Frameshift mutations in peripheral blood as a biomarker for surveillance of Lynch syndrome.

BACKGROUND: Lynch syndrome is a hereditary cancer predisposition syndrome caused by germline mutations in DNA mismatch repair genes, which lead to high microsatellite instability and frameshift mutations at coding mononucleotide repeats in the genome. Recurrent frameshift mutations in these regions are thought to play a central role in the increased risk of various cancers, but no biomarkers are currently available for the surveillance of high microsatellite instability-associated cancers. METHODS: A frameshift mutation-based biomarker panel was developed and validated by targeted next-generation sequencing of supernatant DNA from cultured high microsatellite instability colorectal cancer cells. This panel supported selection of 122 frameshift mutation targets as potential biomarkers. This biomarker panel was then tested using matched tumor, adjacent normal tissue, and buffy coat samples (53 samples) and blood-derived cell-free DNA (cfDNA) (38 samples) obtained from 45 high microsatellite instability and mismatch repair-deficient patients. We also sequenced cfDNA from 84 healthy participants to assess background noise. RESULTS: Recurrent frameshift mutations at coding mononucleotide repeats were detectable not only in tumors but also in cfDNA from high microsatellite instability and mismatch repair-deficient patients, including a Lynch syndrome carrier, with a varying range of target detection (up to 85.2%), whereas they were virtually undetectable in healthy participants. Receiver operating characteristic curve analysis showed high sensitivity and specificity (area under the curve = 0.94) of the investigated panel. CONCLUSIONS: We demonstrated that frameshift mutations can be detected in cfDNA from high microsatellite instability and mismatch repair-deficient patients and asymptomatic carriers. The 122-target frameshift mutation panel described here has promise as a tool for improved surveillance of high microsatellite instability and mismatch repair-deficient patients, with the potential to reduce the frequency of invasive screening methods for this high-cancer-risk cohort.

Organoids and metastatic orthotopic mouse model for mismatch repair-deficient colorectal cancer.

Background: Genome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed. Methods: To better understand the biology of MMRd cancers, elucidate the resistance mechanisms to immune modulation, and develop vaccines and therapeutic testing platforms for this high-risk population, we generated organoids and an orthotopic mouse model from intestine tumors developed in a Msh2-deficient mouse model, and followed with a detailed characterization. Results: The organoids were shown to be of epithelial origin with stem cell features, to have a high frameshift mutation frequency with MSI-H and chromosome instability, and intra- and inter-tumor heterogeneity. An orthotopic model using intra-cecal implantation of tumor fragments derived from organoids showed progressive tumor growth, resulting in the development of adenocarcinomas mixed with mucinous features and distant metastasis in liver and lymph node. Conclusions: The established organoids with characteristics of MSI-H cancers can be used to study MMRd cancer biology. The orthotopic model, with its distant metastasis and expressing frameshift peptides, is suitable for evaluating the efficacy of neoantigen-based vaccines or anticancer drugs in combination with other therapies.

A novel mouse model of PMS2 founder mutation that causes mismatch repair defect due to aberrant splicing.

Hereditary non-polyposis colorectal cancer, now known as Lynch syndrome (LS) is one of the most common cancer predisposition syndromes and is caused by germline pathogenic variants (GPVs) in DNA mismatch repair (MMR) genes. A common founder GPV in PMS2 in the Canadian Inuit population, NM_000535.5: c.2002A>G, leads to a benign missense (p.I668V) but also acts as a de novo splice site that creates a 5 bp deletion resulting in a truncated protein (p.I668*). Individuals homozygous for this GPV are predisposed to atypical constitutional MMR deficiency with a delayed onset of first primary malignancy. We have generated mice with an equivalent germline mutation (Pms2c.1993A>G) and demonstrate that it results in a splicing defect similar to those observed in humans. Homozygous mutant mice are viable like the Pms2 null mice. However, unlike the Pms2 null mice, these mutant mice are fertile, like humans homozygous for this variant. Furthermore, these mice exhibit a significant increase in microsatellite instability and intestinal adenomas on an Apc mutant background. Rectification of the splicing defect in human and murine fibroblasts using antisense morpholinos suggests that this novel mouse model can be valuable in evaluating the efficacy aimed at targeting the splicing defect in PMS2 that is highly prevalent among the Canadian Inuits.

Comparison of Eight Technologies to Determine Genotype at the UGT1A1 (TA)n Repeat Polymorphism: Potential Clinical Consequences of Genotyping Errors?

To ensure accuracy of UGT1A1 (TA)n (rs3064744) genotyping for use in pharmacogenomics-based irinotecan dosing, we tested the concordance of several commonly used genotyping technologies. Heuristic genotype groupings and principal component analysis demonstrated concordance for Illumina sequencing, fragment analysis, and fluorescent PCR. However, Illumina sequencing and fragment analysis returned a range of fragment sizes, likely arising due to PCR "slippage". Direct sequencing was accurate, but this method led to ambiguous electrophoregrams, hampering interpretation of heterozygotes. Gel sizing, pyrosequencing, and array-based technologies were less concordant. Pharmacoscan genotyping was concordant, but it does not ascertain (TA)8 genotypes that are common in African populations. Method-based genotyping differences were also observed in the publication record (p < 0.0046), although fragment analysis and direct sequencing were concordant (p = 0.11). Genotyping errors can have significant consequences in a clinical setting. At the present time, we recommend that all genotyping for this allele be conducted with fluorescent PCR (fPCR).

Pharmacogenomics Implementation at the National Institutes of Health Clinical Center.

The National Institutes of Health Clinical Center (NIH CC) is the largest hospital in the United States devoted entirely to clinical research, with a highly diverse spectrum of patients. Patient safety and clinical quality are major goals of the hospital, and therapy is often complicated by multiple cotherapies and comorbidities. To this end, we implemented a pharmacogenomics program in 2 phases. In the first phase, we implemented genotyping for HLA-A and HLA-B gene variations with clinical decision support (CDS) for abacavir, carbamazepine, and allopurinol. In the second phase, we implemented genotyping for drug-metabolizing enzymes and transporters: SLCO1B1 for CDS of simvastatin and TPMT for CDS of mercaptopurine, azathioprine, and thioguanine. The purpose of this review is to describe the implementation process, which involves clinical, laboratory, informatics, and policy decisions pertinent to the NIH CC.

Loss of δ-catenin function in severe autism.

Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 null mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.

Sequence analysis of the shelterin telomere protection complex genes in dyskeratosis congenita.

BACKGROUND: Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterised by dystrophic nails, abnormal skin pigmentation and oral leukoplakia. Patients are at very high risk of cancer and other medical problems. They have exceedingly short telomeres for their age and approximately 60% have a germline mutation in a gene important in telomere biology (DKC1, TERC, TERT, TINF2, NOP10, or NHP2). The shelterin complex consists of six proteins encoded by TINF2, ACD, POT1, TERF1, TERF2 and TERF2IP, which are essential for telomeric stability. TINF2 mutations are present in 11-25% of patients with DC. METHODS: Bi-directional sequence analysis was conducted of all exons, intron-exon boundaries and the proximal promoter of the other five shelterin genes to determine whether mutations in these genes were associated with DC. Sixteen mutation-negative patients, nine with DC and seven patients with short telomeres and bone marrow failure, were evaluated. RESULTS: Two variants were identified, ACD Ex1+189 G→A and TERF1 Ex9+59 G→A, which were each present in one patient and a healthy parent but absent in 364 controls. Three other variants were rare (<1%) but present in both patients and controls. DISCUSSION: These data suggest that except for TINF2, mutations in shelterin genes are not a common cause of DC.

Cancer and neurologic degeneration in xeroderma pigmentosum: long term follow-up characterises the role of DNA repair.

BACKGROUND: The frequency of cancer, neurologic degeneration and mortality in xeroderma pigmentosum (XP) patients with defective DNA repair was determined in a four decade natural history study. METHODS: All 106 XP patients admitted to the National Institutes of Health from 1971 to 2009 were evaluated from clinical records and follow-up. RESULTS: In the 65 per cent (n=69) of patients with skin cancer, non-melanoma skin cancer (NMSC) was increased 10,000-fold and melanoma was increased 2000-fold in patients under age 20. The 9 year median age at diagnosis of first non-melanoma skin cancer (NMSC) (n=64) was significantly younger than the 22 year median age at diagnosis of first melanoma (n=38)-a relative age reversal from the general population suggesting different mechanisms of carcinogenesis between NMSC and melanoma. XP patients with pronounced burning on minimal sun exposure (n=65) were less likely to develop skin cancer than those who did not. This may be related to the extreme sun protection they receive from an earlier age, decreasing their total ultraviolet exposure. Progressive neurologic degeneration was present in 24% (n=25) with 16/25 in complementation group XP-D. The most common causes of death were skin cancer (34%, n=10), neurologic degeneration (31%, n=9), and internal cancer (17%, n=5). The median age at death (29 years) in XP patients with neurodegeneration was significantly younger than those XP patients without neurodegeneration (37 years) (p=0.02). CONCLUSION: This 39 year follow-up study of XP patients indicates a major role of DNA repair genes in the aetiology of skin cancer and neurologic degeneration.