Processed pseudogenes (PΨgs) are disabled gene copies that are transcribed and may affect expression of paralogous genes. Moreover, their insertion in the genome can disrupt the structure or the ...regulatory region of a gene, affecting its expression level. These events have been identified as occurring mutations during cancer development, thus being able to identify PΨgs and their location will improve their impact on diagnostic testing, not only in cancer but also in inherited disorders.
We have implemented PΨFinder (P-psy-finder), a tool that identifies PΨgs, annotates known ones and predicts their insertion site(s) in the genome. The tool screens alignment files and provides user-friendly summary reports and visualizations. To demonstrate its applicability, we scanned 218 DNA samples from patients screened for hereditary colorectal cancer. We detected 423 PΨgs distributed in 96% of the samples, comprising 7 different parent genes. Among these, we confirmed the well-known insertion site of the SMAD4-PΨg within the last intron of the SCAI gene in one sample. While for the ubiquitous CBX3-PΨg, present in 82.6% of the samples, we found it reversed inserted in the second intron of the C15ORF57 gene.
PΨFinder is a tool that can automatically identify novel PΨgs from DNA sequencing data and determine their location in the genome with high sensitivity (95.92%). It generates high quality figures and tables that facilitate the interpretation of the results and can guide the experimental validation. PΨFinder is a complementary analysis to any mutational screening in the identification of disease-causing mutations within cancer and other diseases.
Lynch syndrome caused by constitutional mismatch‑repair defects is one of the most common hereditary cancer syndromes with a high risk for colorectal, endometrial, ovarian and urothelial cancer. ...Lynch syndrome is caused by mutations in the mismatch repair (MMR) genes i.e., MLH1, MSH2, MSH6 and PMS2. After 20 years of genetic counseling and genetic testing for Lynch syndrome, we have compiled the mutation spectrum in Sweden with the aim to provide a population-based perspective on the contribution from the different MMR genes, the various types of mutations and the influence from founder mutations. Mutation data were collected on a national basis from all laboratories involved in genetic testing. Mutation analyses were performed using mainly Sanger sequencing and multiplex ligation-dependent probe amplification. A total of 201 unique disease-predisposing MMR gene mutations were identified in 369 Lynch syndrome families. These mutations affected MLH1 in 40%, MSH2 in 36%, MSH6 in 18% and PMS2 in 6% of the families. A large variety of mutations were identified with splice site mutations being the most common mutation type in MLH1 and frameshift mutations predominating in MSH2 and MSH6. Large deletions of one or several exons accounted for 21% of the mutations in MLH1 and MSH2 and 22% in PMS2, but were rare (4%) in MSH6. In 66% of the Lynch syndrome families the variants identified were private and the effect from founder mutations was limited and predominantly related to a Finnish founder mutation that accounted for 15% of the families with mutations in MLH1. In conclusion, the Swedish Lynch syndrome mutation spectrum is diverse with private MMR gene mutations in two-thirds of the families, has a significant contribution from internationally recognized mutations and a limited effect from founder mutations.
This study assessed the diagnostic yield of high-throughput sequencing methods in a cohort of craniosynostosis (CS) patients not presenting causal variants identified through previous targeted ...analysis.
Whole-genome or whole-exome sequencing (WGS/WES) was performed in a cohort of 59 patients (from 57 families) assessed by retrospective phenotyping as having syndromic or nonsyndromic CS.
A syndromic form was identified in 51% of the unrelated cases. A genetic cause was identified in 38% of syndromic cases, with novel variants detected in
(a rare Alu insertion),
, and
. Additionally, we report two patients with rare recurrent variants in
and
as well as two patients with structural genomic aberrations: one with a 22q13 duplication and one with a complex rearrangement involving chromosome 2 (2p25 duplication including
and deletion of 2q22). Moreover, we identified potentially relevant variants in 87% of the remaining families with no previously detected causal variants, including novel variants in
, and
.
These results confirm WGS/WES as a powerful diagnostic tool capable of either targeted
or broad genomic analysis depending on phenotypic presentation (e.g., classical or unusual forms of syndromic CS).
Immunotherapy by blocking programmed death protein-1 (PD-1) or programmed death protein-ligand1 (PD-L1) with antibodies (PD-1 blockade) has revolutionized treatment options for patients with ...non-small cell lung cancer (NSCLC). However, the benefit of immunotherapy is limited to a subset of patients. This study aimed to investigate the value of combining immune and genetic variables analyzed within 3-4 weeks after the start of PD-1 blockade therapy to predict long-term clinical response.
Blood collected from patients with NSCLC were analyzed for changes in the frequency and concentration of immune cells using a clinical flow cytometry assay. Next-generation sequencing (NGS) was performed on DNA extracted from archival tumor biopsies of the same patients. Patients were categorized as clinical responders or non-responders based on the 9 months' assessment after the start of therapy.
We report a significant increase in the post-treatment frequency of activated effector memory CD4
and CD8
T-cells compared with pre-treatment levels in the blood. Baseline frequencies of B cells but not NK cells, T cells, or regulatory T cells were associated with the clinical response to PD-1 blockade. NGS of tumor tissues identified pathogenic or likely pathogenic mutations in tumor protein P53, Kirsten rat sarcoma virus, Kelch-like ECH-associated protein 1, neurogenic locus notch homolog protein 1, and serine/threonine kinase 11, primarily in the responder group. Finally, multivariate analysis of combined immune and genetic factors but neither alone, could discriminate between responders and non-responders.
Combined analyses of select immune cell subsets and genetic mutations could predict early clinical responses to immunotherapy in patients with NSCLC and after validation, can guide clinical precision medicine efforts.
BackgroundBlockade of PD-1 or PD-L1 (PD-1 blockade) with antibodies has revolutionized treatment options for patients with non-small cell lung cancer particularly with tumors that don’t present any ...targetable mutations. However, the benefit of PD-1 blockade is limited only to a subset of the patients. This study aims to identify biological markers of clinical response in a discovery and validation cohort of patients early on during treatment to distinguish responders from non-responders.MethodsBlood was drawn from patients with non-small cell lung cancer, at pre-treatment and at 4 cycles post-treatment. Immune cell subsets identified in the blood using a well characterized panel of immune markers that could distinguish CD4 and CD8 T cells based on proliferation, expression of immune checkpoint receptors, receptors of exhaustion and activation and cytokine secretion. Blood drawn from patients with non-small cell lung cancer before and after PD-1 blockade was also analyzed using a clinical flow cytometric assay for changes in the frequencies and concentration of immune cell populations particularly naive and memory CD4 and CD8 T cells. Further, next generation sequencing (NGS) was performed on the extracted DNA from archival tumor biopsies from the same patients as the flow cytometric analysis. The iRECIST criteria was used to evaluate the clinical responses with cut-off at 9 months.ResultsIn the responder patients a significant increase in the frequency of activated effector memory CD4 and CD8 T cells, and decreased frequency of central memory CD4 and CD8 T cells was observed in the blood post-treatment compared to pre-treatment. In a separate cohort of 29 patients, we could validate the initial findings and further report that CD8 T cells in non-responders expressed high levels of TNFα, TIGIT and PD-1 compared to non-responders at baseline. Our next-generation sequencing analysis of tumor biopsies identified the presence of pathogenic and likely-pathogenic mutations TP53, KRAS, KEAP1, NOTCH-1 and STK11 in the tumor of patients which correlated with response to PD-1 blockade. A multivariate analysis combining immune and genetic parameters could discriminate responders from non-responders for the discovery cohort of patients.ConclusionsWe report that combined analyses of selected immune cell and genetic parameters could predict early clinical response to PD-1 blockade.
Immunotherapy with PD‐1 and PD‐L1 inhibitors has revolutionized the treatment for patients with NSCLC the last years with increased overall survival and in particular increased number of long‐time ...survivors in patients with metastatic disease. It is now a treatment of choice for patients with distant metastases (stage IV) and in conjunction with chemoradiotherapy for patients with limited spread confined to the chest (stage III). PD‐1 inhibition has been proven to be superior to standard chemotherapy, both as a single treatment and when combined with either chemotherapy or CTLA‐4 inhibition. Despite the success of immunotherapy, the majority of patients do not respond or relapse within a short time frame. Biomarkers that would help to properly select patients with a high likelihood of clinical response to PD‐1 and PD‐L1 inhibitors are scarce and far from optimal, and only one (PD‐L1 expression) has reached clinical practice. Thus for immunotherapy to be effective, the discovery and validation of additional biomarkers is critical for patient selection and prediction of clinical response. In this mini‐review, we give an overview of current clinical management of NSCLC including treatment landscape with regard to immunotherapy, as well as discuss the current genetic and immune cell biomarker studies and their potential for introduction into clinical practice.
The dominantly inherited condition familial adenomatous polyposis (FAP) is caused by germline mutations in the APC gene. Finding the causative mutations has great implications for the families. ...Correlating the genotypes to the phenotypes could help to improve the diagnosis and follow-up of patients.
Mutation screening of APC and the clinical characterization of 96 unrelated FAP patients from the Swedish Polyposis Registry was performed. In addition to generally used mutation screening methods, analyses of splicing-affecting mutations and investigations of the presence of low-frequency mutation alleles, indicating mosaics, have been performed, as well as quantitative real-time polymerase chain reaction to detect lowered expression of APC.
Sixty-one different APC mutations in 81 of the 96 families were identified and 27 of those are novel. We have previously shown that 6 of the 96 patients carried biallelic MUTYH mutations. The 9 mutation-negative cases all display an attenuated or atypical phenotype. Probands with a genotype (codon 1250-1464) predicting a severe phenotype had a median age at diagnosis of 21.8 (range, 11-49) years compared with 34.4 (range, 14-57) years among those with mutations outside this region (P < 0.017). Dense polyposis (> 1000) occurred in 75% of the probands with a severe phenotype compared with 30% in those with mutations outside this region. The morbidity in colorectal cancer among probands was 25% at a mean age of 37.5 years and 29% at a mean age of 46.6 years.
Using a variety of mutation-detection techniques, we have achieved a 100% detection frequency in classical FAP. Probands with APC mutations outside codon 1250-1464, although exhibiting a less-severe phenotype, are at high risk of having a colorectal cancer at diagnosis indicating that age at diagnosis is as important as the severity of the disease for colorectal cancer morbidity.
Background Processed pseudogenes (PΨgs) are disabled gene copies that are transcribed and may affect expression of paralogous genes. Moreover, their insertion in the genome can disrupt the structure ...or the regulatory region of a gene, affecting its expression level. These events have been identified as occurring mutations during cancer development, thus being able to identify PΨgs and their location will improve their impact on diagnostic testing, not only in cancer but also in inherited disorders. Results We have implemented PΨFinder (P-psy-finder), a tool that identifies PΨgs, annotates known ones and predicts their insertion site(s) in the genome. The tool screens alignment files and provides user-friendly summary reports and visualizations. To demonstrate its applicability, we scanned 218 DNA samples from patients screened for hereditary colorectal cancer. We detected 423 PΨgs distributed in 96% of the samples, comprising 7 different parent genes. Among these, we confirmed the well-known insertion site of the SMAD4-PΨg within the last intron of the SCAI gene in one sample. While for the ubiquitous CBX3-PΨg, present in 82.6% of the samples, we found it reversed inserted in the second intron of the C15ORF57 gene. Conclusions PΨFinder is a tool that can automatically identify novel PΨgs from DNA sequencing data and determine their location in the genome with high sensitivity (95.92%). It generates high quality figures and tables that facilitate the interpretation of the results and can guide the experimental validation. PΨFinder is a complementary analysis to any mutational screening in the identification of disease-causing mutations within cancer and other diseases. Keywords: Processed pseudogenes, DNA sequencing, Colorectal cancer, SMAD4, CBX3, C15ORF57, SCAI
PDF
