A critical spotlight on the paradigms of FFPE-DNA sequencing.

In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called 'ERROR-FFPE-DNA' that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes.

Genetic lesions in MYC and STAT3 drive oncogenic transcription factor overexpression in plasmablastic lymphoma.

Plasmablastic lymphoma mutational profile is undescribed. Here we performed a targeted exonic NGS analysis of 30 plasmablastic lymphoma cases with a B cell lymphoma dedicated panel and FISH for the detection of MYC rearrangements. A complete phenotyping of the neoplastic and microenvironment cell populations was also performed. We have identified an enrichment in recurrent genetic events in MYC (69% with MYC translocation or amplification and 3 cases with missense point mutations), PRDM1/Blimp1 and STAT3 mutations. These gene mutations were more frequent in EBV positive disease. Other genetic events included mutations in BRAF, EP300, BCR (CD79A and CD79B), NOTCH pathway (NOTCH2, NOTCH1 and SGK1) and MYD88pL265P. Immunohistochemical analysis showed consistent MYC expression, higher in cases with MYC rearrangements together with phospho-STAT3 (Tyr705) overexpression in cases with STAT3 SH2 domain mutations. Microenvironment populations were heterogeneous and unrelated with EBV, with an enrichment of Tumor Associated Macrophages (TAM) and PD1 positive T cells. PD-L1 was expressed in all cases in the TAM population but only in 5 cases in the neoplastic cells (4 out of 14 EBV positive cases). HLA expression was absent in the majority of PBL cases. In summary, Plasmablastic lymphoma mutational profile is heterogeneous and related with EBV infection. Genetic events in MYC, STAT3 and PRDM1/Blimp1 are more frequent in EBV positive disease. An enrichment in TAM and PD1 reactive T lymphocytes is found in the microenvironment of PBL cases, that express PD-L1 in the neoplastic cells in a fraction of cases.

Variability in porcine microRNA genes and its association with mRNA expression and lipid phenotypes.

BACKGROUND: Mature microRNAs (miRNAs) play an important role in repressing the expression of a wide range of mRNAs. The presence of polymorphic sites in miRNA genes and their corresponding 3'UTR binding sites can disrupt canonical conserved miRNA-mRNA pairings, and thus modify gene expression patterns. However, to date such polymorphic sites in miRNA genes and their association with gene expression phenotypes and complex traits are poorly characterized in pigs. RESULTS: By analyzing whole-genome sequences from 120 pigs and wild boars from Europe and Asia, we identified 285 single nucleotide polymorphisms (SNPs) that map to miRNA loci, and 109,724 SNPs that are located in predicted 7mer-m8 miRNA binding sites within porcine 3'UTR. In porcine miRNA genes, SNP density is reduced compared with their flanking non-miRNA regions. By sequencing the genomes of five Duroc boars, we identified 12 miRNA SNPs that were subsequently genotyped in their offspring (N = 345, Lipgen population). Association analyses of miRNA SNPs with 38 lipid-related traits and hepatic and muscle microarray expression phenotypes recorded in the Lipgen population were performed. The most relevant detected association was between the genotype of the rs319154814 (G/A) SNP located in the apical loop of the ssc-miR-326 hairpin precursor and PPP1CC mRNA levels in the liver (q-value = 0.058). This result was subsequently confirmed by qPCR (P-value = 0.027). The rs319154814 (G/A) genotype was also associated with several fatty acid composition traits. CONCLUSIONS: Our findings show a reduced variability of porcine miRNA genes, which is consistent with strong purifying selection, particularly in the seed region that plays a critical role in miRNA binding. Although it is generally assumed that SNPs mapping to the seed region are those with the most pronounced consequences on mRNA expression, we show that a SNP mapping to the apical region of ssc-miR-326 is significantly associated with hepatic mRNA levels of the PPP1CC gene, one of its predicted targets. Although experimental confirmation of such an interaction is reported in humans but not in pigs, this result highlights the need to further investigate the functional effects of miRNA polymorphisms that are located outside the seed region on gene expression in pigs.

Runs of homozygosity reveal signatures of positive selection for reproduction traits in breed and non-breed horses.

BACKGROUND: Modern horses represent heterogeneous populations specifically selected for appearance and performance. Genomic regions under high selective pressure show characteristic runs of homozygosity (ROH) which represent a low genetic diversity. This study aims at detecting the number and functional distribution of ROHs in different horse populations using next generation sequencing data. METHODS: Next generation sequencing was performed for two Sorraia, one Dülmen Horse, one Arabian, one Saxon-Thuringian Heavy Warmblood, one Thoroughbred and four Hanoverian. After quality control reads were mapped to the reference genome EquCab2.70. ROH detection was performed using PLINK, version 1.07 for a trimmed dataset with 11,325,777 SNPs and a mean read depth of 12. Stretches with homozygous genotypes of >40 kb as well as >400 kb were defined as ROHs. SNPs within consensus ROHs were tested for neutrality. Functional classification was done for genes annotated within ROHs using PANTHER gene list analysis and functional variants were tested for their distribution among breed or non-breed groups. RESULTS: ROH detection was performed using whole genome sequences of ten horses of six populations representing various breed types and non-breed horses. In total, an average number of 3492 ROHs were detected in windows of a minimum of 50 consecutive homozygous SNPs and an average number of 292 ROHs in windows of 500 consecutive homozygous SNPs. Functional analyses of private ROHs in each horse revealed a high frequency of genes affecting cellular, metabolic, developmental, immune system and reproduction processes. In non-breed horses, 198 ROHs in 50-SNP windows and seven ROHs in 500-SNP windows showed an enrichment of genes involved in reproduction, embryonic development, energy metabolism, muscle and cardiac development whereas all seven breed horses revealed only three common ROHs in 50-SNP windows harboring the fertility-related gene YES1. In the Hanoverian, a total of 18 private ROHs could be shown to be located in the region of genes potentially involved in neurologic control, signaling, glycogen balance and reproduction. Comparative analysis of homozygous stretches common in all ten horses displayed three ROHs which were all located in the region of KITLG, the ligand of KIT known to be involved in melanogenesis, haematopoiesis and gametogenesis. CONCLUSIONS: The results of this study give a comprehensive insight into the frequency and number of ROHs in various horses and their potential influence on population diversity and selection pressures. Comparisons of breed and non-breed horses suggest a significant artificial as well as natural selection pressure on reproduction performance in all types of horse populations.

Next generation sequencing gives an insight into the characteristics of highly selected breeds versus non-breed horses in the course of domestication.

BACKGROUND: Domestication has shaped the horse and lead to a group of many different types. Some have been under strong human selection while others developed in close relationship with nature. The aim of our study was to perform next generation sequencing of breed and non-breed horses to provide an insight into genetic influences on selective forces. RESULTS: Whole genome sequencing of five horses of four different populations revealed 10,193,421 single nucleotide polymorphisms (SNPs) and 1,361,948 insertion/deletion polymorphisms (indels). In comparison to horse variant databases and previous reports, we were able to identify 3,394,883 novel SNPs and 868,525 novel indels. We analyzed the distribution of individual variants and found significant enrichment of private mutations in coding regions of genes involved in primary metabolic processes, anatomical structures, morphogenesis and cellular components in non-breed horses and in contrast to that private mutations in genes affecting cell communication, lipid metabolic process, neurological system process, muscle contraction, ion transport, developmental processes of the nervous system and ectoderm in breed horses. CONCLUSIONS: Our next generation sequencing data constitute an important first step for the characterization of non-breed in comparison to breed horses and provide a large number of novel variants for future analyses. Functional annotations suggest specific variants that could play a role for the characterization of breed or non-breed horses.

Host genetic and immune factors drive evasion of HIV-1 pathogenesis in viremic non-progressors.

BACKGROUND: Viremic non-progressors (VNPs) represent an exceptional and uncommon subset of people with HIV-1, characterized by the remarkable preservation of normal CD4+ T cell counts despite uncontrolled viral replication-a trait reminiscent of natural hosts of simian immunodeficiency virus. The mechanisms orchestrating evasion from HIV-1 pathogenesis in human VNPs remain elusive, primarily due to the absence of integrative studies. METHODS: We implemented a novel single-cell and multiomics approach to comprehensively characterize viral, genomic, transcriptomic, and metabolomic factors driving this exceedingly rare disease phenotype in 16 VNPs and 29 HIV+ progressors. FINDINGS: Genetic predisposition to the VNP phenotype was evidenced by a higher prevalence of CCR5Δ32 heterozygosity, which was associated with lower levels of CCR5 expression and a lower frequency of infected cells in peripheral circulation. We also observed reduced levels of plasma markers of intestinal disruption and attenuated interferon responses in VNPs. These factors potentially drive the other phenotypic traits of immune preservation in this population, including the unaltered tryptophan metabolic profile, reduced activation of cytotoxic lymphocytes, and reduced bystander CD4+ T cell apoptosis. CONCLUSIONS: In summary, our comprehensive analysis identified intricate factors collectively associated with the unique immunovirological equilibrium in VNPs, shedding light on potential avenues for therapeutic exploration in managing HIV pathogenesis. FUNDING: The work was supported by funding from the Spanish Ministry of Science and Innovation and the National Institutes of Health (NIH).

Quantification of rare somatic single nucleotide variants by droplet digital PCR using SuperSelective primers.

Somatic single-nucleotide variants (SNVs) occur every time a cell divides, appearing even in healthy tissues at low frequencies. These mutations may accumulate as neutral variants during aging, or eventually, promote the development of neoplasia. Here, we present the SP-ddPCR, a droplet digital PCR (ddPCR) based approach that utilizes customized SuperSelective primers aiming at quantifying the proportion of rare SNVs. For that purpose, we selected five potentially pathogenic variants identified by whole-exome sequencing (WES) occurring at low variant allele frequency (VAF) in at-risk colon healthy mucosa of patients diagnosed with colorectal cancer or advanced adenoma. Additionally, two APC SNVs detected in two cancer lesions were added to the study for WES-VAF validation. SuperSelective primers were designed to quantify SNVs at low VAFs both in silico and in clinical samples. In addition to the two APC SNVs in colonic lesions, SP-ddPCR confirmed the presence of three out of five selected SNVs in the normal colonic mucosa with allelic frequencies ≤ 5%. Moreover, SP-ddPCR showed the presence of two potentially pathogenic variants in the distal normal mucosa of patients with colorectal carcinoma. In summary, SP-ddPCR offers a rapid and feasible methodology to validate next-generation sequencing data and accurately quantify rare SNVs, thus providing a potential tool for diagnosis and stratification of at-risk patients based on their mutational profiling.

Germ line variants in patients with acute myeloid leukemia without a suspicion of hereditary hematologic malignancy syndrome.

Germ line predisposition in acute myeloid leukemia (AML) has gained attention in recent years because of a nonnegligible frequency and an impact on management of patients and their relatives. Risk alleles for AML development may be present in patients without a clinical suspicion of hereditary hematologic malignancy syndrome. In this study we investigated the presence of germ line variants (GVs) in 288 genes related to cancer predisposition in 47 patients with available paired, tumor-normal material, namely bone marrow stroma cells (n = 29), postremission bone marrow (n = 17), and saliva (n = 1). These patients correspond to 2 broad AML categories with heterogeneous genetic background (AML myelodysplasia related and AML defined by differentiation) and none of them had phenotypic abnormalities, previous history of cytopenia, or strong cancer aggregation. We found 11 pathogenic or likely pathogenic variants, 6 affecting genes related to autosomal dominant cancer predisposition syndromes (ATM, DDX41, and CHEK2) and 5 related to autosomal recessive bone marrow failure syndromes (FANCA, FANCM, SBDS, DNAJC21, and CSF3R). We did not find differences in clinical characteristics nor outcome between carriers of GVs vs noncarriers. Further studies in unselected AML cohorts are needed to determine GV incidence and penetrance and, in particular, to clarify the role of ATM nonsense mutations in AML predisposition.

Systematic Collaborative Reanalysis of Genomic Data Improves Diagnostic Yield in Neurologic Rare Diseases.

Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%).

PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions.

The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.

Fine-scale population structure in five rural populations from the Spanish Eastern Pyrenees using high-coverage whole-genome sequence data.

The area of the Spanish Pyrenees is particularly interesting for studying the demographic dynamics of European rural areas given its orography, the main traditional rural condition of its population and the reported higher patterns of consanguinity of the region. Previous genetic studies suggest a gradient of genetic continuity of the area in the West to East axis. However, it has been shown that micro-population substructure can be detected when considering high-quality NGS data and using spatial explicit methods. In this work, we have analyzed the genome of 30 individuals sequenced at 40× from five different valleys in the Spanish Eastern Pyrenees (SEP) separated by less than 140 km along a west to east axis. Using haplotype-based methods and spatial analyses, we have been able to detect micro-population substructure within SEP not seen in previous studies. Linkage disequilibrium and autozygosity analyses suggest that the SEP populations show diverse demographic histories. In agreement with these results, demographic modeling by means of ABC-DL identify heterogeneity in their effective population sizes despite of their close geographic proximity, and suggests that the population substructure within SEP could have appeared around 2500 years ago. Overall, these results suggest that each rural population of the Pyrenees could represent a unique entity.

The Genetic Landscape of Inherited Retinal Diseases in a Mexican Cohort: Genes, Mutations and Phenotypes.

In this work, we aimed to provide the genetic diagnosis of a large cohort of patients affected with inherited retinal dystrophies (IRDs) from Mexico. Our data add valuable information to the genetic portrait in rare ocular diseases of Mesoamerican populations, which are mostly under-represented in genetic studies. A cohort of 144 unrelated probands with a clinical diagnosis of IRD were analyzed by next-generation sequencing using target gene panels (overall including 346 genes and 65 intronic sequences). Four unsolved cases were analyzed by whole-exome sequencing (WES). The pathogenicity of new variants was assessed by in silico prediction algorithms and classified following the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic or likely pathogenic variants were identified in 105 probands, with a final diagnostic yield of 72.9%; 17 cases (11.8%) were partially solved. Eighteen patients were clinically reclassified after a genetic diagnostic test (17.1%). In our Mexican cohort, mutations in 48 genes were found, with ABCA4, CRB1, RPGR and USH2A as the major contributors. Notably, over 50 new putatively pathogenic variants were identified. Our data highlight cases with relevant clinical and genetic features due to mutations in the RAB28 and CWC27 genes, enrich the novel mutation repertoire and expand the IRD landscape of the Mexican population.

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics.

Exome-wide rare variant analysis in familial essential tremor.

INTRODUCTION: Essential tremor (ET) is one of the most common movement disorders. Despite its high prevalence and heritability, its genetic etiology remains elusive with only a few susceptibility genes identified and poorly replicated. Our aim was to find novel candidate genes involved in ET predisposition through whole exome sequencing. METHODS: We studied eight multigenerational families (N = 40 individuals) with an autosomal-dominant inheritance using a comprehensive strategy combining whole exome sequencing followed by case-control association testing of prioritized variants in a separate cohort comprising 521 ET cases and 596 controls. We further performed gene-based burden analyses in an additional dataset comprising 789 ET patients and 770 healthy individuals to investigate whether there was an enrichment of rare deleterious variants within our candidate genes. RESULTS: Fifteen variants co-segregated with disease status in at least one of the families, among which rs749875462 in CCDC183, rs535864157 in MMP10 and rs114285050 in GPR151 showed a nominal association with ET. However, we found no significant enrichment of rare variants within these genes in cases compared with controls. Interestingly, MMP10 protein is involved in the inflammatory response to neuronal damage and has been previously associated with other neurological disorders. CONCLUSIONS: We prioritized a set of promising genes, especially MMP10, for further genetic and functional studies in ET. Our study suggests that rare deleterious coding variants that markedly increase susceptibility to ET are likely to be found in many genes. Future studies are needed to replicate and further infer biological mechanisms and potential disease causality for our identified genes.

Diagnostic value of bone marrow core biopsy patterns in lymphoplasmacytic lymphoma/Waldenström macroglobulinaemia and description of its mutational profiles by targeted NGS.

AIMS: The aim of this study was to describe the characteristics of the bone marrow infiltration found in a series of clinically defined lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinaemia (WM) and IgM-monoclonal gammopathy of undetermined significance (MGUS) and to perform a targeted next-generation sequencing (NGS) for the identification of additional somatic mutations to MYD88p.L265P in LPL/WM. METHODS: We have reviewed a series of 35 bone marrow biopsies from 28 patients with a clinical diagnosis of LPL/WM (24 cases) or MGUS (4 cases). Bone marrow infiltration characteristics by morphology, immunohistochemistry, flow cytometry (FCM), allele-specific real-time PCR for the detection of MYD88p.L265P mutation, targeted exonic amplicon-based NGS of 35 lymphoma-related genes and direct sequencing were analysed. RESULTS: Our findings show that bone marrow trephine biopsy evaluation is superior to FCM in the identification of significant lymphoid infiltrates. A combined paratrabecular and interstitial infiltration pattern is the most common feature in LPL/WM while a patchy interstitial pattern characterises IgM-MGUS cases. MYD88p.L265P mutation was found by allele-specific-PCR in 92% of the LPL cases (22 out of 24) and 25% of IgM-MGUS cases (1 out of 4 cases). In addition to MYD88p.L265P somatic mutations in CXCR4, KMT2D, PRDM1/Blimp1, MYC and ID3 were found by NGS and direct sequencing in 4 cases. CONCLUSIONS: In conclusion, bone marrow core biopsy evaluation is critical in the identification of unequivocal bone marrow infiltration by LPL/WM. In addition to MYD88p.L265P, somatic mutations in CXCR4, KMT2D, PRDM1/Blimp1, MYC and ID3 can appear in a fraction of LPL/WM.

Haemodialysis through a cellulose membrane induces dephosphorylation of CD11b and promotes leukocyte adhesion to endothelial cells.

PURPOSE: To explore modifications in signal mechanisms involving CD11b and leukocyte adhesion in patients under haemodialysis (HD). METHODS: Samples were obtained from uremic patients at baseline, 15 and 120 min of HD from both arterial and venous lines. CD11b expression was studied by flow cytometry. To study signalling mechanisms, CD11b was immunoprecipitated using a specific antibody. Immunoprecipitates were resolved by 8% SDS-PAGE to measure phosphorylation in immunoblots. Leukocyte adhesion was measured after blood perfusion using endothelial cells (EC) as adhesive substrate. Parallel studies were performed with blood from healthy donors. RESULTS: The percentage of CD11b+ cells increased during HD with a cellulose membrane in the venous line at 15 and 120 min (6.2+/-2.9% and 11.0+/-7.1%) and in the arterial line at 120 min (11.5+/-8.5 vs. 3.1+/-1.0% in control P < 0.05). After 120 min HD, CD11b phosphorylation decreased in leukocytes from both arterial (72.6+/-2.9) and venous lines (51.8+/-6.5) vs. basal samples (119.5+/-15.5 P < 0.005). Control leukocytes showed enhanced adhesion to uremic EC compared with control EC (3.0+/-0.3 vs. 2.3+/-1.0 leukocytes x100 EC(-1) P < 0.05). Uremic leukocyte adhesion was enhanced after HD compared with basal samples 4.2+/-0.2 leukocytes/100 EC in the arterial and 4.4+/-0.3 in the venous line; after 120 min vs 2.3+/-1.0 (P < 0.005). CONCLUSION: Leukocyte activation during HD through a cellulose membrane occurs with decreases in CD11b phosphorylation. Activation also induces increases in CD11b expression associated with enhanced leukocyte adhesion to uremic endothelial cells.

A de novo heterozygous missense BSCL2 variant in 2 siblings with intractable developmental and epileptic encephalopathy.

PURPOSE: We present the case of 2 siblings with profound refractory epilepsy and neurological regression that began at the ages of 3 and 6 months. Diagnosis remained elusive despite extensive metabolic and genetic workups, including use of a targeted next-generation sequencing panel for epilepsy genes. METHODS: Whole-exome sequencing was performed for the 2 siblings and their unaffected parents, in addition to fibroblast cell culture, RNA extraction and reverse-transcription, and cDNA PCR. Brain tissue from one of the siblings was collected post-mortem for neuropathological examination, including histology and immunohistochemistry. RESULTS: Ade novo nucleotide change (c.566 T > A; p.(Met189Lys)) in exon 4 of the BSCL2 gene was detected in the 2 siblings, and confirmed by Sanger sequencing. This variant was absent in the parents and in a third, unaffected sibling. CONCLUSION: Given thede novo nature of the variant, its absence from public and in-house databases, our in silico pathogenicity predictions, and co-segregation of the variant with the disease phenotype, we believe that this novel variant is associated with the epileptic encephalopathy phenotype of the 2 siblings. Our findings provide the first evidence of an association between a heterozygous BSCL2 variant and developmental and early infantile epileptic encephalopathy. Further functional studies will be needed to elucidate the pathophysiological mechanisms underlying this new BSCL2-associated phenotype.

Chromosome 12p Amplification in Triple-Negative/BRCA1-Mutated Breast Cancer Associates with Emergence of Docetaxel Resistance and Carboplatin Sensitivity.

Taxanes are the mainstay of treatment in triple-negative breast cancer (TNBC), with de novo and acquired resistance limiting patient's survival. To investigate the genetic basis of docetaxel resistance in TNBC, exome sequencing was performed on matched TNBC patient-derived xenografts (PDX) sensitive to docetaxel and their counterparts that developed resistance in vivo upon continuous drug exposure. Most mutations, small insertions/deletions, and copy number alterations detected in the initial TNBC human metastatic samples were maintained after serial passages in mice and emergence of resistance. We identified a chromosomal amplification of chr12p in a human BRCA1-mutated metastatic sample and the derived chemoresistant PDX, but not in the matched docetaxel-sensitive PDX tumor. Chr12p amplification was validated in a second pair of docetaxel-sensitive/resistant BRCA1-mutated PDXs and after short-term docetaxel treatment in several TNBC/BRCA1-mutated PDXs and cell lines, as well as during metastatic recurrence in a patient with BRCA1-mutated breast cancer who had progressed on docetaxel treatment. Analysis of clinical data indicates an association between chr12p amplification and patients with TNBC/basal-like breast cancer, a BRCA1 mutational signature, and poor survival after chemotherapy. Detection of chr12p amplification in a cohort of TNBC PDX models was associated with an improved response to carboplatin. Our findings reveal tumor clonal dynamics during chemotherapy treatments and suggest that a preexisting population harboring chr12p amplification is associated with the emergence of docetaxel resistance and carboplatin responsiveness in TNBC/BRCA1-mutated tumors. SIGNIFICANCE: Chr12p copy number gains indicate rapid emergence of resistance to docetaxel and increased sensitivity to carboplatin, therefore sequential docetaxel/carboplatin treatment could improve survival in TNBC/BRCA1 patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4258/F1.large.jpg.

Deep analysis of acquired resistance to FGFR1 inhibitor identifies MET and AKT activation and an expansion of AKT1 mutant cells.

The development of acquired resistance (AR) to tyrosine kinase inhibitors (TKIs) of FGFR1 activation is currently not well understood. To gain a deeper insight into this matter in lung cancer, we used the FGFR1-amplified DMS114 cell line and generated multiple clones with AR to an FGFR1-TKI. We molecularly scrutinized the resistant cells, using whole-exome sequencing, RNA sequencing and global DNA methylation analysis. Our results show a de novo activation of AKT and ERK, and a reactivation of mTOR. Furthermore, the resistant cells exhibited strong upregulation and activation of MET, indicating crosstalk between the FGFR1 and MET axes. The resistant cells also underwent a global decrease in promoter hypermethylation of the CpG islands. Finally, we observed clonal expansion of a pre-existing change in AKT1, leading to S266L substitution, within the kinase domain of AKT. Our results demonstrate that AR to FGFR1-TKI involves deep molecular changes that promote the activation of MET and AKT, coupled with common gene expression and DNA methylation profiles. The expansion of a substitution at AKT1 was the only shared genetic change, and this may have contributed to the AR.