Low Copy Number Variants Research Articles

Uptake of Chemicals through the Skin: An Important Role of Filaggrin Gene Variants.

Uptake of Chemicals through the Skin: An Important Role of Filaggrin Gene Variants.

Filaggrin Polymorphisms and the Uptake of Chemicals through the Skin-A Human Experimental Study.

Background:The filaggrin protein is important for skin barrier structure and function. Loss-of-function (null) mutations in the filaggrin gene FLG may increase dermal absorption of chemicals.Objective:The objective of the study was to clarify if dermal absorption of chemicals differs depending on FLG genotype.Method:We performed a quantitative real-time polymerase chain reaction (qPCR)-based genetic screen for loss-of-function mutations (FLG null) in 432 volunteers from the general population in southern Sweden and identified 28 FLG null carriers. In a dermal exposure experiment, we exposed 23 FLG null and 31 wild-type (wt) carriers to three organic compounds common in the environment: the polycyclic aromatic hydrocarbon pyrene, the pesticide pyrimethanil, and the ultraviolet-light absorber oxybenzone. We then used liquid-chromatography mass-spectrometry to measure the concentrations of these chemicals or their metabolites in the subjects’ urine over 48 h following exposure. Furthermore, we used long-range PCR to measure FLG repeat copy number variants (CNV), and we performed population toxicokinetic analysis.Results:Lag times for the uptake and dermal absorption rate of the chemicals differed significantly between FLG null and wt carriers with low (20–22 repeats) and high FLG CNV (23–24 repeats). We found a dose-dependent effect on chemical absorption with increasing lag times by increasing CNV for both pyrimethanil and pyrene, and decreasing area under the urinary excretion rate curve () with increasing CNV for pyrimethanil. FLG null carriers excreted 18% and 110% more metabolite (estimated by ) for pyrimethanil than wt carriers with low and high CNV, respectively.Conclusion:We conclude that FLG genotype influences the dermal absorption of some common chemicals. Overall, FLG null carriers were the most susceptible, with the shortest lag time and highest rate constants for skin absorption, and higher fractions of the applied dose excreted. Furthermore, our results indicate that low FLG CNV resulted in increased dermal absorption of chemicals. https://doi.org/10.1289/EHP7310

Abstract 5899: Development and characterization an NGS multi-cancer panel plus CNV detection; a single-tube, multiplex-PCR based NGS Assay with 309 tiled amplicons

Abstract Introduction: SLIMamp technology allows multiplex-PCR of tiled amplicons in a single tube, which enables targeting of large exons for NGS analysis with a streamlined process. Copy number variants (CNVs) are DNA segments that are present with variation in the number of copies compared to a normal genome. CNVs have a high prevalence in the pathogenesis of cancer and their characterization is important to acquire a more comprehensive picture of the mutations present in a patient sample. Pillar developed a proprietary CNV algorithm and combined it with SLIMamp to develop an integrated multi-cancer plus CNV detection NGS panel which identifies CNVs in the ERBB2, EGFR, MET, and MYC genes. Methods: To assess the ability of the ONCO/Reveal Multi-Cancer with CNV Panel to detect ERBB2 CNV compared to DISH (dual in situ hybridization), libraries were created from 44 well-characterized FFPE breast cancer samples plus 3 Genome in a Bottle (GIAB) samples. 23 tumor samples had known ERBB2 amplification (20 with matched normal tissue), 21 were known non-amplified tumors, and 3 GIAB samples were used as negative controls. Libraries were sequenced on an Illumina MiSeq and data was analyzed by the Pillar Variant Analysis Toolkit (PiVAT). The previous DISH results were not known to Pillar. Results were provided to an independent collaborator who unblinded the study and compared the NGS results to DISH. Results: Overall the assay performed well with a mapping rate of 99.4% ± 2.1%, on-target rate of 99.1% ± 0.4%, and coverage uniformity >0.2x mean coverage of 93.5% ± 4.1%. With respect to CNV calling, the assay and software detected 100% (21/21) of ERBB2 amplification negative samples as confirmed by DISH. For amplification positive samples, the NGS assay detected 100% (23/23) of positive samples. The software correctly identified 91% (21/23) of amplification positive samples. The two samples that were not called by PiVAT had normalized genes counts of 1.2 and 1.4, and also had the lowest DISH scores. All samples with a normalized gene count >1.5 were correctly called by PiVAT. The NGS assay was able to accurately call CNV independent of tumor cellularity with tumor content of 20%-90% not impacting the CNV call. Conclusions: The ONCO/Reveal Multi-Cancer with CNV Panel is a robust assay for the detection of CNVs, SNVs, and Indels of interest across multiple solid tumor cancer types. The workflow is streamlined, with same day loading of finished libraries when starting from as little as 5ng of isolated input DNA. The assay and software demonstrate detection of low CNV with the recommended cutoff set at 1.5. With further optimization of the calling algorithm accurate calling of even lower CNVs may be possible. The detection and identification of CNVs along with other mutations gives a more comprehensive overview of the mutations present in a sample, supporting better clinical management of patients. Citation Format: Nicholas Lodato, Zonghan Liu, Akuah Kontor, Xiaoxi Wu, Lukas Hillmer, Sean Polvino, Yue Ke, Erin Petrilli, Adam Labonte, Geoffrey Richman, Zhaohui Wang. Development and characterization an NGS multi-cancer panel plus CNV detection; a single-tube, multiplex-PCR based NGS Assay with 309 tiled amplicons [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5899.

FunVar: A systematic pipeline to unravel the convergence patterns of genetic variants in ASD, a paradigmatic complex disease.

In recent years, the technological advances for capturing genetic variation in large populations led to the identification of large numbers of putative or disease-causing variants. However, their mechanistic understanding is lagging far behind and has posed new challenges regarding their relevance for disease phenotypes, particularly for common complex disorders. In this study, we propose a systematic pipeline to infer biological meaning from genetic variants, namely rare Copy Number Variants (CNVs). The pipeline consists of three modules that seek to (1) improve genetic data quality by excluding low confidence CNVs, (2) identify disrupted biological processes, and (3) aggregate similar enriched biological processes terms using semantic similarity. The proposed pipeline was applied to CNVs from individuals diagnosed with Autism Spectrum Disorder (ASD). We found that rare CNVs disrupting brain expressed genes dysregulated a wide range of biological processes, such as nervous system development and protein polyubiquitination. The disrupted biological processes identified in ASD patients were in accordance with previous findings. This coherence with literature indicates the feasibility of the proposed pipeline in interpreting the biological role of genetic variants in complex disease development. The suggested pipeline is easily adjustable at each step and its independence from any specific dataset and software makes it an effective tool in analyzing existing genetic resources. The FunVar pipeline is available at https://github.com/lasigeBioTM/FunVar and includes pre and post processing steps to effectively interpret biological mechanisms of putative disease causing genetic variants.

Structural and copy number chromosome abnormalities in canine cutaneous mast cell tumours

Mast cell tumours (MCTs) are the most common skin tumours in dogs. Their clinical behaviour is variable and their aetiology remains largely unknown. We performed a metaphase fluorescence in situ hybridisation (FISH) with whole chromosome painting probes, and interphase FISH with BAC probes for 14 cancer-related genes to reveal clonal structural chromosome rearrangements and copy number variants (CNVs) in canine cutaneous MCTs. The metaphase FISH performed in three MCTs revealed several clonal monosomies and trisomies and two different chromosome rearrangements. No centric fusions were detected. The interphase FISH showed a variety of low frequency CNVs for the individual cancer-related genes. The heterogeneous character of the detected abnormalities indicates increased chromosome instability in canine MCTs. The clonal gain of chromosome 11 was detected in 81% (13/16) of the MCTs. Further research is needed to evaluate the significance of this abnormality as prognostic factor for the survival time or recurrence risk assessments in canine cutaneous MCTs.

Copy number variants and genetic polymorphisms in TBX21, GATA3, Rorc, Foxp3 and susceptibility to Behcet's disease and Vogt-Koyanagi-Harada syndrome.

This study aimed to investigate the role of genetic variants including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) of TBX21, GATA3, Rorc and Foxp3 genes in Behcet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. Genotyping of 25 SNPs was performed by iPLEX system (Sequenom) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). TaqMan real time PCR was used to assess CNVs. The expression of Rorc and Foxp3 were examined by real-time PCR and cytokine production was measured by ELISA. High Rorc CNV was associated with the susceptibility to BD (P = 8.99 × 10−8, OR = 3.0), and low Foxp3 CNV predisposed to BD in female patients (P = 1.92 × 10−5, OR = 3.1). CNVs for the investigated genes were not altered in VKH syndrome. Further functional studies demonstrated that the relative mRNA expression levels of Rorc were increased in individuals with high Rorc copy number, but not for Foxp3. Increased production of IL-1β and IL-6 was found in individuals carrying a high CNV of Rorc. Our study showed that high CNVs of Rorc and low CNVs of Foxp3 confer risk for BD but not for VKH syndrome. The tested 25 SNPs in TBX21, GATA3, Rorc and Foxp3 did not associate with BD and VKH syndrome.

Large transcription units unify copy number variants and common fragile sites arising under replication stress.

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics.

Association of common copy number variants at the glutathione S-transferase genes and rare novel genomic changes with schizophrenia

Copy number variants (CNVs) are a substantial source of human genetic diversity, influencing the variable susceptibility to multifactorial disorders. Schizophrenia is a complex illness thought to be caused by a number of genetic and environmental effects, few of which have been clearly defined. Recent reports have found several low prevalent CNVs associated with the disease. We have used a multiplex ligation-dependent probe amplification-based (MLPA) method to target 140 previously reported and putatively relevant gene-containing CNV regions in 654 schizophrenic patients and 604 controls for association studies. Most genotyped CNVs (95%) showed very low (<1%) population frequency. A few novel rare variants were only present in patients suggesting a possible pathogenic involvement, including 1.39 Mb overlapping duplications at 22q11.23 found in two unrelated patients, and duplications of the somatostatin receptor 5 gene (SSTR5) at 16p13.3 in three unrelated patients. Furthermore, among the few relatively common CNVs observed in patients and controls, the combined analysis of gene copy number genotypes at two glutathione S-transferase (GST) genes, GSTM1 (glutathione S-transferase mu 1) (1p13.3) and GSTT2 (glutathione S-transferase theta 2) (22q11.23), showed a statistically significant association of non-null genotypes at both loci with an additive effect for increased vulnerability to schizophrenia (odds ratio of 1.92; P=0.0008). Our data provide complementary evidences for low prevalent, but highly penetrant chromosomal variants associated with schizophrenia, as well as for common CNVs that may act as susceptibility factors by disturbing glutathione metabolism.