Ultradeep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability

Grassi, Anna; Segala, C.; Iannelli, Fabio; Volorio, Sara; Bertario, Lucio; Radice, Paolo; Bernard, Loris; Ciccarelli, Francesca D.

doi:10.1371/journal.pbio.1000275

Cited by 27 publications

(27 citation statements)

References 54 publications

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“…Consequently, individuals heterozygous for an inactivating MLH1 mutation constitutively present cellular MLH1 levels that are 50% of normal levels. In the healthy tissues of these individuals, MSI is not prominent, but is detectable in in-depth analyses (42,43). This confirms that the intracellular MLH1 concentration in mutation carriers is just sufficient to retain (almost normal) MMR activity.…”

Section: Effect Of Mlh1 Protein Concentrations On Mmr Activitysupporting

confidence: 60%

Expression Defect Size among Unclassified MLH1 Variants Determines Pathogenicity in Lynch Syndrome Diagnosis

Hinrichsen

Brieger

Zeuzem

et al. 2013

Clinical Cancer Research

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Purpose: Lynch syndrome is caused by a germline mutation in a mismatch repair gene, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants with unclear clinical significance. The functionality of these variants can be tested in the laboratory, but the results cannot be used for clinical diagnosis. We therefore aimed to establish a laboratory test that can be applied clinically.Experimental Design: We assessed the expression, stability, and mismatch repair activity of 38 MLH1 missense variants and determined the pathogenicity status of recurrent variants using clinical data.Results: Four recurrent variants were classified as neutral (K618A, H718Y, E578G, V716M) and three as pathogenic (A681T, L622H, P654L). All seven variants were proficient in mismatch repair but showed defects in expression. Quantitative PCR, pulse-chase, and thermal stability experiments confirmed decreases in protein stability, which were stronger in the pathogenic variants. The minimal cellular MLH1 concentration for mismatch repair was determined, which corroborated that strongly destabilized variants can cause repair deficiency. Loss of MLH1 tumor immunostaining is consistently reported in carriers of the pathogenic variants, showing the impact of this protein instability on these tumors.Conclusions: Expression defects are frequent among MLH1 missense variants, but only severe defects cause Lynch syndrome. The data obtained here enabled us to establish a threshold for distinguishing tolerable (clinically neutral) from pathogenic expression defects. This threshold allows the translation of laboratory results for uncertain MLH1 variants into pathogenicity statements for diagnosis, thereby improving the targeting of cancer prevention measures in affected families.

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Section: Effect Of Mlh1 Protein Concentrations On Mmr Activitysupporting

confidence: 60%

Expression Defect Size among Unclassified MLH1 Variants Determines Pathogenicity in Lynch Syndrome Diagnosis

Hinrichsen

Brieger

Zeuzem

et al. 2013

Clinical Cancer Research

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“…S6, left red arrow). Both of these variants were within a region that seems to have increased mutability compared with the average mutability (28). Furthermore, patient #6 had constitutional A/G alleles for SNP rs3910657; however, we identified A/A in CTC7 and G/G in CTC14.…”

Section: Sequencing Of the Ultraconserved Region Ucr41 In Ctcsmentioning

confidence: 56%

“…To address this issue, we exploited ultraconserved regions (UCR), which represent genomic regions that do not accumulate true mutations (28). Thus, all sequence variations in UCRs should represent bona fide errors, making UCRs an ideal control for DNA amplification and sequencing errors (28). Therefore, we sequenced the ultraconserved region UCR41 (ref.…”

Section: Sequencing Of the Ultraconserved Region Ucr41 In Ctcsmentioning

confidence: 99%

Complex Tumor Genomes Inferred from Single Circulating Tumor Cells by Array-CGH and Next-Generation Sequencing

et al. 2013

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Circulating tumor cells (CTC) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here, we conducted the first comprehensive genomic profiling of CTCs using array-comparative genomic hybridization (CGH) and next-generation sequencing. We used the U.S. Food and Drug Administration-cleared CellSearch system, which detected CTCs in 21 of 37 patients (range, 1-202/7.5 mL sample) with stage IV colorectal carcinoma. In total, we were able to isolate 37 intact CTCs from six patients and identified in those multiple colorectal cancer-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 colorectal cancer-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes [e.g., adenomatous polyposis coli (APC), KRAS, or PIK3CA] found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we conducted additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. This study paves the way to use CTCs as a liquid biopsy in patients with cancer, providing more effective options to monitor tumor genomes that are prone to change during progression, treatment, and relapse. Cancer Res; 73(10); 2965-75. Ó2013 AACR.

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“…Some of the generated mutations impart selective growth advantage by overcoming geographical constraints imposed by neighboring tissues, diminished vascular supply, and altered nutrition. By the time a tumor is clinically detected, there are tens to hundreds of thousands of different mutations; clonal lineages within the tumor are geographically demarcated and different genes are mutated in different individuals with the same types of tumors (1). This extensive landscape of subclonal mutations could account for the phenotypic heterogeneity of tumors as well as resistance to therapy (2).…”

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confidence: 99%

Human Cancers Express a Mutator Phenotype: Hypothesis, Origin, and Consequences

Loeb

2016

Cancer Research

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The mutator phenotype hypothesis was postulated more than 40 years ago. It was based on the multiple enzymatic steps required to precisely replicate the 6 billion bases in the human genome each time a normal cell divides. A reduction in this accuracy during tumor progression could be responsible for the striking heterogeneity of malignant cells within a tumor and for the rapidity by which cancers become resistant to therapy.

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Ultradeep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability

Abstract: Ultradeep sequencing of genomes permits the detection of very low-level genomic instability in non-neoplastic tissues of patients with the most common form of inherited colorectal cancer.

Cited by 27 publications

References 54 publications

Expression Defect Size among Unclassified MLH1 Variants Determines Pathogenicity in Lynch Syndrome Diagnosis

Expression Defect Size among Unclassified MLH1 Variants Determines Pathogenicity in Lynch Syndrome Diagnosis

Complex Tumor Genomes Inferred from Single Circulating Tumor Cells by Array-CGH and Next-Generation Sequencing

Human Cancers Express a Mutator Phenotype: Hypothesis, Origin, and Consequences

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