Unknown Variants Research Articles

Identifying novel regulatory effects for clinically relevant genes through the study of the Greek population

BackgroundExpression quantitative trait loci (eQTL) studies provide insights into regulatory mechanisms underlying disease risk. Expanding studies of gene regulation to underexplored populations and to medically relevant tissues offers potential to reveal yet unknown regulatory variants and to better understand disease mechanisms. Here, we performed eQTL mapping in subcutaneous (S) and visceral (V) adipose tissue from 106 Greek individuals (Greek Metabolic study, GM) and compared our findings to those from the Genotype-Tissue Expression (GTEx) resource.ResultsWe identified 1,930 and 1,515 eGenes in S and V respectively, over 13% of which are not observed in GTEx adipose tissue, and that do not arise due to different ancestry. We report additional context-specific regulatory effects in genes of clinical interest (e.g. oncogene ST7) and in genes regulating responses to environmental stimuli (e.g. MIR21, SNX33). We suggest that a fraction of the reported differences across populations is due to environmental effects on gene expression, driving context-specific eQTLs, and suggest that environmental effects can determine the penetrance of disease variants thus shaping disease risk. We report that over half of GM eQTLs colocalize with GWAS SNPs and of these colocalizations 41% are not detected in GTEx. We also highlight the clinical relevance of S adipose tissue by revealing that inflammatory processes are upregulated in individuals with obesity, not only in V, but also in S tissue.ConclusionsBy focusing on an understudied population, our results provide further candidate genes for investigation regarding their role in adipose tissue biology and their contribution to disease risk and pathogenesis.

Anthrenus (s. str.) semipallens sp. nov., a new species from Spain (Coleoptera: Dermestidae: Anthreninae).

During an examination of Spanish Anthrenus spp. held in Andreas Herrmann's private collection, four specimens of a new species were noted: Anthrenus (Anthrenus) semipallens. Images of habitus features, including antenna, are presented and compared with other Anthrenus species thought to occur in Spain. A. semipallens is small so some comparison species could be eliminated courtesy of size. Although A. semipallens doesn't resemble the colour pattern of any other species, the possibility that A. semipallens is an unknown colour variant of a comparison species was considered. The A. semipallens specimens were dissected and the aedeagus compared with aedeagi from all other possible species. There was no similarity. Anthrenus semipallens is a valid new species.

Use of whole exome sequencing for identification of genetic variants related to Growth Hormone Deficiency and Short Stature: A Family-Based Study.

Genetic polymorphisms in genes involved in growth process and Vitamin-D metabolism form a significant etiology behind growth hormone deficiency and short stature. The aim of this study was to explore for known and unknown genes and variants related to growth hormone and short stature in a family based study using whole exome sequencing (WES). This family-based study included a family with members diagnosed with growth hormone deficiency, short stature and Vitamin-D deficiency (four boys affected and four boys non-affected). The participants were recruited from King Abdulaziz University Hospital (Jeddah, Saudi Arabia) and referred to King Fahad Centre for Medical Research (Jeddah, Saudi Arabia from April 2022 to June 2022. The consanguineous parents and one of the affected boys (aged 16 years old) underwent WES. Several variants in RNPC3, ACAN, GC, VDR and LRP2 were identified in index cases but not in controls. Novel frameshift and splice region variants in RNPC3 (c.358dupA, p.Arg120fs) were detected. Other missense variants were also observed including variants in ACAN (c.2591C>T, c.2789G>T, c.2815T>A, c.4207A>G, c.4523A>C and c.7119C>G), GC (rs4588 and rs7041) and LRP2 (rs2075252 and rs1991517). A start loss variant in VDR (rs2228570) with high impact was also observed. Our findings suggest a potential association of these variants with growth hormone deficiency and short stature. In this study, novel pathogenic variants in RNPC3 were revealed as well as other variants in ACAN and in genes related to Vitamin-D metabolism (GC, VDR and LRP2) that some or all might be associated with growth hormone deficiency. Further large-scale studies are required to address the association of these variants with growth hormone deficiency and its subsequent short stature.

Are DDX41 variants of unknown significance and pathogenic variants created equal?

Not available.

A novel SLC25A13 gene splice site variant causes Citrin deficiency in an infant

Citrin deficiency is an autosomal recessive disorder associated with SLC25A13 gene pathogenic variants, with more than a hundred known at present. It manifests in neonates as failure to thrive and acute liver insufficiency. We herein describe a case of a 4-week-old infant who presented with insufficient weight gain and liver failure accompanied by hyperammonemia. She was diagnosed with Citrin deficiency after a thorough biochemical and molecular analysis including amino acid profile, DNA sequencing of genes of interest and RNA splice site evaluation, to reveal a yet unknown damaging variant of the SLC25A13 gene.

Clinical features and magnesium levels: Novel insights in 15q11.2 BP1-BP2 copy number variants.

Investigating copy number variations (CNVs) such as microdeletions or microduplications can significantly contribute to discover the aetiology of neurodevelopmental disorders. 15q11.2 genomic region, including NIPA1 and NIPA2 genes, contains a recurrent but rare CNV, flanked by the break points BP1 and BP2. Both BP1-BP2 microdeletion and microduplication have been associated with intellectual disability (ID), neuropsychiatric/behavioural disturbances and mild clinical features, even if with incomplete penetrance and variable expressivity. The pathogenic role of this CNV is quite unclear though. Unknown variants in other DNA regions and parent-of-origin effect (POE) are some of the mechanisms that have been proposed as an explanation of the wide phenotypic variability. As NIPA1 and NIPA2 encode for proteins that mediate magnesium (Mg2+ ) metabolism, it has been suggested that urinary Mg2+ levels could potentially represent informative and affordable biomarkers for a rapid screening of 15q11.2 duplications or deletions. Furthermore, magnesium supplementation has been proposed as possible therapeutic strategy. Thirty one children with ID and/or other neurodevelopmental disorders carrying either a duplication or a deletion in 15q11.2 BP1-BP2 region have been recruited. When available, blood samples from parents have been analysed to identify the CNV origin. All participants underwent family and medical data collection, physical examination and neuropsychiatric assessment. Electroencephalogram (EEG) and brain magnetic resonance imaging (MRI) scan were performed in 15 children. In addition, 11 families agreed to participate to the assessment of blood and urinary Mg2+ levels. We observed a highly variable phenotypic spectrum of developmental issues encompassing ID in most subjects as well as a variety of behavioural disorders such as autism and attention-deficit disorder/attention-deficit hyperactivity disorder. Dysmorphic traits and malformations were detected only in a minority of the participants, and no clear association with growth anomalies was found. Abnormal brain MRI and/or EEG were reported respectively in 64% and 92% of the subjects. Inheritance assessment highlighted an excess of duplication of maternal origin, while cardiac alterations were detected only in children with 15q11.2 CNV inherited from the father. We found great variability in Mg2+ urinary values, without correlation with 15q11.2 copy numbers. However, the variance of urinary Mg2+ levels largely increases in individuals with 15q11.2 deletion/duplication. This study provides further evidence that 15q11.2 BP1-BP2 CNV is associated with a broad spectrum of neurodevelopmental disorders and POE might be an explanation for clinical variability. However, some issues may question the real impact of 15q11.2 CNV on the phenotype in the carriers: DNA sequencing could be useful to exclude other pathogenic gene mutations. Our results do not support the possibility that urinary Mg2+ levels can be used as biomarkers to screen children with neurodevelopmental disorders for 15q11.2 duplication/deletion. However, there are evidences of correlations between 15q11.2 BP1-BP2 CNV and Mg2+ metabolism and future studies may pave the way to new therapeutic options.

A Case of Pantothenate Kinase-Associated Neurodegeneration (PKAN) in a Patient with One Known Pathologic Variant and One Variant of Unknown Significance (P11-4.006)

<h3>Objective:</h3> Raise interest in the possibility of an unidentified PANK2 gene mutation in the pathogenesis of PKAN <h3>Background:</h3> Pantothenate kinase-associated neurodegeneration (PKAN), previously known as Hallervorden-Spatz Disease, is a neurodegenerative disorder associated with brain iron deposition. PKAN follows an autosomal recessive inheritance pattern and presents in childhood (classical) or around the second decade of life (atypical). Individuals with PKAN typically present with dysarthria, dystonia, speech difficulties, and extrapyramidal symptoms. PKAN has a characteristic “eye of the tiger” sign on MRI with hypointensity of the globus pallidus surrounded by an area of hyperintensity. Pantothenate kinase 2 (PANK2) gene sequencing changes have been identified as markers of PKAN. <h3>Case Description:</h3> The patient discussed is a 27-year-old woman who presented in 2013 for abnormal posturing and cramping of her hands and feet since she was four. Based on the patient’s physical exam, it was believed she was suffering from young-onset Parkinson’s disease or dystonia. However, a trial of Levodopa resulted in no improvement, genetic testing for dystonia was negative, and her brain MRI was read as normal. Due to worsening symptoms, her MRI was reassessed, and the subtle “eye of the tiger” sign was recognized. Further genetic testing showed she was a carrier for two heterozygous PANK2 mutations, one known pathologic variant and one unknown. The patient elected to join a clinical trial as her symptoms progressed. Nine years since presenting, she has completed two clinical trials and is currently on Deferiprone (an iron chelator), Artane, and Baclofen for symptom management. <h3>Design/Methods:</h3> NA <h3>Results:</h3> NA <h3>Conclusions:</h3> This report discusses a patient with PKAN who has one PANK2 mutation of known significance and one unknown variant. Considering PKAN is an autosomal recessive disease, this report suggests a novel PANK2 pathogenic variant. This study also demonstrates how the diagnosis of PKAN can be easily missed due to subtle MRI changes. <b>Disclosure:</b> Dr. Cevallos has nothing to disclose. Miss Rubin has nothing to disclose. Miss Rhinehart has nothing to disclose. Dr. Houghton has nothing to disclose.

Abstract 2058: Identifications of novel variants using system biology-based approaches in NT5C2, its association to relapse acute lymphoblastic leukaemia

Abstract Acute Lymphoblastic Leukaemia (ALL) patients experience relapse despite having a good prognosis; approximately 90% of children and 75% to 85% of adolescents and young adults survive for five years. Chemotherapy resistance and a poor prognosis are linked to relapsed acute lymphoblastic Leukaemia (R-ALL). Evidence suggests that the cytosolic 5 nucleotidase II (NT5C2) gene mutations play a crucial role in the emergence of recurrent ALL; The early R-ALL is usually accompanied by 6-MP resistance, which is typically brought on by gain-of-function mutations impacting NT5C2-driven mutation in relapse mutations by different mechanisms is still unknown. The study aims to identify novel top missense variants of NT5C2 that play a crucial role in the resistance of chemotherapy drugs, altered biological pathways and their impact on the treatment of relapse leukaemia. NT5C2 (5'-Nucleotidase, Cytosolic II) is a Protein Coding gene. This gene induces resistance to chemotherapeutic drug 6 mercaptopurine Which causes leukaemia cell growth and leukaemia-initiating cell activity. The NT5C2 gene variants are extracted from Genome Aggregation Database (gnomAD) and ExAC. The extracted data contained 2280 variants with chromosome number, position reference allele, and altered allele and these were functionally annotated using the ANNOVAR tool. The variants are screened through various computational tools to narrow down based on non-synonymous and stopgain mutations in the exonic region. The predicted 519 variants narrow down by scoring GERP (&amp;gt;2.0) and CADD PHRED (cut-off score &amp;gt;10), ClinVar, SIFT score, Polyphen, PhyloP, PhastCons, LRT score predictions and which indicates the top 10% deleterious variants including reported and unknown variants. The SNPs, insertions, deletions, CNVs or structural variants of top missense variants on genes, transcripts, and protein sequence determined on Variants Effect Predictor on the ensemble. These variants were verified on dbSNP and Clinvar through their variant IDs. The functional prediction of these variants was made to detect their significant role in R-ALL. These findings examine the future role of NT5C2 mutations in the chemoresistance of relapse ALL. This may help to find a new therapeutic combination of drugs to treat relapse acute lymphoblastic leukaemia to improve overall survival. Citation Format: Sugunakar V, Obul Reddy Bandapalli, Ramita Sharma. Identifications of novel variants using system biology-based approaches in NT5C2, its association to relapse acute lymphoblastic leukaemia [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 2058.

Alpha-Synuclein mRNA Level Found Dependent on L444P Variant in Carriers and Gaucher Disease Patients on Enzyme Replacement Therapy.

Gaucher disease (GD) is the most frequent sphingolipidosis, caused by biallelic pathogenic variants in the GBA1 gene encoding for β-glucocerebrosidase (GCase, E.C. 3.2.1.45). The condition is characterized by hepatosplenomegaly, hematological abnormalities, and bone disease in both non-neuronopathic type 1 (GD1) and neuronopathic type 3 (GD3). Interestingly, GBA1 variants were found to be one of the most important risk factors for the development of Parkinson's disease (PD) in GD1 patients. We performed a comprehensive study regarding the two most disease-specific biomarkers, glucosylsphingosine (Lyso-Gb1) and α-synuclein for GD and PD, respectively. A total of 65 patients with GD treated with ERT (47 GD1 patients and 18 GD3 patients), 19 GBA1 pathogenic variant carriers (including 10 L444P carriers), and 16 healthy subjects were involved in the study. Lyso-Gb1 was assessed by dried blood spot testing. The level of α-synuclein as an mRNA transcript, total, and oligomer protein concentration were measured with real-time PCR and ELISA, respectively. α-synuclein mRNA level was found significantly elevated in GD3 patients and L444P carriers. GD1 patients, along with GBA1 carriers of an unknown or unconfirmed variant, as well as healthy controls, have the same low level of α-synuclein mRNA. There was no correlation found between the level of α-synuclein mRNA and age in GD patients treated with ERT, whereas there was a positive correlation in L444P carriers.

Detecting impaired muscle relaxation in myopathies with the use of motor cortical stimulation

Impaired muscle relaxation is a notable feature in specific myopathies. Transcranial magnetic stimulation (TMS) of the motor cortex can induce muscle relaxation by abruptly halting corticospinal drive. Our aim was to quantify muscle relaxation using TMS in different myopathies with symptoms of muscle stiffness, contractures/cramps, and myalgia and explore the technique's diagnostic potential. In men, normalized peak relaxation rate was lower in Brody disease (n = 4) (-3.5 ± 1.3 s-1), nemaline myopathy type 6 (NEM6; n = 5) (-7.5 ± 1.0 s-1), and myotonic dystrophy type 2 (DM2; n = 5) (-10.2 ± 2.0 s-1) compared to healthy (n = 14) (-13.7 ± 2.1 s-1; all P ≤ 0.01) and symptomatic controls (n = 9) (-13.7 ± 1.6 s-1; all P ≤ 0.02). In women, NEM6 (n = 5) (-5.7 ± 2.1 s-1) and McArdle patients (n = 4) (-6.6 ± 1.4 s-1) had lower relaxation rate compared to healthy (n = 10) (-11.7 ± 1.6 s-1; both P ≤ 0.002) and symptomatic controls (n = 8) (-11.3 ± 1.8 s-1; both P ≤ 0.008). TMS-induced muscle relaxation achieved a high level of diagnostic accuracy (area under the curve = 0.94 (M) and 0.92 (F)) to differentiate symptomatic controls from myopathy patients. Muscle relaxation assessed using TMS has the potential to serve as a diagnostic tool, an in-vivo functional test to confirm the pathogenicity of unknown variants, an outcome measure in clinical trials, and monitor disease progression.

MEFV gene mutation spectrum in patients with familial mediterranean fever

MEFV gene mutation spectrum in patients with familial mediterranean fever

Novel pedestrian navigation system based on zero velocity update procedure technology and improved Sage‐Husa adaptive Kalman filter with index fading memory factor

AbstractTo acquire an accurate location on the occasions, such as in an indoor, tunnel, and valley, where satellite navigation signals fail. The paper designs a pedestrian navigation system by using the zero velocity update procedure technology (ZUPT) and Kalman filter to reduce the location error. The measurement noise characteristic (mean and variance) of the micro electro mechanical systems gyros is unknown and time variant, but in traditional studies, it is usually thought and calculated as a constant. So the optimality of the error estimation of the Kalman filter cannot be reached. To address this question, this paper proposes the improved Sage–Husa Adaptive Kalman Filter (SHAKF) based on the index fading memory factor to realise the state estimation of the Kalman filter and navigation error correction. The advantage of improved SHAKF is it can accurately estimate the state vector when the measurement noise is unknown and time variant. To verify the validity of novel navigation methods, walking experiments under outdoor environments and indoor environments are carried out. The results of actual walking experiments demonstrate that the proposed method can effectively reduce the pedestrian location error compared with the traditional ZUPT method. The mean location error is reduced by more than 10%, and the variance of the location error is reduced by more than 5%.

Next-generation sequencing of 12 obesity genes in a Portuguese cohort of patients with overweight and obesity

We examined 12 monogenic obesity genes in 72 Portuguese individuals with overweight and obesity (class 1 and class 2), some of which with suspected genetic obesity, to identify known or unknown potential obesity variants. Genomic DNA was analyzed for variants in genes LEP, LEPR, MC4R, POMC, PCSK1, BDNF, NTRK2, SIM1, SH2B1, UCP3, GCG and ADCY3 through next generation sequencing (NGS). The impact of the rare variants was investigated in the ClinVar database and using in silico tools for prediction of pathogenicity. Four potential pathogenic missense variants were detected at the heterozygous state in five individuals: two in the ADCY3 gene, NM_004036.5:c.1153G > A (p.Val385Ile) (rs756783003) and NM_004036.5:c.1222G > A (p.Gly408Arg) (rs201606553), one in gene SH2B1, NM_001145795.1:c.127C > A (p.Arg43Ser) (rs547678855), and the fourth in gene POMC NM_000939.4:c.706C > G (p.Arg236Gly) (rs28932472), which was found in two individuals. Moreover, six rare variants near splicing sites were also identified, as well as eight rare synonymous variants. In summary, some potential pathogenic rare missense variants were identified, two of them in ADCY3 gene, the most recently identified gene as having a role in monogenic obesity. Further analysis should be performed to confirm the clinical relevance of these variants.

Novel Multidimensional Liquid Chromatography Workflow with In-Loop Enzymatic Digests of Multiple Heart-Cuts for Fast and Flexible Characterization of Biotherapeutic Protein Variants

Multidimensional liquid chromatography (mD-LC) is becoming a powerful tool for complete characterization of individual peaks and protein variants through separation methods such as nondenaturing ion exchange (IEC) or size-exclusion chromatography coupled to reversed-phase (RP) chromatography. The flexibility of commercially available and customized mD-LC systems is still limited in terms of enzymatic peak processing between chromatographic dimensions. In this regard, only a few column-immobilized proteases are available for detailed peak characterization by mD-LC coupled to mass spectrometry (mD-LC-MS). Here, we present a purpose-built and automated multiple heart-cutting mD-LC design with a novel analytical workflow involving in-loop enzymatic heart-cut digestion between the first-dimensional column and transfer to the second dimension before MS or MS/MS analyses. The setup facilitates the spike-in of any enzyme to multiple heart-cuts for multilevel analysis, for example, for peptide mapping, fragment generation, or deglycosylation, to reduce heterogeneity and provide maximum flexibility in terms of incubation time for optimal peak characterization. We demonstrate the application of IEC coupled to RP-LC-MS and automated in-loop deglycosylation and on-column reduction of an IgG antibody combined with upper hinge region cleavage for Fab generation. We further employ mD-LC-MS and mD-LC-MS/MS to assess post-translational modifications of a bispecific antibody and to support molecule selection by evaluating the best downstream purification strategy. The novel design and automated workflow of the mD-LC system described here offers enhanced flexibility for in-solution processing and real-time monitoring of multiple heart-cuts enabling streamlined characterization of unknown biotherapeutic charge and size variants.

A Real-Time Hybrid Approach to Combat In-Browser Cryptojacking Malware

Cryptojacking is a type of computer piracy in which a hacker uses a victim’s computer resources, without their knowledge or consent, to mine for cryptocurrency. This is made possible by new memory-based cryptomining techniques and the growth of new web technologies such as WebAssembly, allowing mining to occur within a browser. Most of the research in the field of cryptojacking has focused on detection methods rather than prevention methods. Some of the detection methods proposed in the literature include using static and dynamic features of in-browser cryptojacking malware, along with machine learning algorithms such as Support Vector Machine (SVM), Random Forest (RF), and others. However, these methods can be effective in detecting known cryptojacking malware, but they may not be able to detect new or unknown variants. The existing prevention methods are shown to be effective only against web-assembly (WASM)-based cryptojacking malware and cannot handle mining service-providing scripts that use non-WASM modules. This paper proposes a novel hybrid approach for detecting and preventing web-based cryptojacking. The proposed approach performs the real-time detection and prevention of in-browser cryptojacking malware, using the blacklisting technique and statistical code analysis to identify unique features of non-WASM cryptojacking malware. The experimental results show positive performances in the ease of use and efficiency, with the detection accuracy improved from 97% to 99.6%. Moreover, the time required to prevent already known malware in real time can be decreased by 99.8%.

Optimized Detection of Unknown MET Exon 14 Skipping Mutations in Routine Testing for Patients With Non-Small-Cell Lung Cancer.

MET exon 14 (METex14) skipping is an actionable biomarker in non-small-cell lung cancer. However, MET variants are highly complex and diverse, and not all variants lead to exon 14 skipping. Assessing the skipping effect of unknown variants is still a key issue in molecular diagnosis. We retrospectively collected MET variants around exon 14 from 4,233 patients with non-small-cell lung cancer who underwent next-generation sequencing testing using DNA, as well as two published data sets. Among the 4,233 patients, 44 unique variants including 29 novel variants (65.9%) were discovered from 53 patients. Notably, 31 samples (58.5%) failed RNA verification. Using RNA verification, nine novel skipping variants and five nonskipping variants were confirmed. We further used SpliceAI with the delta score cutoff of 0.315 to aid the classification of novel variants (sensitivity = 98.88% and specificity = 100%). When applied to the reported variants, we also found three wrongly classified nonskipping variants. Finally, an optimized knowledge-based interpretation procedure for clinical routine was built according to the mutation type and location, and five more skipping mutations from the 13 unknown variants were determined, which improved the population determination rate to 0.92%. This study discovered more METex14 skipping variants and optimized an innovative approach that could be adapted for the interpretation of infrequent or novel METex14 variants timely without experimental validation.

P905 Predictive pharmacogenetic risk of pancreatitis in Inflammatory Bowel Disease patients treated with thiopurines: a case-control study from the ENEIDA registry

Abstract Background Treatment with thiopurines may be associated with different adverse effects, including acute pancreatitis. Risk factors for developing pancreatitis due to thiopurines in patients with Inflammatory Bowel Disease (IBD) are not clearly identified, with underlying genetic predisposition being a possible cause. Our aim was to evaluate predictive pharmacogenetic risk of pancreatitis in IBD patients treated with thiopurines. Methods IBD patients treated with thiopurines were identified from the prospectively maintained ENEIDA registry of the Spanish Working Group on Crohn’s Disease and Ulcerative Colitis (GETECCU). We included as cases those patients who met the Atlanta diagnostic criteria for pancreatitis and had an imaging test that ruled out biliary origin. We included as controls those patients without pancreatitis after at least two years of treatment with thiopurines. Blood samples were collected, and DNA was extracted from leukocytes for all participants and, subsequently, pooled samples were sequenced. Sequencing of a panel of genes (CASR, CEL, CDLN2, CFTR, CPA1, CTRC, PRSS1, and SPINK1) was carried out in the NextSeq500 platform. Variants detected were classified as pathogenic, variants of unknown significance, and benign according to the American College of Medical Genetics and Genomics (ACMGG) guidelines. Results Ninety-five cases and 105 controls were enrolled, 57% were women. Pancreatitis was diagnosed at a median age of 39±13 years old. No patient had previous history of chronic or acute pancreatitis. There were no differences between thiopurine-treated patients with and without pancreatitis in age, sex, and age at IBD onset. We identified 81 benign variants (50 in cases and 67 in controls) and a total of 35 distinct rare pathogenic and unknown significance variants (10 in CEL, 21 in CFTR, 1 in CDLN2, and 3 in CPA1). Of these 35 variants (12 in cases and 18 in controls), 6 were classified as pathogenic (1 in cases and 5 in controls), belonging to the CEL and CFTR genes, and 30 as variants of uncertain significance (21 in cases and 22 in controls). There were no significant differences between the groups of cases and controls, both in totals and by genes. None of the cases or controls carried pancreatitis-predisposing variants within the CASR, CPA1, PRSS1, and SPINK1 genes. None of the analysed samples with pancreatitis carried a pathogenic CFTR mutation. Four different variants of unknown significance were detected in the CDLN and CPA1 genes. One of them was in the CDLN gene in a single patient with pancreatitis, and 3 variants in the CPA1 gene in 5 control individuals. Conclusion In patients with IBD, there is no predisposition to thiopurine-induced pancreatitis associated with genes known to cause pancreatitis.

Analysis of mutational history of multidrug-resistantgenotypes with a mutagenetic tree model.

Human immunodeficiency virus (HIV) can develop resistance to all antiretroviral drugs. Multidrug resistance, however, is a rare event in modern HIV treatment, but can be life-threatening, particular in patients with very long therapy histories and in areas with limited access to novel drugs. To understand the evolution of multidrug resistance, we analyzed the EuResist database to uncover the accumulation of mutations over time. We hypothesize that the accumulation of resistance mutations is not acquired simultaneously and randomly across viral genotypes but rather tends to follow a predetermined order. The knowledge of this order might help to elucidate potential mechanisms of multidrug resistance.Our evolutionary model shows an almost monotonic increase of resistance with each acquired mutation, including less well-known nucleosidereverse transcriptase (RT) inhibitor-related mutations like K223Q, L228H, and Q242H. Mutations within the integrase (IN) (T97A, E138A/K G140S, Q148H, N155H) indicate high probability of multidrug resistance. Hence, these IN mutations also tend to be observed together with mutations in the protease (PR) and RT. We followed up with an analysis of the mutation-specific error rates of our model given the data. We identified several mutations with unusual rates (PR: M41L, L33F, IN: G140S). This could imply the existence of previously unknown virus variants in the viral quasispecies. In conclusion, our bioinformatics model supports the analysis and understanding of multidrug resistance.

A Computational Framework for Comprehensive Genomic Profiling in Solid Cancers: The Analytical Performance of a High-Throughput Assay for Small and Copy Number Variants.

In January 2022, our institution launched a comprehensive cancer genome profiling program on 10 cancer types using a non-IVD solution named the TruSight Oncology 500 Assay provided by Illumina®. The assay analyzes both DNA and RNA, identifying Single-Nucleotide Variants (SNV)s and Insertion-Deletion (InDel) in 523 genes, as well as known and unknown fusions and splicing variants in 55 genes and Copy Number Alterations (CNVs), Mutational Tumor Burden (MTB) and Microsatellite Instability (MSI). According to the current European IVD Directive 98/79/EC, an internal validation was performed before running the test. A dedicated open-source bioinformatics pipeline was developed for data postprocessing, panel assessment and embedding in high-performance computing framework using the container technology to ensure scalability and reproducibility. Our protocols, applied to 71 DNA and 64 RNA samples, showed full agreement between the TruSight Oncology 500 assay and standard approaches, with only minor limitations, allowing to routinely perform our protocol in patient screening.

Molecular cytology: Opportunities and challenges

Predictive marker (re-)analysis of tumor material can be areal obstacle in several tumor entities, like non-small cell lung cancer (NSCLC), due to difficult anatomic conditions and small biopsy samples. As reported in the literature, cytological samples comprise excellent starting material for predictive marker analysis like fluorescence in situ hybridization and next generation sequencing. As for formalin-fixed paraffin-embedded tissue samples, rigorous quality control and standardized laboratory operating procedures are mandatory. Further advantages of cytological specimens are the rapid and straightforward inspection of representativeness, for example by rapid on-site evaluation (ROSE). Another striking advantage is that the fresh cellular material from smears and serous cavity fluids can be used for the generation of two- and three-dimensional cell culture models. Hereby, in addition to the conventional biomarker testing, complex complementary functional genomic assays can also be applied, for example, to assess the effects of multiple variants in one sample and unknown variants of tumor driver genes and tumor suppressor genes. This information may provide additional vulnerabilities of the tumor to be considered for the therapy decision, for example in the molecular tumor board.