Generation Sequencing Methodologies Research Articles

Binary classification of copy number alteration profiles in liquid biopsy with potential clinical impact in advanced NSCLC

Liquid biopsy has recently emerged as an important tool in clinical practice particularly for lung cancer patients. We retrospectively evaluated cell-free DNA analyses performed at our Institution by next generation sequencing methodology detecting the major classes of genetic alterations. Starting from the graphical representation of chromosomal alterations provided by the analysis software, we developed a support vector machine classifier to automatically classify chromosomal profiles as stable (SCP) or unstable (UCP). High concordance was found between our binary classification and tumor fraction evaluation performed using shallow whole genome sequencing. Among clinical features, UCP patients were more likely to have ≥ 3 metastatic sites and liver metastases. Longitudinal assessment of chromosomal profiles in 33 patients with lung cancer receiving immune checkpoint inhibitors (ICIs) showed that only patients that experienced early death or hyperprogressive disease retained or acquired an UCP within 3 weeks from the beginning of ICIs. UCP was not observed following ICIs among patients that experienced progressive disease or clinical benefit. In conclusion, our binary classification, applied to whole copy number alteration profiles, could be useful for clinical risk stratification during systemic treatment for non-small cell lung cancer patients.

Molecular characterization of novel and rare DNA variants in patients with galactosemia

Introduction: Galactosemia is an inherited disorder caused by mutations in the three genes that encode enzymes implicated in galactose catabolism. Currently, the only available treatment for galactosemia is life-long dietary restriction of galactose/lactose, and despite treatment, it might result in long-term complications.Methods: Here, we present five cases of newborn patients with elevated galactose levels, identified in the context of the newborn screening program. Genetic analysis concerned a next generation sequencing (NGS) methodology covering the exons and adjacent splice regions of the GALT, GALK1, and GALE genes.Results: Our approach led to the identification of eight rare nonsynonymous DNA variants. Four of these variants, namely, p.Arg204Gln and p.Met298Ile in GALT, p.Arg68Leu in GALK1, and p.Ala180Thr in GALE, were already recorded in relevant databases, yet their clinical significance is uncertain. The other four variants, namely, p.Phe245Leu in GALT, p.Gly193Glu in GALK1, and p.Ile266Leu and p.Ala216Thr in the GALE gene, were novel. In silico analysis of the possible effect of these variants in terms of protein function and stability was performed using a series of bioinformatics tools, followed by visualization of the substituted amino acids within the protein molecule. The analysis revealed a deleterious and/or destabilizing effect for all the variants, supported by multiple tools in each case.Discussion: These results, given the extreme rarity of the variants and the specific phenotype of the respective cases, support a pathogenic effect for each individual variant. Altogether, our study shows that targeted NGS methodologies may offer a time- and cost-effective approach for the genetic investigation of galactosemia and can assist in elucidating the complex genetic background of this disorder.

P-240 High mitochondrial DNA content in Day6 blastocyst may impair ongoing pregnancy rate

Abstract Study question Is there an association between mitochondrial DNA (mtDNA) content and Ongoing Pregnancy Rate (OPR) in D5 or D6 euploid blastocyst? Summary answer Day6 OPR is decreased in high mtDNA content blastocyst. Day5 and Day6 mtDNA levels are independent of maternal age or standard morphology. What is known already Preimplantation development is an energy-demanding process and mitochondrial ATP production is crucial for cellular activity in fast replicating cells. Mitochondrial content in preimplantation embryo has been proposed as a marker of embryo potential in term of viability and implantation success though the dynamics and distribution of mitochondria in human embryo is still debated. In recent years, it has been suggested that low mtDNA levels were associated with euploid chromosomal complement and higher implantation rate while other studies failed to find a correlation between mtDNA content and reproductive outcome. Study design, size, duration This study is a retrospective cohort analysis from 2020 to 2022 including 343 Day5 and 187 D6 single euploid blastocyst transfer. Primary endpoint was OPR beyond pregnancy week 16. Day5 and Day6 frozen-thawed transfer were divided based on blastocyst mtDNA content in four groups: Day5-high, Day5-low, D6-high, D6-low. Secondary endpoints were relationship between mtDNA content, maternal age and standard morphology in all obtained blastocysts. Statistical differences were compared by Chi-Square test. Participants/materials, setting, methods 771 couples performed IVF cycles with next-generation sequencing (NGS)-preimplantation genetic testing of aneuploidy (PGT-A) (age 19-47yrs; Mean age 37.8yrs). Following culture in sequential media 700 euploid blastocyst were obtained and mitochondrial and chromosomal DNA copy number variation were examined simultaneously with next generation sequencing (NGS) methodology. mtDNA copy numbers was based on the observed ratios of sequence coverages between mtDNA and nuclear DNA. Main results and the role of chance In our clinical setting OPR is similar in Day5 vs. Day6 transfers (55% vs. 49% n.s.). However when mtDNA content is considered D6-low OPR is comparable to D5-high or low (57.3%; 53.3%; 57,3% respectively n.s.) while D6-high transfers yield significantly decreased OPR (31,2% p < 0.05). When stratified by age only Advanced Maternal Age (AMA) patients ( > = 38yrs) showed this decrease at a significant level (Day6 high OPR 20% p < 0,05).This result in transferred blastocyst is obtained despite high:low ratio of all biopsied blastocyst is constant in Day5 (1:1) (47%-53%) and markedly decreased in Day6 (1:4) (25%-75%) either overall or in younger (n.s.) and AMA patients (n.s.). Moreover when standard morphology distribution is evaluated according to Gardner classification criteria top, fair and poor quality blastocysts display the same 1:1 high:low blastocysts mtDNA ratio in Day5 and 1:4 in Day 6 overall and in younger and AMA patients. Hence, the mtDNA blastocyst content is independent of patients’ age and blastocyst quality. The origin of Day6 OPR decrease could therefore depend on the number of Day6-high blastocyst transferred in AMA patients and ultimately in the number of blastocyst available to transfer for each patient. Limitations, reasons for caution Due to the small numbers of Day6-high mtDNA blastocyst, a larger sample size is required to confirm these preliminary findings. Moreover, the retrospective nature of the study may introduce some bias mainly in patients’ characteristics and the strategy of embryo selected for transfer. Wider implications of the findings While Day5-high blastocyst yield higher OPR the persistence of higher mtDNA levels in Day6 blastocyst may hamper their implantation potential. Our findings may help to select the embryo to transfer to maximize transfer success. D6 blastocysts with low mtDNA content should be preferred whenever a choice is possible. Trial registration number Not Applicable

Abstract 6610: Increasing blood volumes to detect minimal residual disease in neoadjuvant-treated early breast cancer patients

Abstract Breast cancer (BC) is the most frequent neoplasia affecting women worldwide normally detected at early stages. In this regard, early diagnosis drastically decreases mortality, however, around 20% of these patients will later relapse. This is mainly caused by undetectable molecular residual disease (MRD) not eliminated by standard primary treatments. Therefore, it is crucial to detect the after-treatment MRD to stratify the patients by their risk of relapse. Liquid biopsies have emerged as non-invasive method to obtain information about tumors and improve clinical cancer management. Regarding this, much has been hypothesized about utilizing high blood volumes to overcome the necessity of complex and resource-intensive next generation sequencing (NGS) methodologies to detect highly diluted blood tumor components in localized cancers. Herein, we employed a combined analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) together with high blood volumes and single-assay droplet digital PCR (ddPCR) to detect MRD with ultra-high sensitivity. We prospectively assayed 124 samples extracted at baseline, post-neoadjuvant therapy (NAT), post-surgery and a follow-up on a six-monthly basis. A median of 76.40 mL of blood to detect CTCs and 40 mL of plasma to detect ctDNA per patient from 19 BC women were used in this study. ddPCR assays were performed with a median of 14 partitions per determination to detect ctDNA and 12 partitions for CTCs. Overall, ctDNA, CTCs and ctDNA and/or CTCs were detected in 84.21%, 66.66% and 89.47% respectively in the pre-treatment blood samples. MRD (ctDNA and/or CTCs) was detected in 73.68% of the after NAT blood samples. On the other hand, it was detected in 46.66% and 70.00% of the post-surgery and follow-up samples respectively. Post-NAT MRD was detected in 57.14% (4/7) and 83.33% (10/12) of patients with and without pathological complete response pCR respectively. To note, the discordant patients achieving pCR in tissue with detectable MRD in blood were high-risk BC. Importantly, in one of the two patients without pCR and no MRD detected, not enough sample were available to complete the analysis. The other discordant patient presented a localized disease with residual cancer burden value of 1 and no lymph nodes affected. In 1 out of 19 (5.26%) patient clinically relapsed with a positive MRD detection 6 months earlier. Applying this methodology, we observed a sensitivity of 0.004% in ctDNA detection and 0.224 CTCs per mL of blood. Overall, this novel methodology greatly improves sensitivity for ctDNA and CTCs detection in treatment-naïve early BC. In addition, MRD was successfully detected in post-treatment samples antedating clinical relapse by 6 months in one patient. This prospective study is potentially demonstrating that using high blood volumes and a single-assay ddPCR is a cost-effective strategy to monitor localized BC and predict relapses. Citation Format: Alfonso Alba-Bernal, Ana Godoy-Ortiz, María Emilia Domínguez-Recio, Begoña Jimenez-Rodriguez, María Elena Quirós-Ortega, Esperanza López-López, Guillermo Carbajosa-Antona, Jesús Peralta-Linero, Luis Vicioso, Estefanía Bellagarza-García, Guadalupe Dolores Garrido-Ruiz, Cynthia Robles-Podadera, Alicia Garrido-Aranda, María Dunia Roldán-Díaz, Jesús Velasco-Suelto, Rocío Lavado-Valenzuela, Martina Álvarez, Nuria Ribelles, Javier Pascual, Emilio Alba, Iñaki Comino-Méndez. Increasing blood volumes to detect minimal residual disease in neoadjuvant-treated early breast cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6610.

Dental implant surfaces and their interaction with the oral microbiome

• Differences between periodontitis and peri-implantitis aetiology remain unclear • Optimal peri-implantitis modelling potentially requires implant-specific microbiota • Complex biofilms provide greater functional understanding of surface impact • Contact effect, anti-adhesion & antimicrobial release strategies can reduce biofilm • An amalgamation of approaches may offer the greatest anti-biofilm potential This review aims to collate the current knowledge in the field of antimicrobial surfaces on dental implant materials, focusing on microbial population and functional responses, predominantly from omics-based studies. Extensive searching of Scopus and Pubmed databases informed a narrative review on the antimicrobial impact of implant surfaces on the complex oral microbiome. The awareness of this issue has led to considerable research resources being directed towards the augmentation of implant surfaces to counteract microbial colonization. Whilst the implant material itself has a direct influence on bacterial adhesion and viability, the surface finish and putative antimicrobial coatings are critical to countering early biofilm formation. Multiple modes of surface modification have been developed to counteract early colonization, including direct physical contact effect, such as anti-adhesion strategies and extract effects, through antimicrobial release chemistry or material leaching. These concepts deploy different techniques, including nano-texturing, surface chemistry alteration and controlled release, each with a diverse set of benefits and drawbacks. Novel surface finishes and coatings require investigation with regards to their influence on oral biofilms, whether on individual bacterial species or against mature biofilms. The search for optimal implant surfaces is necessary for the reduction of the peri-implantitis burden and the longevity of dental implants. To date, next generation sequencing methodologies, enabling a greater depth of understanding of the complex interactions between oral microorganisms, host response, and implant surface coatings are under used in this area of research.

Molecular detection and characterisation of the first Japanese encephalitis virus belonging to genotype IV acquired in Australia.

A fatal case of Japanese encephalitis (JE) occurred in a resident of the Tiwi Islands, in the Northern Territory of Australia in February 2021, preceding the large JE outbreak in south-eastern Australia in 2022. This study reports the detection, whole genome sequencing and analysis of the virus responsible (designated JEV/Australia/NT_Tiwi Islands/2021). Reverse transcription quantitative PCR (RT-qPCR) testing was performed on post-mortem brain specimens using a range of JE virus (JEV)-specific assays. Virus isolation from brain specimens was attempted by inoculation of mosquito and mammalian cells or embryonated chicken eggs. Whole genome sequencing was undertaken using a combination of Illumina next generation sequencing methodologies, including a tiling amplicon approach. Phylogenetic and selection analyses were performed using alignments of the Tiwi Islands JEV genome and envelope (E) protein gene sequences and publicly available JEV sequences. Virus isolation was unsuccessful and JEV RNA was detected only by RT-qPCR assays capable of detecting all JEV genotypes. Phylogenetic analysis revealed that the Tiwi Islands strain is a divergent member of genotype IV (GIV) and is closely related to the 2022 Australian outbreak virus (99.8% nucleotide identity). The Australian strains share highest levels of nucleotide identity with Indonesian viruses from 2017 and 2019 (96.7-96.8%). The most recent common ancestor of this Australian-Indonesian clade was estimated to have emerged in 2007 (95% HPD range: 1998-2014). Positive selection was detected using two methods (MEME and FEL) at several sites in the E and non-structural protein genes, including a single site in the E protein (S194N) unique to the Australian GIV strains. This case represents the first detection of GIV JEV acquired in Australia, and only the second confirmed fatal human infection with a GIV JEV strain. The close phylogenetic relationship between the Tiwi Islands strain and recent Indonesian viruses is indicative of the origin of this novel GIV lineage, which we estimate has circulated in the region for several years prior to the Tiwi Islands case.

An insight into gill microbiome of Eastern Mediterranean wild fish by applying next generation sequencing

Bacterial diseases of marine fish inflict significant economic damage to fisheries and aquaculture and pose an increasing risk to public health. When addressing fish disease, an accumulating body of research suggests adding another factor to the classic epidemiological triangle of host-environment-pathogen: the microbiome. The gills, being a gateway into the fish body and bearing an important role in fish homeostasis, have been found to be a proxy of the gut microbiota as well as reflecting the microbial communities of surrounding water. In this study, 16S rRNA amplicons of bacterial DNA extracted from the gills of 89 asymptomatic specimens of three wild fish species (Pagrus caeruleostictus, Scomber colias and Saurida lessepsianus) were sequenced using Next Generation Sequencing methodology (NGS). Data analyses revealed the presence of 41 potentially pathogenic species, including several zoonotic agents. Five genera known to include widespread and potentially pathogenic species were chosen for further investigation: Photobacterium, Shewanella, Staphylococcus, Streptococcus and Vibrio. Of these, Photobacterium and Shewanella proved the most prevalent and abundant, making up 30.2% and 11.3% of the Bluespotted seabream (P. caeruleostictus) gill microbiome alone. Photobacterium damselae and Shewanella baltica were most common at the species level. The remaining genera - Vibrio, Staphylococcus and Streptococcus – were less prevalent, and at a species level were comprised of only 1−4% potentially pathogenic representatives. Gill microbiomes exhibited host species specificity, with strong correlations between certain bacterial taxonomic groups. No definite obligatory pathogenic bacteria were found in this study, and it was suggested that pathogenic species are present as either covert pathobionts or as opportunists of the fish found to host them.

HLA allele-specific expression: Methods, disease associations, and relevance in hematopoietic stem cell transplantation.

Varying HLA allele-specific expression levels are associated with human diseases, such as graft versus host disease (GvHD) in hematopoietic stem cell transplantation (HSCT), cytotoxic T cell response and viral load in HIV infection, and the risk of Crohn’s disease. Only recently, RNA-based next generation sequencing (NGS) methodologies with accompanying bioinformatics tools have emerged to quantify HLA allele-specific expression replacing the quantitative PCR (qPCR) -based methods. These novel NGS approaches enable the systematic analysis of the HLA allele-specific expression changes between individuals and between normal and disease phenotypes. Additionally, analyzing HLA allele-specific expression and allele-specific expression loss provide important information for predicting efficacies of novel immune cell therapies. Here, we review available RNA sequencing-based approaches and computational tools for NGS to quantify HLA allele-specific expression. Moreover, we explore recent studies reporting disease associations with differential HLA expression. Finally, we discuss the role of allele-specific expression in HSCT and how considering the expression quantification in recipient-donor matching could improve the outcome of HSCT.

The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

Genomic surveillance of SARS-CoV-2 Spike gene by sanger sequencing.

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.

The complete chloroplast genome of Scutellaria meehanioides (Lamiaceae) from Shaanxi Province, China

Scutellaria meehanioides C.Y.Wu is a medicinal perennial herb native to China. The complete chloroplast genome sequence of the S. meehanioides was determined and assembled using next generation sequencing methodologies. The complete genome is 152,484 base pairs (bp) in length and has an overall GC content of 38.4%. The chloroplast genome contains, a large single-copy region (LSC) of 83,859 bp, small single-copy region (SSC) of 17,467 bp and a pair of inverted repeats (IRs) of 24,029 bp. The genome of S. meehanioides contains 133 genes, including 87 protein-coding, 38 tRNA,and 8 rRNA genes. Phylogenetic analysis fully resolved S. meehanioides in a clade with S. orthocalyx. This study provides useful information for future genetic study of S. meehanioides.

Cross-talk between next generation sequencing methodologies to identify genomic signatures of esophageal cancer.

The asymptomatic behaviour of esophageal cancerous cells at early stages develops advanced clinical presentation of the disease, resulting in poor prognosis and curbed intervention of therapeutic modalities. The endeavours to detect diagnostic and prognostic markers have been proven futile at the clinical platform. While several biomarkers have been investigated, including CYFRA 21-1, carcinoembryonic antigen and squamous cell carcinoma antigen, their sensitivity has not proved consistently satisfactory across the various stages of esophageal cancer. Hence, there is an impending requirement of biomarkers for early diagnosis and better prognosis. In the recent past, next generation sequencing (NGS) tool has emerged as an important tool to highlight the hallmarks of esophageal cancer (EC). This review summarizes the changes/mutations occurred in tumor cells during carcinogenesis and addresses the contribution of NGS techniques, viz. whole genome sequencing (WGS), RNA-Sequencing and Exome sequencing (ES), in EC. Additionally, this review highlights the connection between the findings from these techniques. An effort has been made to emphasize the genes affected and involved signaling pathway in EC. Further, investigations of these mutated genes would not only shed light on the relevant genes to be studied but also help in the better management and cure through personalized therapy.

Usefulness of NGS for Diagnosis of Dominant Beta-Thalassemia and Unstable Hemoglobinopathies in Five Clinical Cases.

Unstable hemoglobinopathies (UHs) are rare anemia disorders (RADs) characterized by abnormal hemoglobin (Hb) variants with decreased stability. UHs are therefore easily precipitating, causing hemolysis and, in some cases, leading to dominant beta-thalassemia (dBTHAL). The clinical picture of UHs is highly heterogeneous, inheritance pattern is dominant, instead of recessive as in more prevalent major Hb syndromes, and may occur de novo. Most cases of UHs are not detected by conventional testing, therefore diagnosis requires a high index of suspicion of the treating physician. Here, we highlight the importance of next generation sequencing (NGS) methodologies for the diagnosis of patients with dBTHAL and other less severe UH variants. We present five unrelated clinical cases referred with chronic hemolytic anemia, three of them with severe blood transfusion dependent anemia. Targeted NGS analysis was performed in three cases while whole exome sequencing (WES) analysis was performed in two cases. Five different UH variants were identified correlating with patients’ clinical manifestations. Four variants were related to the beta-globin gene (Hb Bristol—Alesha, Hb Debrousse, Hb Zunyi, and the novel Hb Mokum) meanwhile one case was caused by a mutation in the alpha-globin gene leading to Hb Evans. Inclusion of alpha and beta-globin genes in routine NGS approaches for RADs has to be considered to improve diagnosis’ efficiency of RAD due to UHs. Reducing misdiagnoses and underdiagnoses of UH variants, especially of the severe forms leading to dBTHAL would also facilitate the early start of intensive or curative treatments for these patients.

A review of the use of next generation sequencing methodologies to identify biomarkers of resistance to CDK4/6 inhibitors in ER+/HER2- breast cancer.

The development of cyclin-dependent kinases (CDK) 4 and 6 inhibitors represented a substantial breakthrough in the treatment of estrogen receptor positive (ER+), human epidermal growth factor receptor 2 (HER2) negative metastatic breast cancer. These drugs showed a significant clinical benefit in pivotal clinical trials. However, resistance eventually occurs, leading to disease progression. Next Generation Sequencing methodologies have been employed to investigate predictive biomarkers of response or resistance to CDK4/6 inhibitors. Whole exome and targeted sequencing of solid and liquid biopsies have revealed several possible genomic alterations associated with resistance. Notably, genomic alterations identified by DNA-sequencing did not fully recapitulate the entire landscape of resistance to CDK4/6 inhibitors. Gene expression analysis, such as RNA-Seq methodologies, have provided insights into transcriptional profiles and may need further application. Herein, we report the main findings derived from the use of NGS analysis in the context of resistance to CDK4/6 inhibitors in ER + breast cancer.

The Dimensions of Primary Mitochondrial Disorders.

The concept of a mitochondrial disorder was initially described in 1962, in a patient with altered energy metabolism. Over time, mitochondrial energy metabolism has been discovered to be influenced by a vast number of proteins with a multitude of functional roles. Amongst these, defective oxidative phosphorylation arose as the hallmark of mitochondrial disorders. In the premolecular era, the diagnosis of mitochondrial disease was dependent on biochemical criteria, with inherent limitations such as tissue availability and specificity, preanalytical and analytical artifacts, and secondary effects. With the identification of the first mitochondrial disease-causing mutations, the genetic complexity of mitochondrial disorders began to unravel. Mitochondrial dysfunctions can be caused by pathogenic variants in genes encoded by the mitochondrial DNA or the nuclear DNA, and can display heterogenous phenotypic manifestations. The application of next generation sequencing methodologies in diagnostics is proving to be pivotal in finding the molecular diagnosis and has been instrumental in the discovery of a growing list of novel mitochondrial disease genes. In the molecular era, the diagnosis of a mitochondrial disorder, suspected on clinical grounds, is increasingly based on variant detection and associated statistical support, while invasive biopsies and biochemical assays are conducted to an ever-decreasing extent. At present, there is no uniform biochemical or molecular definition for the designation of a disease as a “mitochondrial disorder”. Such designation is currently dependent on the criteria applied, which may encompass clinical, genetic, biochemical, functional, and/or mitochondrial protein localization criteria. Given this variation, numerous gene lists emerge, ranging from 270 to over 400 proposed mitochondrial disease genes. Herein we provide an overview of the mitochondrial disease associated genes and their accompanying challenges.

Extensive CFTR sequencing through NGS in Brazilian individuals with cystic fibrosis: unravelling regional discrepancies in the country

BackgroundThe Brazilian population has a tri-hybrid composition with a high degree of ethnic admixture. We hypothesized that Brazilian individuals with CF from different Brazilian regions have a specific distribution of CFTR variants. MethodsIndividuals with CF with data available in the Patient Registry and without an established genotype were submitted to CFTR sequencing by Next Generation Sequencing (NGS) methodology, and results were anonymously incorporated to the Registry Database. Genotyping results were expressed as ‘positive’, ‘inconclusive’ or ‘negative’. Logistic regression models were performed to investigate the association between demographic/clinical variables and genotyping results. Mediation analysis was conducted to estimate direct and indirect effects of Brazilian region on a binary positive genotyping response. ResultsIn October 2017, data from 4,654 individuals with CF were available, and 3,104(66.7%) of them had a genotyping result. A total of 236 variants (114 new variants) were identified, with F508del identified in 46% of the alleles tested. Genotyping revealed 2,002(64.5%) individuals positive, 757(24.4%) inconclusive and 345(11.1%) negative. Distribution of genotype categories was markedly different across Brazilian Regions, with greater proportions of negative individuals in the North (45%) and Northeast (26%) regions. Newborn screening (CF-NBS) and age at diagnosis were identified as mediators of the effect of Brazilian region on a positive genotyping result. ConclusionsThis large initiative of CFTR genotyping showed significant regional discrepancies in Brazil, probably related to socio-economic conditions, lack of adequate CF-NBS and poor access to reliable sweat testing. These results may be useful to indicate Regions where CF care demands more attention.

Clinical and Genetic Overview of Paroxysmal Movement Disorders and Episodic Ataxias

Paroxysmal movement disorders (PMDs) are rare neurological diseases typically manifesting with intermittent attacks of abnormal involuntary movements. Two main categories of PMDs are recognized based on the phenomenology: Paroxysmal dyskinesias (PxDs) are characterized by transient episodes hyperkinetic movement disorders, while attacks of cerebellar dysfunction are the hallmark of episodic ataxias (EAs). From an etiological point of view, both primary (genetic) and secondary (acquired) causes of PMDs are known. Recognition and diagnosis of PMDs is based on personal and familial medical history, physical examination, detailed reconstruction of ictal phenomenology, neuroimaging, and genetic analysis. Neurophysiological or laboratory tests are reserved for selected cases. Genetic knowledge of PMDs has been largely incremented by the advent of next generation sequencing (NGS) methodologies. The wide number of genes involved in the pathogenesis of PMDs reflects a high complexity of molecular bases of neurotransmission in cerebellar and basal ganglia circuits. In consideration of the broad genetic and phenotypic heterogeneity, a NGS approach by targeted panel for movement disorders, clinical or whole exome sequencing should be preferred, whenever possible, to a single gene approach, in order to increase diagnostic rate. This review is focused on clinical and genetic features of PMDs with the aim to (1) help clinicians to recognize, diagnose and treat patients with PMDs as well as to (2) provide an overview of genes and molecular mechanisms underlying these intriguing neurogenetic disorders.

Mutations in Mycobacterium tuberculosis Isolates with Discordant Results for Drug-Susceptibility Testing in Peru.

Evaluation of resistance to antituberculosis drugs is routinely performed with genotypic or phenotypic methods; however, discordance can be seen between these different methodologies. Our objective was to identify mutations that could explain discordant results in the evaluation of susceptibility to rifampicin and isoniazid between molecular and phenotypic methods, using whole genome sequencing (WGS). Peruvian strains showing sensitive results in the GenoType MTBDRplus v2.0 test and resistant results in the proportions in the agar-plaque test for isoniazid or rifampin were selected. Discordance was confirmed by repeating both tests, and WGS was performed, using the Next Generation Sequencing methodology. Obtained sequences were aligned “through reference” (genomic mapping) using the program BWA with the algorithm “mem”, using as a reference the genome of the M. tuberculosis H37Rv strain. Discordance was confirmed in 14 strains for rifampicin and 21 for isoniazid, with 1 strain in common for both antibiotics, for a total of 34 unique strains. The most frequent mutation in the rpoB gene in the discordant strains for rifampicin was V170F. The most frequent mutations in the discordant strains for isoniazid were katG R463L, kasA G269S, and Rv1592c I322V. Several other mutations are reported. This is the first study in Latin America addressing mutations present in strains with discordant results between genotypic and phenotypic methods to rifampicin and isoniazid. These mutations could be considered as future potential targets for genotypic tests for evaluation of susceptibility to these drugs.

Next Generation Sequencing and Bioinformatics Methodologies for Infectious Disease Research and Public Health: Approaches, Applications, and Considerations for Development of Laboratory Capacity.

Next generation sequencing (NGS) combined with bioinformatics has successfully been used in a vast array of analyses for infectious disease research of public health relevance. For instance, NGS and bioinformatics approaches have been used to identify outbreak origins, track transmissions, investigate epidemic dynamics, determine etiological agents of a disease, and discover novel human pathogens. However, implementation of high-quality NGS and bioinformatics in research and public health laboratories can be challenging. These challenges mainly include the choice of the sequencing platform and the sequencing approach, the choice of bioinformatics methodologies, access to the appropriate computation and information technology infrastructure, and recruiting and retaining personnel with the specialized skills and experience in this field. In this review, we summarize the most common NGS and bioinformatics workflows in the context of infectious disease genomic surveillance and pathogen discovery, and highlight the main challenges and considerations for setting up an NGS and bioinformatics-focused infectious disease research public health laboratory. We describe the most commonly used sequencing platforms and review their strengths and weaknesses. We review sequencing approaches that have been used for various pathogens and study questions, as well as the most common difficulties associated with these approaches that should be considered when implementing in a public health or research setting. In addition, we provide a review of some common bioinformatics tools and procedures used for pathogen discovery and genome assembly, along with the most common challenges and solutions. Finally, we summarize the bioinformatics of advanced viral, bacterial, and parasite pathogen characterization, including types of study questions that can be answered when utilizing NGS and bioinformatics.

1534P - TP53 mutations predicts worse prognosis in EGFR-mutated NSCLC patients receiving TKIs in first- or further line of treatment

BackgroundNon-small cell lung cancer (NSCLC) patients carrying epidermal growth factor receptor (EGFR) mutations are sensitive to tyrosine kinase inhibitors (TKIs); nonetheless, about 20% of patients show primary resistance to TKIs. We previously demonstrated the association between TP53 mutations and primary resistance to first-line TKIs in EGFR-mutated adenocarcinoma (ADC) patients. Here we analyze a validation case series of ADC patients treated with first-line TKIs to confirm our previous data, and we assess the association between TP53 status and the prognosis of ADC patients treated with third generation TKIs. MethodsWe considered 136 ADC EGFR-mutated patients treated with a TKI in the first-line setting. Also, we analyzed 42 patients who developed T790M mutation and were treated with a third generation TKI in second- or further line setting. EGFR and TP53 mutation analyses were performed by Sanger Sequencing or Next Generation Sequencing methodologies. TP53 mutations were evaluated in relation to disease control rate (DCR: complete response [CR], partial response [PR] or stable disease [SD]), objective response rate (ORR: CR, PR), progression-free survival (PFS) and overall survival (OS). ResultsOf 136 patients, we found 42 (31%) TP53 mutations: 12 mutations in exon 5 (28.6%), 6 in exon 6 (14.3%), 13 in exon 7 (30.9%), and 11 in exon 8 (26.2%). DCR and ORR were 31.8% and 32.5% in TP3-mutated patients with respect to 68.6% and 67.4% in TP53 wild-type (wt) patients, respectively. Exon 8 TP53-mutated patients had a worse PFS with respect to TP53wt patients (HR 3.19 [1.62-6.27], p=0.001), but not OS. Moreover, we assessed TP53 status in 42 patients who developed a T790M mutation and received a third generation TKI in second or additional lines of treatment. In 30 patients with evaluable clinical response, DCR and ORR were 62.5% and 25% in TP53 mutated patients, with respect to 77.2% and 54.5% in the TP53wt patients, respectively. Data analyses of PFS and OS are ongoing. ConclusionsEGFR-mutated NSCLC patients with concomitant TP53 mutations had a worse clinical response to first-, second-, and third generation TKIs, administrated in any line of treatment. Legal entity responsible for the studyIstituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.