Maple syrup urine disease diagnosis in Brazilian patients by massive parallel sequencing.

Biallelic pathogenic variants cause maple syrup urine disease (MSUD) in one of the branched-chain α-keto acid dehydrogenase (BCKDH) complex genes (BCKDHA, BCKDHB, DBT, DLD, and PPM1K) leading to the accumulation of leucine, isoleucine, and valine. This study aimed to perform a molecular diagnosis of Brazilian patients with MSUD using gene panels and massive parallel sequencing. Eighteen Brazilian patients with a biochemical diagnosis of MSUD were analyzed by massive parallel sequencing in the Ion PGM Torrent Server using a gene panel with the BCKDHA, BCKDHB, and DBT genes. The American College of Medical Genetics and Genomics guidelines were used to determine variant pathogenicity. Thirteen patients had both variants found by massive parallel sequencing, whereas 3 patients had only one variant found. In 2 patients, the variants were not found by this analysis. These 5 patients required additional Sanger sequencing to confirm their genotype. Twenty-five pathogenic variants were identified in the 3 MSUD-related genes (BCKDHA, BCKDHB, and DBT). Most variants were present in the BCKDHB gene, and no common variants were found. Nine novel variants were observed: c.922 A > G, c.964C > A, and c.1237 T > C in the BCKDHA gene; and c.80_90dup, c.384delA, c.478 A > T, c.528C > G, c.977 T > C, and c.1039-2 A > G in the BCKDHB gene. All novel variants were classified as pathogenic. Molecular modeling of the novel variants indicated that the binding of monomers was affected in the BCKDH complex tetramer, which could lead to a change in the stability and activity of the enzyme. Massive parallel sequencing with targeted gene panels seems to be a cost-effective method that can provide a molecular diagnosis of MSUD.

The first case of a point pathogenic variant in the RREB1 gene in Noonan-like Rasopathy.

The RREB1 is a zinc finger transcription factor that plays a role in regulating gene expression and inactivating MAPK signalling components. To date, no pathogenic variant in the RREB1 gene has been associated with any disease, but several cases of 6p terminal deletions affecting the RREB1 gene have been reported. In this study, we report the first case of RREB1-associated Noonan-like RASopathy caused by a pathogenic variant within this gene. Genetic testing included whole-genome sequencing (WGS) of the proband and Sanger sequencing of the proband, his parents, and his sibling. The proband had a de novo c.2677del, p.(Ala893Argfs*20) variant, likely resulting in RREB1 haploinsufficiency. Comparative analysis of patients with microdeletions, including in the RREB1 gene, confirmed shared clinical traits while highlighting unique features, such as blue sclerae and absence of cardiac anomalies. This study reinforces previous data on RREB1 haploinsufficiency as the driver of a new Noonan-like RASopathy variant, which includes intellectual disability, delayed motor skills, short stature, short neck, and distinctive facial dysmorphisms as key clinical indicators. These findings shed light on this RREB1-related syndrome and underscore the necessity for further investigation into the functional consequences of RREB1 mutations.

Spitting in the wind?-The challenges of RNA sequencing for biomarker discovery from saliva.

Forensic trace contextualization, i.e., assessing information beyond who deposited a biological stain, has become an issue of great and steadily growing importance in forensic genetic casework and research. The human transcriptome encodes a wide variety of information and thus has received increasing interest for the identification of biomarkers for different aspects of forensic trace contextualization over the past years. Massively parallel sequencing of reverse-transcribed RNA ("RNA sequencing") has emerged as the gold standard technology to characterize the transcriptome in its entirety and identify RNA markers showing significant expression differences not only between different forensically relevant body fluids but also within a single body fluid between forensically relevant conditions of interest. Here, we analyze the quality and composition of four RNA sequencing datasets (whole transcriptome as well as miRNA sequencing) from two different research projects (the RNAgE project and the TrACES project), aiming at identifying contextualizing forensic biomarker from the forensically relevant body fluid saliva. We describe and characterize challenges of RNA sequencing of saliva samples arising from the presence of oral bacteria, the heterogeneity of sample composition, and the confounding factor of degradation. Based on these observations, we formulate recommendations that might help to improve RNA biomarker discovery from the challenging but forensically relevant body fluid saliva.

The Unholy Grail of cancer screening: or is it just about the Benjamins?

The biotechnology company Grail developed a non-invasive blood test (Galleri test) which is claimed to detect 50 types of cancer at early and potentially curable stages. The initially promising results from prospective studies, and the anticipated financial success of Grail led the sequencing giant Illumina to purchase Grail for $8 billion (2021). Following this event, Grail collaborated with the UK National Health System to further clarify the test's capability, in a 3-year prospective trial, along with the standard of care. At the end of the first year, UK-NHS announced that they will stop the trial due to unsatisfactory clinical performance and until they analyze the data for the first year (which already enrolled 140,000 participants). Legal and financial issues between the interested parties are currently in flux. The Grail case is reminiscent of the multi-billion Theranos scandal, which sent a few people to jail and led to company dissolution. We previously expressed concerns about the sensitivity and specificity of the Galleri test. In this Perspective, we revisit the hyped technology, and we provide new suggestions on the use of this test.

Functional studies associate novel DUOX2 gene variants detected in heterozygosity to Crohn's disease.

PURPOSE: Crohn's disease is a chronic gastrointestinal inflammatory disease with possible extraintestinal symptoms. There are predisposing genetic factors and even monogenic variants of the disorder. One of the possible genetic factors are variants of the DUOX2 gene. The protein product of the DUOX2 gene is a dual oxidase enzyme producing H2O2 in the bowel. Reduced H2O2 levels impact mucosal homeostasis and contribute to the development of inflammatory bowel disease. Thus far, only 19 patients with IBD with the DUOX2 variants have been described. METHODS: Here we present a case report of an adolescent female diagnosed at eleven years of age with IBD that was subsequently reclassified as Crohn's disease. She was treated with immunosuppressants and biological therapy but experienced additional complications. Her peripheral blood lymphocyte DNA was studied using massive parallel sequencing. Detected variants were functionally studied. RESULTS: Whole exome sequencing found two novel DUOX2 gene variants: a de novo variant c.3646C>T; p.R1216W and a maternally inherited variant c.3391G>A; p.A1131T which were initially classified as variants of unknown significance. However, follow-up functional studies demonstrated that both DUOX2 variants led to impaired H2O2 generation, which led to their reclassification to the likely pathogenic class according to the ACMG.net. Therefore, we conclude that these variants are causative for the disease. CONCLUSIONS: Identifying novel variants in patients with Crohn's disease and their families is important for precision medicine approaches and understanding of the pathogenesis of likely "monogenic" rare forms of inflammatory bowel disease.

Barriers and facilitators to next-generation sequencing use in United States oncology settings: a systematic review.

Aim: Next-generation sequencing (NGS) of solid tumors can inform treatment decisions; however, uptake remains low. This objective of this systematic review was to identify barriers to and facilitators of NGS in US oncology settings.Materials & methods: Embase and MEDLINE were searched in March 2023 for articles published from 2012 to 2023 on barriers and facilitators of NGS adoption for solid tumors. Surveys, interviews and observational studies were eligible. Studies on genetic testing for hereditary cancers and non-US studies were excluded. The Motheral scale, Joanna Briggs Institute critical appraisal checklist and McGill Mixed Methods Appraisal Tool were used to assess study quality. Data were synthesized narratively.Results: Twenty-one studies were included. Study participants were clinicians, payers and administrators. Key barriers included complex reimbursement processes and uncertainties around clinical utility. Including recommendations for NGS in clinical practice guidelines was a key facilitator, although insurance policies were often more restrictive than guideline recommendations.Conclusion: Uptake of NGS is increasing but barriers remain. Changes to the current reimbursement frameworks are needed to increase access to NGS. The impact of implementing the 2018 National Coverage Determination, which allows access to NGS for all Medicare beneficiaries with advanced cancer, is not yet evident in the published literature.

[Box: see text].

Implementing Massive Parallel Sequencing into Biliary Samples Obtained through Endoscopic Retrograde Cholangiopancreatography for Diagnosing Malignant Bile Duct Strictures.

Despite advancements in radiologic, laboratory, and pathological evaluations, differentiating between benign and malignant bile duct strictures remains a diagnostic challenge. Recent developments in massive parallel sequencing (MPS) have introduced new opportunities for early cancer detection and management, but these techniques have not yet been rigorously applied to biliary samples. We prospectively evaluated the Oncomine Comprehensive Assay (OCA) and the Oncomine Pan-Cancer Cell-Free Assay (OPCCFA) using biliary brush cytology and bile fluid obtained via endoscopic retrograde cholangiopancreatography from patients with bile duct strictures. The diagnostic performance of MPS testing was assessed and compared to the pathological findings of biliary brush cytology and primary tissue. Mutations in TP53, BRAF, CTNNB1, SMAD4, and K-/N-RAS identified in biliary brush cytology samples were also detected in the corresponding bile fluid samples from patients with extrahepatic cholangiocarcinoma. These mutations were also identified in the bile fluid samples, but with variant allele frequencies lower than those in the corresponding biliary brush cytology samples. In control patients diagnosed with gallstones, neither the biliary brush cytology samples nor the bile fluid samples showed any pathogenic mutations classified as tier 1 or 2. Our study represents a prospective investigation into the role of MPS-based molecular testing in evaluating bile duct strictures. MPS-based molecular testing shows promise in identifying actionable genomic alterations, potentially enabling the stratification of patients for targeted chemotherapeutic treatments. Future research should focus on integrating OCA and OPCCFA testing, as well as similar MPS-based assays, into existing surveillance and management protocols for patients with bile duct strictures.

Fungal Integrated Histomolecular Diagnosis Using Targeted Next-Generation Sequencing on Formalin-Fixed Paraffin-Embedded Tissues.

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Lamb-Shaffer syndrome: 20 Spanish patients and literature review expands the view of neurodevelopmental disorders caused by SOX5 haploinsufficiency.

Lamb-Shaffer Syndrome (LSS; OMIM #616803; ORPHA #313892; ORPHA #313884) is an infrequent genetic disorder that affects multiple aspects of human development especially those related to the development of the nervous system. LSS is caused by variants in the SOX5 gene. At the molecular level, SOX5 gene encodes for a transcription factor containing a High Mobility Group (HMG) DNA-Binding domain with relevant functions in brain development in different vertebrate species. Clinical features of Lamb-Shaffer syndrome may include intellectual disability, delayed speech and language development, attention deficits, hyperactivity, autism spectrum disorder, visual problems and seizures. Additionally, patients with the syndrome may present distinct facial dimorphism such as a wide mouth with full lips, small chin, broad nasal bridge, and deep-set eyes. Other physical features that have been reported in some patients include short stature, scoliosis, and joint hypermobility. Here, we report the clinical and molecular characterization of a Spanish LSS cohort of new 20 patients and review all the patients published so far which amount for 111 patients. The most frequent features included developmental delay, intellectual disability, visual problems, poor speech development and facial dysmorphic features. Strikingly, pain insensitivity and hypermetropia seems to be more frequent than previously reported, based on the frequency seen in the Spanish cohort. Eighty-three variants have been reported so far, single nucleotide variants (SNV) and copy number variants represent 47% and 53%, respectively, from the total of variants reported. Similarly to previous reports, the majority of the SNVs variants of the novel patients reported herein fall in the HMG domain of the protein. However, new variants, affecting other functional domains, were also detected. In conclusion, LLS is a rare genetic disorder mostly characterized by a wide range of developmental and neurological symptoms. Early diagnosis would allow to start of care programs, clinical follow up, prospective studies and appropriate genetic counseling, to promote clinical and social improvement to have profound lifelong benefits for patients and their families. Further research is needed to better understand the underlying mechanisms of the syndrome related to SOX5 haploinsufficiency.

A case of haemolytic disease of the fetus and newborn attributed to a novel antigen in the RHAG blood group system.

BACKGROUND AND OBJECTIVES: A newborn presented with jaundice in Thailand. The cord red cells tested positive by direct antiglobulin test (DAT) for an unknown maternal red cell antibody. Initial blood group sequencing suggested that the infant carried a novel variant RHAG c.140T>C, responsible for a low-prevalence antigen in the RHAG blood group system (ISBT 030). We report here on testing of samples from the infant's parents and older sibling to define a new antigen in the RHAG system. MATERIALS AND METHODS: Massive parallel sequencing (MPS) using a custom-designed panel was performed on all four family members. Extended serological testing was also performed to determine whether family members with the same variant as the infant showed reactivity with the antibody in the maternal plasma. RESULTS: We identified a novel single nucleotide variant (SNV) (RHAG c.140T>C, p.[Phe47Ser]) in samples from three of the four family members tested (the infant, the older sibling and the father). The variant was not detected in the mother's sample. Maternal plasma showed positive agglutination with all family members tested; however, when tested with routine panel cells, no reactivity was observed. CONCLUSION: This case study showed that the presence of the novel variant (RHAG c.140T>C), encoding a p.(Phe47Ser) change in the RhAG glycoprotein, was the apparent cause of incompatibility between maternal plasma and that of red cells from the proband, father and older sibling of the proband. We propose this variant to be a new low-prevalence antigen in the RHAG blood group system.

Improving kinship probability in analysis of ancient skeletons using identity SNPs and MPS technology.

In forensic kinship analysis and human identification cases, analysis of STRs is the gold standard. When badly preserved ancient DNA is used for kinship analysis, short identity SNPs are more promising for successful amplification. In this work, kinship analysis was performed on two skeletons from the Early Middle Ages. The surface contaminants of petrous bones were removed by chemical cleaning and UV irradiation; DNA was isolated through full demineralization and purified in an EZ1 Advanced XL machine. The PowerQuant kit was used to analyze DNA yield and degradation, and on average, 17 ng DNA/g of petrous bone was obtained. Both skeletons were typed in duplicate for STR markers using the Investigator EssplexPlus SE QS kit, and comparison of partial consensus genotypes showed shared allelic variants at most loci amplified, indicating close kinship. After statistical calculation, the full-sibling kinship probability was too low for kinship confirmation, and additional analyses were performed with PCR-MPS using the Precision ID Identity Panel. The HID Ion Chef Instrument was used to prepare the libraries and for templating and the Ion GeneStudio S5 System for sequencing. Analysis of identity SNPs produced full genetic profiles from both skeletons. For combined likelihood ratio (LR) calculation, the product rule was used, combining LR for STRs and LR for SNPs, and a combined LR of 3.3 × 107 (corresponding to a full-sibling probability of 99.999997%) was calculated. Through the SNP PCR-MPS that followed the STR analysis, full-sibling kinship between the ancient skeletons excavated from an early medieval grave was confirmed.

Small RNA Profiling in an HTLV-1-Infected Patient with Acute Adult T-Cell Leukemia-Lymphoma at Diagnosis and after Maintenance Therapy: A Case Study.

Small RNAs (sRNAs) are epigenetic regulators of essential biological processes associated with the development and progression of leukemias, including adult T-cell leukemia/lymphoma (ATLL) caused by human T-cell lymphotropic virus type 1 (HTLV-1), an oncogenic human retrovirus originally discovered in a patient with adult T-cell leukemia/lymphoma. Here, we describe the sRNA profile of a 30-year-old woman with ATLL at the time of diagnosis and after maintenance therapy with the aim of correlating expression levels with response to therapy.

Cost-Effective Next Generation Sequencing-Based STR Typing with Improved Analysis of Minor, Degraded and Inhibitor-Containing DNA Samples.

Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.

Advances in sarcoma molecular diagnostics.

Sarcomas are cancers of mesenchymal origin with the potential to arise in diverse anatomic locations. With over 80 subtypes, which often demonstrate overlapping morphologies, sarcomas frequently require ancillary testing to enable accurate classification. Pathognomonic driver mutations can often be leveraged for diagnostic purposes and include fusion genes, amplification events, and recurrent point mutations. Until relatively recently, the major clinical molecular diagnostic tests have been karyotyping, fluorescence in situ hybridization, and polymerase chain reaction; however, these techniques have a number of limitations. Recent technological advances have led to the development of more comprehensive assays with higher throughput, thereby replacing the need for a suite of single gene tests. These approaches include next-generation sequencing, fluorescent bar code hybridization, and DNA methylation profiling, among others. Herein, we review the application of recently developed techniques relevant to the diagnosis of sarcomas, and emerging assays with the potential for future development and clinical implementation.

A versatile 5' RACE-Seq methodology for the accurate identification of the 5' termini of mRNAs.

BACKGROUND: Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. However, 5' ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The implementation of the template-switching mechanism at the reverse transcription stage along with 5' rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5' ends of cDNAs. In the current study, we developed a 5' RACE-seq method by coupling a custom template-switching and 5' RACE assay with targeted nanopore sequencing, to accurately unveil 5' termini of mRNA targets. RESULTS: The optimization of the described 5' RACE-seq method was accomplished using the human BCL2L12 as control gene. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5' RACE. Collectively, our results support the existence of two distinct 5' termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. As proof of concept, we implemented the described 5' RACE-seq methodology to investigate the 5' UTRs of several kallikrein-related peptidases (KLKs) gene family members. Our results confirmed the existence of multiple annotated 5' UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones. CONCLUSIONS: In this work we present an in-house developed 5' RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. This approach enables the broad and in-depth study of 5' UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits.

Molecular Profiling of Tumor Tissue in Mexican Patients with Colorectal Cancer.

Colorectal cancer is a heterogeneous disease with multiple genomic changes that influence the clinical management of patients; thus, the search for new molecular targets remains necessary. The aim of this study was to identify genetic variants in tumor tissues from Mexican patients with colorectal cancer, using massive parallel sequencing. A total of 4813 genes were analyzed in tumoral DNA from colorectal cancer patients, using the TruSight One Sequencing panel. From these, 192 variants with clinical associations were found distributed in 168 different genes, of which 46 variants had not been previous reported in the literature or databases, although genes harboring those variants had already been described in colorectal cancer. Enrichment analysis of the affected genes was performed using Reactome software; pathway over-representation showed significance for disease, signal transduction, and immune system subsets in all patients, while exclusive subsets such as DNA repair, autophagy, and RNA metabolism were also found. Those characteristics, whether individual or shared, could give tumors specific capabilities for survival, aggressiveness, or response to treatment. Our results can be useful for future investigations targeting specific characteristics of tumors in colorectal cancer patients. The identification of exclusive or common pathways in colorectal cancer patients could be important for better diagnosis and personalized cancer treatment.

Isometric artifacts from polymerase chain reaction-massively parallel sequencing analysis of short tandem repeat loci: An emerging issue from a new technology?

The recent introduction of polymerase chain reaction (PCR)-massively parallel sequencing (MPS) technologies in forensics has changed the approach to allelic short tandem repeat (STR) typing because sequencing cloned PCR fragments enables alleles with identical molecular weights to be distinguished based on their nucleotide sequences. Therefore, because PCR fidelity mainly depends on template integrity, new technical issues could arise in the interpretation of the results obtained from the degraded samples. In this work, a set of DNA samples degraded in vitro was used to investigate whether PCR-MPS could generate "isometric drop-ins" (IDIs; i.e., molecular products having the same length as the original allele but with a different nucleotide sequence within the repeated units). The Precision ID GlobalFiler NGS STR panel kit was used to analyze 0.5 and 1 ng of mock samples in duplicate tests (for a total of 16 PCR-MPS analyses). As expected, several well-known PCR artifacts (such as allelic dropout, stutters above the threshold) were scored; 95 IDIs with an average occurrence of 5.9 IDIs per test (min: 1, max: 11) were scored as well. In total, IDIs represented one of the most frequent artifacts. The coverage of these IDIs reached up to 981 reads (median: 239 reads), and the ratios with the coverage of the original allele ranged from 0.069 to 7.285 (median: 0.221). In addition, approximately 5.2% of the IDIs showed coverage higher than that of the original allele. Molecular analysis of these artifacts showed that they were generated in 96.8% of cases through a single nucleotide change event, with the C > T transition being the most frequent (85.7%). Thus, in a forensic evaluation of evidence, IDIs may represent an actual issue, particularly when DNA mixtures need to be interpreted because they could mislead the operator regarding the number of contributors. Overall, the molecular features of the IDIs described in this work, as well as the performance of duplicate tests, may be useful tools for managing this new class of artifacts otherwise not detected by capillary electrophoresis technology.

Digital gene expression analysis of NSCLC-patients reveals strong immune pressure, resulting in an immune escape under immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are currently one of the most promising therapy options in the field of oncology. Although the first pivotal ICI trial results were published in 2011, few biomarkers exist to predict their therapy outcome. PD-L1 expression and tumor mutational burden (TMB) were proven to be sometimes-unreliable biomarkers. We have previously suggested the analysis of processing escapes, a qualitative measurement of epitope structure alterations under immune system pressure, to provide predictive information on ICI response. Here, we sought to further validate this approach and characterize interactions with different forms of immune pressure. METHODS: We identified a cohort consisting of 48 patients with advanced non-small cell lung cancer (NSCLC) treated with nivolumab as ICI monotherapy. Tumor samples were subjected to targeted amplicon-based sequencing using a panel of 22 cancer-associated genes covering 98 mutational hotspots. Altered antigen processing was predicted by NetChop, and MHC binding verified by NetMHC. The NanoString nCounter® platform was utilized to provide gene expression data of 770 immune-related genes. Patient data from 408 patients with NSCLC were retrieved from The Cancer Genome Atlas (TCGA) as a validation cohort. RESULTS: The two immune escape mechanisms of PD-L1 expression (TPS score) (n = 18) and presence of altered antigen processing (n = 10) are mutually non-exclusive and can occur in the same patient (n = 6). Both mechanisms have exclusive influence on different genes and pathways, according to differential gene expression analysis and gene set enrichment analysis, respectively. Interestingly, gene expression patterns associated with altered processing were enriched in T cell and NK cell immune activity. Though both mechanisms influence different genes, they are similarly linked to increased immune activity. CONCLUSION: Pressure from the immune system will lay the foundations for escape mechanisms, leading to acquisition of resistance under therapy. Both PD-L1 expression and altered antigen processing are induced similarly by pronounced immunoactivity but in different context. The present data help to deepen our understanding of the underlying mechanisms behind those immune escapes.

Development and validation of a novel 133-plex forensic STR panel (52 STRs and 81 Y-STRs) using single-end 400 bp massive parallel sequencing.

Short tandem repeats (STRs) are the preferred genetic markers in forensic DNA analysis, routinely measured by capillary electrophoresis (CE) method based on the fragment length features. While, the massive parallel sequencing (MPS) technology could simultaneously target a large number of intriguing forensic STRs, bypassing the intrinsic limitations of amplicon size separation and accessible fluorophores in CE, which is efficient and promising for enabling the identification of forensic biological evidence. Here, we developed a novel MPS-based Forensic Analysis System Multiplecues SetB Kit of 133-plex forensic STR markers (52 STRs and 81 Y-STRs) and one Y-InDel (M175) based on multiplex PCR and single-end 400 bp sequencing strategy. This panel was subjected to developmental validation studies according to the SWGDAM Validation Guidelines. Approximately 2185 MPS-based reactions using 6 human DNA standards and 8 male donors were conducted for substrate studies (filter paper, gauze, cotton swab, four different types of FTA cards, peripheral venous blood, saliva, and exfoliated cells), sensitivity studies (from 2 ng down to 0.0625 ng), mixture studies (two-person DNA mixtures), PCR inhibitor studies (seven commonly encountered PCR inhibitors), species specificity studies (11 non-human species), and repeatability studies. Results of concordance studies (413 Han males and 6 human DNA standards) generated by STRait Razor and in-house Python scripts indicated 99.98% concordance rate in STR calling relative to CE for STRs between 41,900 genotypes at 100 STR markers. Moreover, the limitations of present studies, the nomenclature rules and forensic MPS applications were also described. In conclusion, the validation studies based on ~ 2200 MPS-based and ~ 2500 CE-based DNA profiles demonstrated that the novel MPS-based panel meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities, and the STR nomenclature rules should be further regulated to integrate the inconformity between MPS-based and CE-based methods.

Intellectual disability genomics: current state, pitfalls and future challenges.

Intellectual disability (ID) can be caused by non-genetic and genetic factors, the latter being responsible for more than 1700 ID-related disorders. The broad ID phenotypic and genetic heterogeneity, as well as the difficulty in the establishment of the inheritance pattern, often result in a delay in the diagnosis. It has become apparent that massive parallel sequencing can overcome these difficulties. In this review we address: (i) ID genetic aetiology, (ii) clinical/medical settings testing, (iii) massive parallel sequencing, (iv) variant filtering and prioritization, (v) variant classification guidelines and functional studies, and (vi) ID diagnostic yield. Furthermore, the need for a constant update of the methodologies and functional tests, is essential. Thus, international collaborations, to gather expertise, data and resources through multidisciplinary contributions, are fundamental to keep track of the fast progress in ID gene discovery.