performed using Pearson test when both variables were normally distributed and Spearman test when at least one of the variables was not normally distributed. Significance level was set at Data are presented as mean ± standard error, unless otherwise indicated.Regional Aβ-plaque burden and number of neurofibrillary tangles did not show significant differences between resilient and demented brains at Braak III-IV stages.Recent studies have stressed that regional burden of amyloid plaques and NFTs may be a better predictor of cognition than the mere presence or absence of those lesions . It is wp = 0.33), even though a trend towards higher NFT counts was observed in this region in the demented group  did not show statistically significant differences between resilient and demented brains in either temporal pole or visual cortex, suggesting that neither plaque scores nor regional Aβ-plaque loads were predictive of the different cognitive fate of those two groups Fig. a. Quantioup Fig. b. As expoup Fig. c.Fig. 2RTotal number of astrocytes, microglial cells and oligodendrocytes in the cortical and subcortical ROIs did not differ between control, resilient, and demented brains at Braak III-IV stages.Our results showed that total number and density of astrocytes (ALDH1L1), microglial cells (IBA1) and oligodendrocytes (Olig2), as reported by their respective constitutive markers in cortical and subcortical ROIs did not significantly differ between controls, resilient and demented groups Fig. . These dPhenotypic changes in glial cells are an early phenomenon that precede overt tau tangle pathology in demented individuals at Braak III-IV stages.Although total cell numbers remained stable, there was a phenotypic change in astrocytes in the temporal pole in the setting of dementia with a significantly higher number of GFAP + activated astrocytes compared to both controls and resilient brains Fig. . Interesp = 0.40 and p = 0.51 in temporal pole and visual cortex respectively; HLA-DR p = 0.46 and p = 0.60 in temporal pole and visual cortex, respectively) or homeostatic microglial markers .A significantly higher number of pro-inflammatory microglial cells, as reported by CD68 antibody Fig. , and a dWhether these early glial phenotypic changes observed in visual cortex of demented individuals may set a path that facilitates subsequent pathogenic tau conversion/deposition and neuronal damage or represent a response to the presence of soluble pathogenic tau aggregates in Braak V-VI regions prior to overt tau tangle pathology remains uncertain . It is pEarly markers of cellular DNA damage precede overt tau tangle pathology and correlate with phenotypic changes in glia of demented individuals at Braak III-IV stages.p = 0.0006/ < 0.0001; CD68: R = 0.44/0.46 and p = 0.0009/0.0005; HLA-DR: R = 0.32/0.07 and p = 0.02/0.6, respectively).Gamma H2AX (ɣH2AX) is a well-established biomarker of early cellular damage. Previous studies have shown that DNA damage resulting in double stranded DNA breakages initiates the phosphorylation of histone variant H2A at the Serine 139 site to generate ɣH2AX . We quanp = 0.43 in temporal pole) or PMIs . Of note, ɣH2AX immunolabelling was observed in neurons as well as in glial cells in demented brains  , which did not reach significance when correlating TMEM119 and ɣH2AX positive cells in the cortex. This suggests that loss of homeostatic microglia in the WM is an early brain change that occurs in advance of classic tau tangle pathology and may potentially contribute to cortical cell damage and loss of brain function in demented brains. It is also possible that the higher burden of overall cerebrovascular risk observed in the demented individuals might be contributing to early glial derangements in the WM of these individuals even in the absence of significant differences in vascular insults across groups. Of note, identical changes in cortical and subcortical glial phenotypes and their correlation with markers of cortical cell damage were also present when analyses were limited to the subset of individuals with PART (not shown).Because the clinical symptoms characterizing the dementing disorder of AD are predominantly cortical rather than subcortical in nature, we evaluated whether the subcortical glial changes observed were associated with cortical cellular vulnerability (ɣH2AX). Interestingly, we found a significant inverse association between number of TMEM119 + microglial cells in the WM and number of ɣH2AX + cells in immediately adjacent temporal and visual cortical regions  – involving both increased pro-inflammatory and decreased homeostatic responses – occur in advance of overt NFT formation along the pathologically defined tau pathway, strongly correlate with markers of cortical cell damage, and more accurately predict than tangles themselves the cognitive status of individuals who are at tau Braak III-IV stages at autopsy. These results favor a model where early glial dysfunction in cortical and subcortical regions may be a key element of the tissue injury response that determines the cognitive fate of individuals who harbor intermediate stages of classic AD tau tangle pathology in their brains. Our data also suggest that the absence of such aberrant glial cell responses in resilient brains may reflect maintenance of homeostatic regulation of neuroinflammation and contribute to the preservation of cell integrity as a mechanism for resilience. If true, this would highlight possible new targets for intervention in the face of plaques and tangles.Current schemes for assessing disease burden in AD examine the distribution, abundance and characteristics of plaques and tangles, yielding an estimate of the likelihood of cognitive impairment. While this is highly predictive for most individuals, in some instances there is a mismatch between lesions and symptoms. Some individuals harbor a substantial burden of these lesions at autopsy which would be expected to have had deleterious consequences on their brain function, but remain at their cognitive baseline. This phenomenon of absence of overt cognitive deterioration in the face of neuropathologic lesions expected to have had substantial clinical consequences is the process we and others have termed ‘resilience’ to AD pathology . While resilient brains with a high burden of AD pathology meeting criteria for high probability of AD at autopsy (moderate-frequent neuritic plaques and tangle Braak stage V-VI) according to NIA-AA guidelines  represenTrying to understand the relationship between plaques, tangles and cognition can be confounded by a wide range of variables, including individual differences in the regional burden of lesions , geneticIn keeping with the above literature, the present study primarily focused on detailed assessments of glial phenotypes of brains from individuals who harbor intermediate stages of NFT pathology at autopsy (Braak stage III-IV) but exhibited opposite antemortem clinical outcomes . It is well known that tangle pathology appears in human AD brains in a hierarchically spatial consistent manner, starting in the transenthorinal/entorhinal cortex (Braak stage I-II), and ultimately culminating in the primary visual cortex (Braak stage VI), resulting in overt tau pathology and cell damage along the same pathway. Thus, intermediate Braak stages (Braak stages III-IV) provide a unique window of opportunity to investigate other brain changes occurring in later regions in the Braak tau pathway  that precede tau tangle accumulation and may ultimately drive, rather than tangles themselves, neurodegeneration and change from baseline function.Importantly, brains with comorbidities like TDP43 and alpha-synuclein were excluded from the present study to avoid additional potential confounders contributing to the heterogeneity in the clinical expression of tangle Braak III-IV stages. We confirmed that the regional burdens of both plaques and tangles in the ROIs studied  were well matched between demented and resilient brains thus ruling out the most parsimonious explanation that a higher regional load of lesions in the demented could explain the divergent clinical expression of the two groups. Even though multiple studies, including our own, have shown that the number of NFTs tends to correlate much better than Aβ plaques with the severity and duration of clinical symptoms and the amount of neuronal loss , some nePrevious studies have convincingly shown that activation of astrocytes and microglial cells are present early and linearly increase around plaques and tangles with disease progression in clinically manifest AD brains . AdditioThis study is not without caveats. First, while observations derived from autopsy studies like the present one may allow to infer information about the hierarchical order of pathological brain changes, their temporo-spatial relationships, and the resulting impact on antemortem brain function, the cross-sectional nature of such studies precludes drawing conclusions on specific underlying molecular mechanisms involved. Secondly, we cannot rule out the possibility that distinct tau strains may exist in demented and resilient brains with different biological properties including their propensity to form toxic soluble tau species (tau seeding activity) prior to the development of overt tau pathology, or the intensity of phosphorylation of different tau phosphor-epitopes that may result in their differential glial cell responses and clinical disease expression. Alternatively, the possibility also exists that the aberrant early changes in the glial phenotype in the grey and white matter of demented brains that precede tangle formation may be the real culprits of neuronal and glial damage and impaired brain function, and that preservation of physiological phenotypes of astrocytes and microglial cells would be needed for a human brain to cope with the challenges of Aβ and tau aggregation and deposition and thus preserve its normal function. Future studies will be conducted to address these important questions.In sum, the present study demonstrates that loss of homeostatic phenotypes of microglia and rise in pro-inflammatory astrocytes and microglial cells in the cortex and WM precede NFT formation along the well-defined tau pathological pathways in demented individuals who are at intermediate stages of classic tangle burden (Braak stages III-IV). These changes in glial phenotypes closely correlate with early markers of DNA damage in neurons and glia and accurately distinguish demented individuals from those exhibiting preserved cognition at a similar tau Braak stage at autopsy. These novel findings have potential important implications to guide the development of new biomarkers and also to develop novel therapies that can prevent cognitive impairment in the presence of classic AD lesion burden (e.g. plaques and tangles). The development of surrogate in vivo markers capable of detecting early changes in the glial phenotypes and signs of early neuronal and glial vulnerability in advance of the predicted spreading of tau aggregation between anatomically connected brain regions has the potential to more precisely identify who will develop clinical symptoms of dementia among asymptomatic amyloid and tau positive elderly at intermediate stages of pathology and over what time frame, providing valuable guidance on the need and optimal timing for personalized preventive intervention.


EXO1 rs1047840G>A and CAMKK2 rs1653586G>T, were significantly associated with worse chemotherapy response  and worse OS  in multivariate analyses. Significantly increased luciferase activity was noted in EXO1 rs1047840 A allele compared to G allele. In conclusion, two SNPs in miRNA binding sites, especially EXO1 rs1047840G>A, were associated with the chemotherapy response and survival outcome in lung adenocarcinoma patients treated with pemetrexed.Pemetrexed is an anti-folate agent which is one of the most frequently used chemotherapy agents for non-squamous non-small cell lung cancer (NSCLC) patients. However, clinical response to pemetrexed chemotherapy and survival outcome of patients varies significantly. We evaluated whether the genetic variants in miRNA target sites may affect the treatment outcome of pemetrexed chemotherapy in lung adenocarcinoma patients. One hundred SNPs in miRNA binding regions in cancer-related genes were obtained from the crosslinking, ligation, and sequencing of hybrids (CLASH) and CancerGenes database, and the associations with the response to pemetrexed chemotherapy and survival outcomes were investigated in 314 lung adenocarcinoma patients. Two polymorphisms,  A huge effort has been made to improve the prognosis of lung cancer patients over the past decades, and the treatment of lung cancer has progressed remarkably by recent innovations of targeted therapy and immunotherapy Pemetrexed is an anti-folate agent that inhibits cell replication and growth through interrupting synthesis of DNA and RNA by targeting three enzymes involved in purine and pyrimidine synthesis: thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) MicroRNA (miRNA) is a small (containing about 22 nucleotides) single-stranded non-coding RNA molecule that functions as endogenous negative regulators by binding to the 3'-untranslated region (UTR) of target mRNA causing degradation or translational inhibition of the target in situ cancers including carcinoma in situ of the cervix or breast. Patients who were diagnosed with stage III/IV or had recurred disease after curative surgery were treated with either pemetrexed plus cisplatin for up to 4 to 6 cycles as first line chemotherapy regimen with/without pemetrexed maintenance therapy, or pemetrexed alone as second or further line chemotherapy. Because pemetrexed maintenance therapy was approved in 2009 in Korea, patients who were treated with pemetrexed plus cisplatin before 2009 did not underwent pemetrexed maintenance therapy even if their disease had not progressed after four cycles of first-line pemetrexed/cisplatin chemotherapy A total of 314 lung adenocarcinoma patients with available genomic DNA samples who underwent pemetrexed based chemotherapy at Kyungpook National University Hospital (KNUH) in Daegu, Korea, between March 2007 and July 2015, were enrolled. We excluded patients who had any active malignancies ≤ 3 years before the diagnosis of lung cancer, except resected non-melanoma skin cancer and http://compbio.uthsc.edu/miRSNP) http://cbio.mskcc.org/cancergenes) r2 ≥ 0.8). Genotyping was performed using the iPLEX® Assay and MassARRAY® System .PolymiRTS database 3.0 . EXO1 coding sequence containing rs1047840G or rs1047840A and CAMKK2 3'UTR sequence containing rs1653586G or rs1653586T were synthesized using PCR from human genomic DNA and cloned into the psiCHECKTM-2 vector. In the PolymiRTS database, we found that miR-30e and miR-185 were experimentally demonstrated to interact with the target sites on mRNAs of EXO1 and CAMKK2, respectively. Each construct was then co-transfected with miRNA  into H1299 cells following the manufacturer's instructions. After 24 hours of incubation, Renilla luciferase activities were measured using the firefly luciferase activities as a normalization control.Luciferase reporter assay was conducted to determine whether 2 test. Genotypes for SNPs were analyzed as three-group categorical variable, and analyzed under dominant, recessive, and codominant models. The association between clinical variables or genotypes and response to chemotherapy was tested by odds ratio (OR) and 95% confidence interval (CI) using unconditional logistic regression. Kaplan-Meier method was used to estimate survival analysis, and log-rank test was used to compare the difference in OS according to different clinical variables or genotypes. Hazard ratio (HR) and 95% CI were estimated using multivariate Cox proportional hazards model. Variables with P value < 0.05 were considered statistically significant. The statistical data were obtained using Statistical Analysis System for Windows, version 9.4 .Deviations from Hardy-Weinberg equilibrium was measured by a goodness-of-fit χAssociations between clinical parameters and the response to chemotherapy and survival outcomes are presented in Table P < 0.05) or low call rates (< 95%). Among these, 7 SNPs were significantly associated with the response to pemetrexed (Table EXO1 rs1047840G>A and CAMKK2 rs1653586G>T) were significantly associated with both chemotherapy response and survival outcome. The EXO1 rs1047840G>A and CAMKK2 rs1653586G>T were significantly associated with worse chemotherapy response  and worse OS  in multivariate analyses .Next, to investigate whether the EXO1 rs1047840G>A and CAMKK2 rs1653586G>T, could predict the response to pemetrexed chemotherapy and survival outcome. The luciferase assay suggested that rs1047840G>A alters the binding of miRNA and consequently increases the expression level of EXO1. These findings suggest that the two SNPs, especially EXO1 rs1047840G>A, could be used as potential biomarkers to predict therapeutic outcomes in lung adenocarcinoma patients treated with pemetrexed chemotherapy, which may help to establish an optimal personalized treatment strategy.In this study, we investigated whether the treatment outcomes of pemetrexed chemotherapy in lung adenocarcinoma patients are different according to the genotypes of genetic polymorphisms in miRNA target sites. The study revealed that two SNPs in miRNA binding sites, EXO1 could impair DNA repair processes which can cause replication stress followed by genomic instability and development of cancer EXO1 genetic variants have been associated with the risk and prognosis of different types of cancers, including lung cancer EXO1 overexpression and its relation to the resistance to chemotherapy or radiotherapy and poor prognosis was reported in multiple types of cancers EXO1 may lead to the resistance to pemetrexed chemotherapy. In this study, EXO1 rs1047840G>A was associated with worse clinical outcomes in lung adenocarcinoma patients after pemetrexed chemotherapy and linked to increased expression of EXO1. Based on our results, it is postulated that the rs1047840-induced change in miRNA binding efficiency may increase the expression level of EXO1, resulting in worse response to pemetrexed chemotherapy and survival outcomes. Although many previous studies have investigated EXO1 polymorphism, especially rs1047840, in lung cancer, most of those studies evaluated the relationship between EXO1 rs1047840 and the risk of lung cancer . Regarding the chemotherapy outcomes, one study by R.Li et al. showed the association between EXO1 rs9350 and survival of patients receiving platinum-based chemotherapy in NSCLC (37). To the best of our knowledge, this is the first study to suggest a predictive role of EXO1 rs1047840 in the therapeutic response to pemetrexed chemotherapy and also survival in lung adenocarcinoma patients. In addition, by the SNP selection using a database for miRNA binding site variants, we could suggest that the rs1047840-induced change in miRNA binding efficiency may be a mechanism of the association between EXO1 rs1047840 and lung cancer. Alternatively, the EXO1 rs1047840G>A is a non-synonymous SNP (E589K) with an amino acid change, which may affect EXO1 protein functions. Further studies are required to understand the role of EXO1 in the mechanism of pemetrexed resistance. On the other hand, potential oncogenic roles of EXO1 have also been suggested, which may be another possible mechanism of the association between EXO1 rs1047840G>A and worse clinical outcomes after pemetrexed chemotherapy. Expression of degradation-resistant EXO1 could result in DNA hyper-resection in homologous recombination, which severely compromised DSBR and lead to chromosomal instability et al. showed that EXO1 was overexpressed in lung adenocarcinoma tissues and the high expression of EXO1 was associated with poor prognosis EXO1 expression and decreased tumor infiltrating B cells and CD4+ T cells, suggesting that EXO1 may induce an immune-suppressive tumor microenvironment in lung adenocarcinoma. Given that immune checkpoint blockade combined with pemetrexed-platinum doublet chemotherapy has been approved as the standard first-line treatment for lung adenocarcinoma, it will be worth to further evaluate EXO1 rs1047840G>A as a biomarker to predict clinical outcomes of combined immunotherapy and pemetrexed chemotherapy.Human exonuclease 1 (EXO1) is a member of the Rad2/XPG family of nucleases which exhibits 5'→3' exonuclease, 5' flap endonuclease, and 5'→3' RNase H activity  33, 35, . RegardiCAMKK2 rs1653586G>T was significantly related with the worse response to pemetrexed and survival outcome in lung adenocarcinoma patients. Calmodulin (CaM) is an intracellular calcium receptor which controls the downstream calcium/calmodulin kinase (CaMK) cascade that has been associated with various metabolic diseases CAMKK2 is generally upregulated in several cancers, including prostate cancer, and high expression of CAMKK2 was correlated with poor overall survival in hepatocellular carcinoma and glioma CAMKK2 potentiated the effect of carboplatin in ovarian cancer by modulating Akt pathway CAMKK2 in the pathogenesis and prognosis of lung cancer has yet to be clarified. Further study is required to understand the molecular mechanism of the association between the genetic variants and clinical outcomes.In this study, EXO1 rs1047840 and CAMKK2 rs1653586 in different subgroups of patients, i.e. stage III/IV and postsurgical recurrence groups, this study is underpowered for stratification analyses or propensity score matching. Therefore, a well-designed and properly powered study is warranted to validate our findings.There are some limitations in this study. First, because pemetrexed maintenance regimen was not approved in the early part of the enrollment period, maintenance therapy was not prescribed for some patients who are considered eligible for the maintenance therapy by the current treatment guidelines. Although maintenance therapy was adjusted for in the multivariate analysis, the prognostic differences between patients with and without pemetrexed maintenance therapy could not be ignored. Second, as a single center retrospective study, the treatment other than pemetrexed in the course of treatment including targeted agents could not be strictly controlled in this study, which should be considered in the interpretation of the survival analysis. Third, the study cohort is relatively small. Although stratified analyses may show the clinical significance of EXO1 rs1047840G>A, were associated with the treatment response to pemetrexed chemotherapy and survival in lung adenocarcinoma patients. Evaluation of genetic variants in miRNA binding sites may be useful for refining therapeutic decisions in the treatment of lung adenocarcinoma by helping to identify subgroups of patients who will benefit from pemetrexed chemotherapy. To verify EXO1 rs1047840G>A and CAMKK2 rs1653586G>T as biomarkers for predicting clinical outcomes, the results of this study need to be further tested in a larger population with diverse ethnicity.In conclusion, two SNPs in miRNA binding sites, especially Supplementary table.Click here for additional data file.