Recommended Guidelines for Validation, Quality Control, and Reporting of TP53 Variants in Clinical Practice.

Accurate assessment of TP53 gene status in sporadic tumors and in the germline of individuals at high risk of cancer due to Li-Fraumeni Syndrome (LFS) has important clinical implications for diagnosis, surveillance, and therapy. Genomic data from more than 20,000 cancer genomes provide a wealth of information on cancer gene alterations and have confirmed TP53 as the most commonly mutated gene in human cancer. Analysis of a database of 70,000 TP53 variants reveals that the two newly discovered exons of the gene, exons 9ß and 9γ, generated by alternative splicing, are the targets of inactivating mutation events in breast, liver, and head and neck tumors. Furthermore, germline rearrange-ments in intron 1 of TP53 are associated with LFS and are frequently observed in sporadic osteosarcoma. In this context of constantly growing genomic data, we discuss how screening strategies must be improved when assessing TP53 status in clinical samples. Finally, we discuss how TP53 alterations should be described by using accurate nomenclature to avoid confusion in scientific and clinical reports. Cancer Res; 77(6); 1250-60. ©2017 AACR.

Synonymous Somatic Variants in Human Cancer Are Not Infamous: A Plea for Full Disclosure in Databases and Publications.

Single-nucleotide variants (SNVs) are the most frequent genetic changes found in human cancer. Most driver alterations are missense and nonsense variants localized in the coding region of cancer genes. Unbiased cancer genome sequencing shows that synonymous SNVs (sSNVs) can be found clustered in the coding regions of several cancer oncogenes or tumor suppressor genes suggesting purifying selection. sSNVs are currently underestimated, as they are usually discarded during analysis. Furthermore, several public databases do not display sSNVs, which can lead to analytical bias and the false assumption that this mutational event is uncommon. Recent progress in our understanding of the deleterious consequences of these sSNVs for RNA stability and protein translation shows that they can act as strong drivers of cancer, as demonstrated for several cancer genes such as TP53 or BCL2L12. It is therefore essential that sSNVs be properly reported and analyzed in order to provide an accurate picture of the genetic landscape of the cancer genome.

Recommendations for analyzing and reporting TP53 gene variants in the high-throughput sequencing era.

The architecture of TP53, the most frequently mutated gene in human cancer, is more complex than previously thought. Using TP53 variants as clinical biomarkers to predict response to treatment or patient outcome requires an unequivocal and standardized procedure toward a definitive strategy for the clinical evaluation of variants to provide maximum diagnostic sensitivity and specificity. An intronic promoter and two novel exons have been identified resulting in the expression of multiple transcripts and protein isoforms. These regions are additional targets for mutation events impairing the tumor suppressive activity of TP53. Reassessment of variants located in these regions is needed to refine their prognostic value in many malignancies. We recommend using the stable Locus Reference Genomic reference sequence for detailed and unequivocal reports and annotations of germ line and somatic alterations on all TP53 transcripts and protein isoforms according to the recommendations of the Human Genome Variation Society. This novel and comprehensive description framework will generate standardized data that are easy to understand, analyze, and exchange across various cancer variant databases. Based on the statistical analysis of more than 45,000 variants in the latest version of the UMD TP53 database, we also provide a classification of their functional effects ("pathogenicity").

Sdhd and SDHD/H19 knockout mice do not develop paraganglioma or pheochromocytoma.

BACKGROUND: Mitochondrial succinate dehydrogenase (SDH) is a component of both the tricarboxylic acid cycle and the electron transport chain. Mutations of SDHD, the first protein of intermediary metabolism shown to be involved in tumorigenesis, lead to the human tumors paraganglioma (PGL) and pheochromocytoma (PC). SDHD is remarkable in showing an 'imprinted' tumor suppressor phenotype. Mutations of SDHD show a very high penetrance in man and we postulated that knockout of Sdhd would lead to the development of PGL/PC, probably in aged mice. METHODOLOGY/PRINCIPAL FINDINGS: We generated a conventional knockout of Sdhd in the mouse, removing the entire third exon. We also crossed this mouse with a knockout of H19, a postulated imprinted modifier gene of Sdhd tumorigenesis, to evaluate if loss of these genes together would lead to the initiation or enhancement of tumor development. Homozygous knockout of Sdhd results in embryonic lethality. No paraganglioma or other tumor development was seen in Sdhd KO mice followed for their entire lifespan, in sharp contrast to the highly penetrant phenotype in humans. Heterozygous Sdhd KO mice did not show hyperplasia of paraganglioma-related tissues such as the carotid body or of the adrenal medulla, or any genotype-related pathology, with similar body and organ weights to wildtype mice. A cohort of Sdhd/H19 KO mice developed several cases of profound cardiac hypertrophy, but showed no evidence of PGL/PC. CONCLUSIONS: Knockout of Sdhd in the mouse does not result in a disease phenotype. H19 may not be an initiator of PGL/PC tumorigenesis.

Deep sequencing to reveal new variants in pooled DNA samples.

We evaluated massive parallel sequencing and long-range PCR (LRP) for rare variant detection and allele frequency estimation in pooled DNA samples. Exons 2 to 16 of the MUTYH gene were analyzed in breast cancer patients with Illumina's (Solexa) technology. From a pool of 287 genomic DNA samples we generated a single LRP product, while the same LRP was performed on 88 individual samples and the resulting products then pooled. Concentrations of constituent samples were measured with fluorimetry for genomic DNA and high-resolution melting curve analysis (HR-MCA) for LRP products. Illumina sequencing results were compared to Sanger sequencing data of individual samples. Correlation between allele frequencies detected by both methods was poor in the first pool, presumably because the genomic samples amplified unequally in the LRP, due to DNA quality variability. In contrast, allele frequencies correlated well in the second pool, in which all expected alleles at a frequency of 1% and higher were reliably detected, plus the majority of singletons (0.6% allele frequency). We describe custom bioinformatics and statistics to optimize detection of rare variants and to estimate required sequencing depth. Our results provide directions for designing high-throughput analyses of candidate genes.

Improving sequence variant descriptions in mutation databases and literature using the Mutalyzer sequence variation nomenclature checker.

Unambiguous and correct sequence variant descriptions are of utmost importance, not in the least since mistakes and uncertainties may lead to undesired errors in clinical diagnosis. We developed the Mutation Analyzer (Mutalyzer) sequence variation nomenclature checker (www.lovd.nl/mutalyzer; last accessed 13 September 2007) for automated analysis and correction of sequence variant descriptions using reference sequences from any organism. Mutalyzer handles most variation types: substitution, deletion, duplication, insertion, indel, and splice-site changes following current recommendations of the Human Genome Variation Society (HGVS). Input is a GenBank accession number or an uploaded reference sequence file in GenBank format with user-modified annotation, an HGNC gene symbol, and the variant (single or in a batch file). Mutalyzer generates variant descriptions at DNA level, the level of all annotated transcripts and the deduced outcome at protein level. To validate Mutalyzer's performance and to investigate the sequence variant description quality in locus-specific mutation databases (LSDBs), more than 11,000 variants in the PAH, BIC BRCA2, and HbVar databases were analyzed, showing that 87%, 25%, and 38%, respectively, were error-free and following the recommendations. Low recognition rates in BIC and HbVar (38% and 51%, respectively) were due to lack of a well-annotated genomic reference sequence (HbVar) or noncompliance to the guidelines (BRCA2). Provided with well-annotated genomic reference sequences, Mutalyzer is very effective for the curation of newly discovered sequence variation descriptions and existing LSDB data. Mutalyzer will be linked to the Leiden Open source Variation Database (LOVD) (www.LOVD.nl; last accessed 13 September 2007) and is the first module of a sequence variant effect prediction package.

Mutation analysis of SDHB and SDHC: novel germline mutations in sporadic head and neck paraganglioma and familial paraganglioma and/or pheochromocytoma.

BACKGROUND: Germline mutations of the SDHD, SDHB and SDHC genes, encoding three of the four subunits of succinate dehydrogenase, are a major cause of hereditary paraganglioma and pheochromocytoma, and demonstrate that these genes are classic tumor suppressors. Succinate dehydrogenase is a heterotetrameric protein complex and a component of both the Krebs cycle and the mitochondrial respiratory chain (succinate:ubiquinone oxidoreductase or complex II). METHODS: Using conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing to analyse genomic DNA from peripheral blood lymphocytes, here we describe the mutation analysis of the SDHB and SDHC genes in 37 patients with sporadic (i.e. no known family history) head and neck paraganglioma and five pheochromocytoma and/or paraganglioma families. RESULTS: Two sporadic patients were found to have a SDHB splice site mutation in intron 4, c.423+1G>A, which produces a mis-spliced transcript with a 54 nucleotide deletion, resulting in an 18 amino acid in-frame deletion. A third patient was found to carry the c.214C>T (p.Arg72Cys) missense mutation in exon 4 of SDHC, which is situated in a highly conserved protein motif that constitutes the quinone-binding site of the succinate: ubiquinone oxidoreductase (SQR) complex in E. coli. Together with our previous results, we found 27 germline mutations of SDH genes in 95 cases (28%) of sporadic head and neck paraganglioma. In addition all index patients of five families showing hereditary pheochromocytoma-paraganglioma were found to carry germline mutations of SDHB: four of which were novel, c.343C>T (p.Arg115X), c.141G>A (p.Trp47X), c.281G>A (p.Arg94Lys), and c.653G>C (p.Trp218Ser), and one reported previously, c.136C>T, p.Arg46X. CONCLUSION: In conclusion, these data indicate that germline mutations of SDHB and SDHC play a minor role in sporadic head and neck paraganglioma and further underline the importance of germline SDHB mutations in cases of familial pheochromocytoma-paraganglioma.

The SDH mutation database: an online resource for succinate dehydrogenase sequence variants involved in pheochromocytoma, paraganglioma and mitochondrial complex II deficiency.

BACKGROUND: The SDHA, SDHB, SDHC and SDHD genes encode the subunits of succinate dehydrogenase (succinate: ubiquinone oxidoreductase), a component of both the Krebs cycle and the mitochondrial respiratory chain. SDHA, a flavoprotein and SDHB, an iron-sulfur protein together constitute the catalytic domain, while SDHC and SDHD encode membrane anchors that allow the complex to participate in the respiratory chain as complex II. Germline mutations of SDHD and SDHB are a major cause of the hereditary forms of the tumors paraganglioma and pheochromocytoma. The largest subunit, SDHA, is mutated in patients with Leigh syndrome and late-onset optic atrophy, but has not as yet been identified as a factor in hereditary cancer. DESCRIPTION: The SDH mutation database is based on the recently described Leiden Open (source) Variation Database (LOVD) system. The variants currently described in the database were extracted from the published literature and in some cases annotated to conform to current mutation nomenclature. Researchers can also directly submit new sequence variants online. Since the identification of SDHD, SDHC, and SDHB as classic tumor suppressor genes in 2000 and 2001, studies from research groups around the world have identified a total of 120 variants. Here we introduce all reported paraganglioma and pheochromocytoma related sequence variations in these genes, in addition to all reported mutations of SDHA. The database is now accessible online. CONCLUSION: The SDH mutation database offers a valuable tool and resource for clinicians involved in the treatment of patients with paraganglioma-pheochromocytoma, clinical geneticists needing an overview of current knowledge, and geneticists and other researchers needing a solid foundation for further exploration of both these tumor syndromes and SDHA-related phenotypes.

Somatic loss of maternal chromosome 11 causes parent-of-origin-dependent inheritance in SDHD-linked paraganglioma and phaeochromocytoma families.

Germline mutations in succinate dehydrogenase subunits B, C and D (SDHB, SDHC and SDHD), genes encoding subunits of mitochondrial complex II, cause hereditary paragangliomas and phaeochromocytomas. In SDHB (1p36)- and SDHC (1q21)-linked families, disease inheritance is autosomal dominant. In SDHD (11q23)-linked families, the disease phenotype is expressed only upon paternal transmission of the mutation, consistent with maternal imprinting. However, SDHD shows biallelic expression in brain, kidney and lymphoid tissues (Baysal et al., 2000). Moreover, consistent loss of the wild-type (wt) maternal allele in SDHD-linked tumours suggests expression of the maternal SDHD allele in normal paraganglia. Here we demonstrate exclusive loss of the entire maternal chromosome 11 in SDHD-linked paragangliomas and phaeochromocytomas, suggesting that combined loss of the wt SDHD allele and maternal 11p region is essential for tumorigenesis. We hypothesize that this is driven by selective loss of one or more imprinted genes in the 11p15 region. In paternally, but not in maternally derived SDHD mutation carriers, this can be achieved by a single event, that is, non-disjunctional loss of the maternal chromosome 11. Thus, the exclusive paternal transmission of the disease can be explained by a somatic genetic mechanism targeting both the SDHD gene on 11q23 and a paternally imprinted gene on 11p15.5, rather than imprinting of SDHD.

Hereditary paraganglioma due to the SDHD M1I mutation in a second Chinese family: a founder effect?

OBJECTIVES/HYPOTHESIS: Hereditary paraganglioma is a rare condition that is inherited in an autosomal-dominant fashion. Four distinct loci have been associated with hereditary paraganglioma, including the SDHD, SDHC, and SDHB genes and a locus at 11q13. The SDHD, SDHC, and SDHB genes code for subunits of succinate dehydrogenase, which forms part of the mitochondrial respiratory chain. SDHD mutations are widely distributed along the gene with no apparent hot spots, although a founder effect has been described in the Dutch population. METHODS: Following a prior report of the SDHD M1I mutation in an Australian Chinese family, a second Chinese family with the same mutation is reported. The proband developed bilateral head and neck paragangliomas at age 34 years and a functioning adrenal pheochromocytoma and two extra-adrenal abdominal paragangliomas 7 years later. His brother had unilateral head and neck paraganglioma at age 39 years. Given the multicentricity of the proband's tumor and the familial clustering of paragangliomas, a clinical diagnosis of hereditary paraganglioma was made, and the proband was tested for a mutation in the SDHD gene. RESULTS: The proband was found to be heterozygous for the SDHD MII mutation that removes the start codon, and his brother subsequently tested positive for the same mutation. The family is not related to the Australian Chinese family. CONCLUSION: The finding suggests the possibility of a founder effect in the Chinese population and warrants further investigation.