Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis.

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

Challenges and approaches to calibrating patient phenotype as evidence for cancer gene variant classification under ACMG/AMP guidelines.

Since first publication of the American College of Medical Genetics and Genomics/Association for Medical Pathology (ACMG/AMP) variant classification guidelines, additional recommendations for application of certain criteria have been released (https://clinicalgenome.org/docs/), to improve their application in the diagnostic setting. However, none have addressed use of the PS4 and PP4 criteria, capturing patient presentation as evidence towards pathogenicity. Application of PS4 can be done through traditional case-control studies, or "proband counting" within or across clinical testing cohorts. Review of the existing PS4 and PP4 specifications for Hereditary Cancer Gene Variant Curation Expert Panels revealed substantial differences in the approach to defining specifications. Using BRCA1, BRCA2 and TP53 as exemplar genes, we calibrated different methods proposed for applying the "PS4 proband counting" criterion. For each approach, we considered limitations, non-independence with other ACMG/AMP criteria, broader applicability, and variability in results for different datasets. Our findings highlight inherent overlap of proband-counting methods with ACMG/AMP frequency codes, and the importance of calibration to derive dataset-specific code weights that can account for potential between-dataset differences in ascertainment and other factors. Our work emphasizes the advantages and generalizability of logistic regression analysis over simple proband-counting approaches to empirically determine the relative predictive capacity and weight of various personal clinical features in the context of multigene panel testing, for improved variant interpretation. We also provide a general protocol, including instructions for data formatting and a web-server for analysis of personal history parameters, to facilitate dataset-specific calibration analyses required to use such data for germline variant classification.

Germline variants of DNA repair and immune genes in lymphoma from lymphoma-cancer families.

The genetic predisposition to lymphoma is not fully understood. We identified 13 lymphoma-cancer families (2011-2021), in which 27 individuals developed lymphomas and 26 individuals had cancers. Notably, male is the predominant gender in lymphoma patients, whereas female is the predominant gender in cancer patients (p = .019; OR = 4.72, 95% CI, 1.30-14.33). We collected samples from 18 lymphoma patients, and detected germline variants through exome sequencing. We found that germline protein truncating variants (PTVs) were enriched in DNA repair and immune genes. Totally, we identified 31 heterozygous germline mutations (including 12 PTVs) of 25 DNA repair genes and 19 heterozygous germline variants (including 7 PTVs) of 14 immune genes. PTVs of ATM and PNKP were found in two families, respectively. We performed whole genome sequencing of diffuse large B cell lymphomas (DLBCLs), translocations at IGH locus and activation of oncogenes (BCL6 and MYC) were verified, and homologous recombination deficiency was detected. In DLBCLs with germline PTVs of ATM, deletion and insertion in CD58 were further revealed. Thus, in lymphoma-cancer families, we identified germline defects of both DNA repair and immune genes in lymphoma patients.

Germline genetic variants in Turkish familial multiple myeloma/monoclonal gammopathy of undetermined significance cases.

Multiple myeloma (MM) is a haematological malignancy primarily affecting the elderly, with a striking male predilection and ethnic disparities in incidence. Familial predisposition to MM has long been recognized, but the genetic underpinnings remain elusive. This study aimed to investigate germline variants in Turkish families with recurrent MM cases. A total of 37 MM-affected families, comprising 77 individuals, were included. Targeted next-generation sequencing analysis yielded no previously reported rare variants. Whole exome sequencing analysis in 11 families identified rare disease-causing variants in various genes, some previously linked to familial MM and others not previously associated. Notably, genes involved in ubiquitination, V(D)J recombination and the PI3K/AKT/mTOR pathway were among those identified. Furthermore, a specific variant in BNIP1 (rs28199) was found in 13 patients across nine families, indicating its potential significance in MM pathogenesis. While this study sheds light on genetic variations in familial MM in Turkey, its limitations include sample size and the absence of in vivo investigations. In conclusion, familial MM likely involves a polygenic inheritance pattern with rare, disease-causing variants in various genes, emphasizing the need for international collaborative efforts to unravel the intricate genetic basis of MM and develop targeted therapies.

nPoRe: n-polymer realigner for improved pileup-based variant calling.

Despite recent improvements in nanopore basecalling accuracy, germline variant calling of small insertions and deletions (INDELs) remains poor. Although precision and recall for single nucleotide polymorphisms (SNPs) now exceeds 99.5%, INDEL recall remains below 80% for standard R9.4.1 flow cells. We show that read phasing and realignment can recover a significant portion of false negative INDELs. In particular, we extend Needleman-Wunsch affine gap alignment by introducing new gap penalties for more accurately aligning repeated n-polymer sequences such as homopolymers ([Formula: see text]) and tandem repeats ([Formula: see text]). At the same precision, haplotype phasing improves INDEL recall from 63.76 to [Formula: see text] and nPoRe realignment improves it further to [Formula: see text].

Variant Classification Concordance using the ACMG-AMP Variant Interpretation Guidelines across Nine Genomic Implementation Research Studies.

Harmonization of variant pathogenicity classification across laboratories is important for advancing clinical genomics. The two CLIA-accredited Electronic Medical Record and Genomics Network sequencing centers and the six CLIA-accredited laboratories and one research laboratory performing genome or exome sequencing in the Clinical Sequencing Evidence-Generating Research Consortium collaborated to explore current sources of discordance in classification. Eight laboratories each submitted 20 classified variants in the ACMG secondary finding v.2.0 genes. After removing duplicates, each of the 158 variants was annotated and independently classified by two additional laboratories using the ACMG-AMP guidelines. Overall concordance across three laboratories was assessed and discordant variants were reviewed via teleconference and email. The submitted variant set included 28 P/LP variants, 96 VUS, and 34 LB/B variants, mostly in cancer (40%) and cardiac (27%) risk genes. Eighty-six (54%) variants reached complete five-category (i.e., P, LP, VUS, LB, B) concordance, and 17 (11%) had a discordance that could affect clinical recommendations (P/LP versus VUS/LB/B). 21% and 63% of variants submitted as P and LP, respectively, were discordant with VUS. Of the 54 originally discordant variants that underwent further review, 32 reached agreement, for a post-review concordance rate of 84% (118/140 variants). This project provides an updated estimate of variant concordance, identifies considerations for LP classified variants, and highlights ongoing sources of discordance. Continued and increased sharing of variant classifications and evidence across laboratories, and the ongoing work of ClinGen to provide general as well as gene- and disease-specific guidance, will lead to continued increases in concordance.

Benchmarking variant callers in next-generation and third-generation sequencing analysis.

DNA variants represent an important source of genetic variations among individuals. Next- generation sequencing (NGS) is the most popular technology for genome-wide variant calling. Third-generation sequencing (TGS) has also recently been used in genetic studies. Although many variant callers are available, no single caller can call both types of variants on NGS or TGS data with high sensitivity and specificity. In this study, we systematically evaluated 11 variant callers on 12 NGS and TGS datasets. For germline variant calling, we tested DNAseq and DNAscope modes from Sentieon, HaplotypeCaller mode from GATK and WGS mode from DeepVariant. All the four callers had comparable performance on NGS data and 30× coverage of WGS data was recommended. For germline variant calling on TGS data, we tested DNAseq mode from Sentieon, HaplotypeCaller mode from GATK and PACBIO mode from DeepVariant. All the three callers had similar performance in SNP calling, while DeepVariant outperformed the others in InDel calling. TGS detected more variants than NGS, particularly in complex and repetitive regions. For somatic variant calling on NGS, we tested TNscope and TNseq modes from Sentieon, MuTect2 mode from GATK, NeuSomatic, VarScan2, and Strelka2. TNscope and Mutect2 outperformed the other callers. A higher proportion of tumor sample purity (from 10 to 20%) significantly increased the recall value of calling. Finally, computational costs of the callers were compared and Sentieon required the least computational cost. These results suggest that careful selection of a tool and parameters is needed for accurate SNP or InDel calling under different scenarios.

Functional genomics for curation of variants in telomere biology disorder associated genes: A systematic review.

PURPOSE: Patients with an underlying telomere biology disorder (TBD) have variable clinical presentations, and they can be challenging to diagnose clinically. A genomic diagnosis for patients presenting with TBD is vital for optimal treatment. Unfortunately, many variants identified during diagnostic testing are variants of uncertain significance. This complicates management decisions, delays treatment, and risks nonuptake of potentially curative therapies. Improved application of functional genomic evidence may reduce variants of uncertain significance classifications. METHODS: We systematically searched the literature for published functional assays interrogating TBD gene variants. When possible, established likely benign/benign and likely pathogenic/pathogenic variants were used to estimate the assay sensitivity, specificity, positive predictive value, negative predictive value, and odds of pathogenicity. RESULTS: In total, 3131 articles were screened and 151 met inclusion criteria. Sufficient data to enable a PS3/BS3 recommendation were available for TERT variants only. We recommend that PS3 and BS3 can be applied at a moderate and supportive level, respectively. PS3/BS3 application was limited by a lack of assay standardization and limited inclusion of benign variants. CONCLUSION: Further assay standardization and assessment of benign variants are required for optimal use of the PS3/BS3 criterion for TBD gene variant classification.

Molecular basis of carotid body tumor and associated clinical features in Japan identified by genomic, immunohistochemical, and clinical analyses.

Carotid body tumor (CBT) is classified as a paraganglioma (PGL). Here, we report the genetic background, protein expression pattern, and clinical findings of 30 Japanese CBT cases. Germline pathogenic or likely pathogenic (P/LP) variants of genes encoding succinate dehydrogenase subunits (SDHs) were detected in 15 of 30 cases (50%). The SDHB variants were the most frequently detected, followed by SDHA and SDHD variants. One case with SDHAF2 variant was bilateral CBT, and other two multiple PGL cases were not detected P/LP variants. The three cases with germline variants that could be tested did not have somatic P/LP variants of the same genes. Immunohistochemical analysis showed negative SDHB signals in CBT tissues in five cases with germline P/LP variants of SDHB, SDHD, or SDHA. In addition, SDHB signals in CBT tissues were negative in four of nine cases without germline P/LP variants of SDHs. These findings suggest the involvement of unidentified molecular mechanisms affecting SDHs.

Germline variants associated with breast cancer in Khakass women of North Asia.

INTRODUCTION: Variants in the BRCA1/2 genes are responsible for familial breast cancer. Numerous studies showed a different spectrum of BRCA variants among breast cancer patients of different Ethnicity origin. In the available literature, no previous research has focused on breast cancer-associated variants among the Khakass people (the indigenous people of the Russian Federation). METHODS: Twenty-six Khakass breast cancer patients were enrolled in the study. Genomic DNA was isolated from blood samples and used to prepare libraries using a Hereditary Cancer Solution kit. Next-generation sequencing (NGS) was performed using the MiSeq System (Illumina, USA). RESULTS: In our study, 12% of patients (3/26) carried a single pathogenic variant; 54% of patients (14/26) carried variants of uncertain significance (VUS) or conflicting variants; and 35% of patients (9/26) did not carry any clinically significant variants. Germline pathogenic variant in the ATM gene (rs780619951, NC_000011.10:g.108259022C > T) was identified in two unrelated patients with a family history of cancer (7.6%, 2/26). The pathogenic truncating variant in the ATM gene (p. R805* or c.2413C > T) leads to the nonfunctional version of the protein. This variant has been earlier reported in individuals with a family history of breast cancer. CONCLUSIONS: Our pilot study describes the germline variant in the ATM gene associated with breast cancer in Khakass women of North Asia.

Identification of Three Human POLH Germline Variants Defective in Complementing the UV- and Cisplatin-Sensitivity of POLH-Deficient Cells.

DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1-432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (kcat/Km) for dATP insertion opposite the 3'-T and 5'-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants-substantially reduced in TLS activity-failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may increase the individual susceptibility to UV irradiation and cisplatin chemotherapy.

Genomic analysis of familial pancreatic cancers and intraductal papillary mucinous neoplasms: A cross-sectional study.

Environmental and genetic factors play a critical role in the pathogenesis of pancreatic cancer, which is likely to follow a multistep process that includes intraductal papillary mucinous neoplasm. The pathogenesis of familial pancreatic cancer has been reported; however, epidemiological characteristics and causative genes remain unclear. This study aimed to determine the relationship between the family history of pancreatic cancer and tumor malignancy and identify novel susceptible germline variants of pancreatic cancer. We performed an epidemiologic study at our institute on a cohort of 668 patients with intraductal papillary mucinous neoplasm and 242 with pancreatic cancer but without associated intraductal papillary mucinous neoplasm stratified by family history of pancreatic cancer. Whole-exome sequencing was conducted for 10 patients from seven families with familial pancreatic cancer and intraductal papillary mucinous neoplasm. We found that patients who had intraductal papillary mucinous neoplasm with positive family history of pancreatic cancer within first-degree relatives were more likely to develop malignancy in a shorter period than those without family history. Duplicate frameshift variants in TET2 c.3180dupG (p.Pro1061fs) and ASXL1 c.1934dupG (p.Gly646fs) in one family and POLN c.1194dupT (p.Glu399fs) in another were identified as pathogenic truncating germline variants which were previously recognised susceptibility genes. Moreover, PDIA2 c.1403C>T (p.Pro468Leu) and DPYSL4 c.926C>A (p.Pro309Gln) were shared in four and two patients, respectively. In particular, PDIA2 was identified as a novel candidate for one of the deleterious variants of familial pancreatic cancer.

Germline Variants Impact Somatic Events during Tumorigenesis.

Cancer is characterized by diverse genetic alterations in both germline and somatic genomes that disrupt normal biology and provide a selective advantage to cells during tumorigenesis. Germline and somatic genomes have been extensively studied independently, leading to numerous biological insights. Analyses integrating data from both genomes have identified genetic variants impacting somatic events in tumors, including hotspot driver mutations. Interactions among specific germline variants and somatic events influence cancer subtypes, treatment response, and clinical outcomes. Investigation of these complex interactions is increasing our understanding of aberrant pathways in tumors that may uncover novel therapeutic targets. Here, we review the literature describing the role of germline genetic variants in promoting the selection and generation of specific mutations during tumorigenesis.

Whole exome sequencing identifies novel germline variants of SLC15A4 gene as potentially cancer predisposing in familial colorectal cancer.

About 15% of colorectal cancer (CRC) patients have first-degree relatives affected by the same malignancy. However, for most families the cause of familial aggregation of CRC is unknown. To identify novel high-to-moderate-penetrance germline variants underlying CRC susceptibility, we performed whole exome sequencing (WES) on four CRC cases and two unaffected members of a Polish family without any mutation in known CRC predisposition genes. After WES, we used our in-house developed Familial Cancer Variant Prioritization Pipeline and identified two novel variants in the solute carrier family 15 member 4 (SLC15A4) gene. The heterozygous missense variant, p. Y444C, was predicted to affect the phylogenetically conserved PTR2/POT domain and to have a deleterious effect on the function of the encoded peptide/histidine transporter. The other variant was located in the upstream region of the same gene (GRCh37.p13, 12_129308531_C_T; 43 bp upstream of transcription start site, ENST00000266771.5) and it was annotated to affect the promoter region of SLC15A4 as well as binding sites of 17 different transcription factors. Our findings of two distinct variants in the same gene may indicate a synergistic up-regulation of SLC15A4 as the underlying genetic cause and implicate this gene for the first time in genetic inheritance of familial CRC.

An updated counseling framework for moderate-penetrance colorectal cancer susceptibility genes.

PURPOSE: With the recent guideline change for individuals at average risk for colorectal cancer (CRC) to initiate colonoscopy at the age of 45 years, there is a need to provide an updated counseling framework for individuals with variants in moderate-penetrance CRC susceptibility genes. METHODS: Population age-specific incidence rates for CRC were obtained from the 2014-2018 US Surveillance, Epidemiology, and End Results Program cancer statistics. Average-risk multipliers derived from a systematic meta-analysis were used to calculate the 5-year and cumulative lifetime risks for specific genetic variants associated with a moderate risk for CRC: NM_007194.4(CHEK2):c.1100del (p.Thr367fs), NM_007194.4(CHEK2):c.470T>C (p.Ile157Thr), NM_000038.6(APC):c.3920T>A (p.Ile1307Lys) and monoallelic MUTYH. RESULTS: When an individual at average risk would initiate colonoscopy at age 45 years, a CRC risk of 0.39% is reached. For CHEK2 1100delC, CHEK2 I157T, and APC I1307K heterozygotes, this same level of risk is reached (or nearly reached) by age 40 to 45 years. For individuals with a monoallelic MUTYH variant, the CRC risk is 0.46% by age 45 to 49 years, similar to individuals at average risk. CONCLUSION: These updated calculations support recommendations to initiate earlier colonoscopy surveillance for CHEK2 and APC I1307K germline variant heterozygotes. However, earlier surveillance is not indicated for individuals with monoallelic MUTYH germline variants in the absence of family history.

A practical guide to interpreting germline variants that drive hematopoietic malignancies, bone marrow failure, and chronic cytopenias.

PURPOSE: The American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines for germline variant interpretation are implemented as a broad framework by standardizing variant interpretation. These rules were designed to be specified, but this process has not been performed for most of the 200 genes associated with inherited hematopoietic malignancies, bone marrow failure, and cytopenias. Because guidelines on how to perform these gene specifications are lacking, variant interpretation is less reliable and reproducible. METHODS: We have used a variety of methods such as calculations of minor allele frequencies, quasi-case-control studies to establish thresholds, proband counting, and plotting of receiver operating characteristic curves to compare different in silico prediction tools to design recommendations for variant interpretation. RESULTS: We herein provide practical recommendations for the creation of thresholds for minor allele frequencies, in silico predictions, counting of probands, identification of functional domains with minimal benign variation, use of constraint Z-scores and functional evidence, prediction of nonsense-mediated decay, and assessment of phenotype specificity. CONCLUSION: These guidelines can be used by anyone interpreting variants associated with inherited hematopoietic malignancies, bone marrow failure, and cytopenias to develop criteria for reliable, accurate, and reproducible germline variant interpretation.

Genetic drivers of Cushing's disease: Frequency and associated phenotypes.

PURPOSE: Cushing's disease (CD) is often explained by a single somatic sequence change. Germline defects, however, often go unrecognized. We aimed to determine the frequency and associated phenotypes of genetic drivers of CD in a large cohort. METHODS: We studied 245 unrelated patients with CD (139 female, 56.7%), including 230 (93.9%) pediatric and 15 (6.1%) adult patients. Germline exome sequencing was performed in 184 patients; tumor exome sequencing was also done in 27 of them. A total of 43 germline samples and 92 tumor samples underwent Sanger sequencing of specific genes. Rare variants of uncertain significance, likely pathogenic (LP), or pathogenic variants in CD-associated genes, were identified. RESULTS: Germline variants (13 variants of uncertain significance, 8 LP, and 11 pathogenic) were found in 8 of 19 patients (42.1%) with positive family history and in 23 of 226 sporadic patients (10.2%). Somatic variants (1 LP and 7 pathogenic) were found in 20 of 119 tested individuals (16.8%); one of them had a coexistent germline defect. Altogether, variants of interest were identified at the germline level in 12.2% of patients, at the somatic level in 7.8%, and coexisting germline and somatic variants in 0.4%, accounting for one-fifth of the cohort. CONCLUSION: We report an estimate of the contribution of multiple germline and somatic genetic defects underlying CD in a single cohort.

RNF43 pathogenic Germline variant in a family with colorectal cancer.

The role of RNF43 as a cause of an inherited predisposition to colorectal cancer (CRC) is yet to be fully explored. This report presents our findings of two individuals with CRC from a single family carrying a likely-pathogenic inherited germline variant in RNF43. The proband (III:1) and the proband's mother (II:2) were diagnosed with mismatch repair proficient CRCs at the age of 50 years and 65 years, respectively. Both patients had BRAFV600E mutated colon tumours, indicating that the CRCs arose in sessile serrated lesions. The germline variant RNF43:c.375+1G>A was identified in both patients. RNA studies showed that this variant resulted in an aberrantly spliced transcript, which was predicted to encode RNF43:p.Ala126Ilefs*50 resulting in premature termination of protein synthesis and was classified as a likely-pathogenic variant. Our report adds further evidence to the hereditary role of RNF43 as a tumour suppressor gene in colorectal tumorigenesis and supports the inclusion of RNF43 as a gene of interest in the investigation of CRC predispositions outside the setting of serrated polyposis.

Posterior fossa ependymoma in neurodevelopmental syndrome caused by a de novo germline pathogenic POLR2A variant.

Ependymoma is the third most common pediatric brain tumor. Predisposition to develop ependymomas has been reported in different hereditary diseases, but the pathogenic variants related to the familial syndromes have rarely been detected in sporadic ependymomas. De novo variants in POLR2A, the gene encoding the largest subunit of RNA polymerase II, cause a neurodevelopmental disorder with a wide range of clinical manifestations, characterized by severe infantile-onset hypotonia, developmental delay, feeding difficulties, palatal anomalies, and facial dysmorphisms. As somatic events, POLR2A mutations represent a recurrent somatic lesion in benign meningiomas. Here we describe a case of ependymoma in a 2-year-old male with a de novo pathogenic variant in POLR2A predicted to impair proper interaction of the subunit with transcription-elongation factor TFIIS, whose function is required for back-tracking of the enzyme due to elongation blocks or nucleotide misincorporation, and expected to result in an increased error and reduced elongation rates. To date, ependymoma has never been reported in patients harboring pathogenic POLR2A variants. Further information is required to explore the possibility of a differential clinical and functional impact of the pathogenic POLR2A variants and the eventual inclusion of the POLR2A neurodevelopmental disorder among the cancer predisposition syndromes with the possible development of ependymomas.

Somatic and Germline Variant Calling from Next-Generation Sequencing Data.

Re-sequencing of the human genome by next-generation sequencing (NGS) has been widely applied to discover pathogenic genetic variants and/or causative genes accounting for various types of diseases including cancers. The advances in NGS have allowed the sequencing of the entire genome of patients and identification of disease-associated variants in a reasonable timeframe and cost. The core of the variant identification relies on accurate variant calling and annotation. Numerous algorithms have been developed to elucidate the repertoire of somatic and germline variants. Each algorithm has its own distinct strengths, weaknesses, and limitations due to the difference in the statistical modeling approach adopted and read information utilized. Accurate variant calling remains challenging due to the presence of sequencing artifacts and read misalignments. All of these can lead to the discordance of the variant calling results and even misinterpretation of the discovery. For somatic variant detection, multiple factors including chromosomal abnormalities, tumor heterogeneity, tumor-normal cross contaminations, unbalanced tumor/normal sample coverage, and variants with low allele frequencies add even more layers of complexity to accurate variant identification. Given the discordances and difficulties, ensemble approaches have emerged by harmonizing information from different algorithms to improve variant calling performance. In this chapter, we first introduce the general scheme of variant calling algorithms and potential challenges at distinct stages. We next review the existing workflows of variant calling and annotation, and finally explore the strategies deployed by different callers as well as their strengths and caveats. Overall, NGS-based variant identification with careful consideration allows reliable detection of pathogenic variant and candidate variant selection for precision medicine.