Malinda L. Butz

Risks of Lynch Syndrome Cancers for MSH6 Mutation Carriers

BACKGROUND Germline mutations in MSH6 account for 10%-20% of Lynch syndrome colorectal cancers caused by hereditary DNA mismatch repair gene mutations. Because there have been only a few studies of mutation carriers, their cancer risks are uncertain. METHODS We identified 113 families of MSH6 mutation carriers from five countries that we ascertained through family cancer clinics and population-based cancer registries. Mutation status, sex, age, and histories of cancer, polypectomy, and hysterectomy were sought from 3104 of their relatives. Age-specific cumulative risks for carriers and hazard ratios (HRs) for cancer risks of carriers, compared with those of the general population of the same country, were estimated by use of a modified segregation analysis with appropriate conditioning depending on ascertainment. RESULTS For MSH6 mutation carriers, the estimated cumulative risks to ages 70 and 80 years, respectively, were as follows: for colorectal cancer, 22% (95% confidence interval [CI] = 14% to 32%) and 44% (95% CI = 28% to 62%) for men and 10% (95% CI = 5% to 17%) and 20% (95% CI = 11% to 35%) for women; for endometrial cancer, 26% (95% CI = 18% to 36%) and 44% (95% CI = 30% to 58%); and for any cancer associated with Lynch syndrome, 24% (95% CI = 16% to 37%) and 47% (95% CI = 32% to 66%) for men and 40% (95% CI = 32% to 52%) and 65% (95% CI = 53% to 78%) for women. Compared with incidence for the general population, MSH6 mutation carriers had an eightfold increased incidence of colorectal cancer (HR = 7.6, 95% CI = 5.4 to 10.8), which was independent of sex and age. Women who were MSH6 mutation carriers had a 26-fold increased incidence of endometrial cancer (HR = 25.5, 95% CI = 16.8 to 38.7) and a sixfold increased incidence of other cancers associated with Lynch syndrome (HR = 6.0, 95% CI = 3.4 to 10.7). CONCLUSION We have obtained precise and accurate estimates of both absolute and relative cancer risks for MSH6 mutation carriers.

Next-Generation Stool DNA Test Accurately Detects Colorectal Cancer and Large Adenomas

BACKGROUND & AIMS Technical advances have led to stool DNA (sDNA) tests that might accurately detect neoplasms on both sides of the colorectum. We assessed colorectal neoplasm detection by a next-generation sDNA test and effects of covariates on test performance. METHODS We performed a blinded, multicenter, case-control study using archived stool samples collected in preservative buffer from 252 patients with colorectal cancer (CRC), 133 with adenomas ≥ 1 cm, and 293 individuals with normal colonoscopy results (controls); two-thirds were randomly assigned to a training set and one-third to a test set. The sDNA test detects 4 methylated genes, a mutant form of KRAS, and the α-actin gene (as a reference value) using quantitative, allele-specific, real-time target and signal amplification; it also quantifies hemoglobin. We used a logistical model to analyze data. RESULTS The sDNA test identified 85% of patients with CRC and 54% of patients with adenomas ≥1 cm with 90% specificity. The test had a high rate of detection for all nonmetastatic stages of CRC (aggregate 87% detection rate for CRC stages I-III). Detection rates increased with adenoma size: 54% ≥ 1 cm, 63% >1 cm, 77% >2 cm, 86% >3 cm, and 92% >4 cm (P < .0001). Based on receiver operating characteristic analysis, the rate of CRC detection was slightly greater for the training than the test set (P = .04), whereas the rate of adenoma detection was comparable between sets. Sensitivities for detection of CRC and adenoma did not differ with lesion site. CONCLUSIONS Early-stage CRC and large adenomas can be detected throughout the colorectum and with high levels of accuracy by the sDNA test. Neoplasm size, but not anatomical site, affected detection rates. Further studies are needed to validate the findings in a larger population and optimize the sDNA test.

A Common Trinucleotide Repeat Expansion within the Transcription Factor 4 (TCF4, E2-2) Gene Predicts Fuchs Corneal Dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a common, familial disease of the corneal endothelium and is the leading indication for corneal transplantation. Variation in the transcription factor 4 (TCF4) gene has been identified as a major contributor to the disease. We tested for an association between an intronic TGC trinucleotide repeat in TCF4 and FECD by determining repeat length in 66 affected participants with severe FECD and 63 participants with normal corneas in a 3-stage discovery/replication/validation study. PCR primers flanking the TGC repeat were used to amplify leukocyte-derived genomic DNA. Repeat length was determined by direct sequencing, short tandem repeat (STR) assay and Southern blotting. Genomic Southern blots were used to evaluate samples for which only a single allele was identified by STR analysis. Compiling data for 3 arms of the study, a TGC repeat length >50 was present in 79% of FECD cases and in 3% of normal controls cases (p<0.001). Among cases, 52 of 66 (79%) subjects had >50 TGC repeats, 13 (20%) had <40 repeats and 1 (2%) had an intermediate repeat length. In comparison, only 2 of 63 (3%) unaffected control subjects had >50 repeats, 60 (95%) had <40 repeats and 1 (2%) had an intermediate repeat length. The repeat length was greater than 1000 in 4 FECD cases. The sensitivity and specificity of >50 TGC repeats identifying FECD in this patient cohort was 79% and 96%, respectively Expanded TGC repeat was more specific for FECD cases than the previously identified, highly associated, single nucleotide polymorphism, rs613872 (specificity = 79%). The TGC trinucleotide repeat expansion in TCF4 is strongly associated with FECD, and a repeat length >50 is highly specific for the disease This association suggests that trinucleotide expansion may play a pathogenic role in the majority of FECD cases and is a predictor of disease risk.

Heterogenous MSH6 loss is a result of microsatellite instability within MSH6 and occurs in sporadic and hereditary colorectal and endometrial carcinomas.

Mismatch-repair (MMR) immunohistochemistry is used to detect tumor MMR deficiency associated with high-level microsatellite instability (MSI). Rare tumors show heterogenous loss of mutS homolog 6 (MSH6) with immunohistochemistry, defined by areas of retained staining and separate areas of complete loss of staining. To investigate the clinical interpretation of this phenomenon, we identified 22 cases of heterogenous MSH6 loss interpreted at Mayo Clinic from January 2001 through December 2012 and reviewed histologic features, MSH6 and other MMR immunohistochemistry, and accompanying MSI testing results (n=20). Heterogenous MSH6 loss was seen in colorectal carcinoma (n=18), endometrial carcinoma (n=3), and sebaceous neoplasm (n=1). In the 18 colorectal carcinoma cases, it accompanied complete loss of mutL homolog 1 (MLH1) or PMS2, or both. Heterogenous MSH6 loss was characterized by MSI and MSH6 C8 tract instability in treatment-naive cases and showed mucinous or signet-ring zones in one quarter of cases. Two cases status post neoadjuvant chemoradiation showed heterogenous MSH6 loss but were microsatellite and C8 tract stable. C8 tracts were unstable in 2 of 4 MSH6-associated Lynch syndrome (LS) tumors, but all 4 showed complete MSH6 loss on immunohistochemistry. Further, 12 such MSH6-associated LS cases showed complete MSH6 loss. In conclusion, heterogenous MSH6 loss is uncommon, usually caused by instability in MSH6 exon 5 polycytosine tract, and not associated with germline MSH6 mutation. Although heterogenous MSH6 loss provides evidence against germline MSH6 mutation, patients whose tumors exhibit this immunolabeling pattern may have LS due to a defect in a different MMR gene.

SERPINA1 Full-Gene Sequencing Identifies Rare Mutations Not Detected in Targeted Mutation Analysis

Genetic α-1 antitrypsin (AAT) deficiency is characterized by low serum AAT levels and the identification of causal mutations or an abnormal protein. It needs to be distinguished from deficiency because of nongenetic causes, and diagnostic delay may contribute to worse patient outcome. Current routine clinical testing assesses for only the most common mutations. We wanted to determine the proportion of unexplained cases of AAT deficiency that harbor causal mutations not identified through current standard allele-specific genotyping and isoelectric focusing (IEF). All prospective cases from December 1, 2013, to October 1, 2014, with a low serum AAT level not explained by allele-specific genotyping and IEF were assessed through full-gene sequencing with a direct sequencing method for pathogenic mutations. We reviewed the results using American Council of Medical Genetics criteria. Of 3523 cases, 42 (1.2%) met study inclusion criteria. Pathogenic or likely pathogenic mutations not identified through clinical testing were detected through full-gene sequencing in 16 (38%) of the 42 cases. Rare mutations not detected with current allele-specific testing and IEF underlie a substantial proportion of genetic AAT deficiency. Full-gene sequencing, therefore, has the ability to improve accuracy in the diagnosis of AAT deficiency.

C9orf72 Repeat Expansion Frequency among Patients with Huntington Disease Genetic Testing

Background: European studies identified the C9orf72 repeat expansion as the most frequent genetic alteration in patients with Huntington disease (HD)-like phenotypes but negative HD genetic testing. Objective: To investigate C9orf72 repeat expansion frequency in individuals tested for HD in a North American tertiary referral laboratory. Methods: Three hundred and seventy-three cases (115 positive and 258 negative for HD) were evaluated by genotyping PCR, with follow-up Southern blot and 5′ repeat methylation status assessment by combined repeat-primed and methylation-specific PCR in a subset. Results: Three cases (all HD-negative) tested positive: 2 had > 2,000 repeats and were methylated, 1 had 80–100 repeats and was unmethylated. Two cases (1 HD-positive and 1 HD-negative) had intermediate alleles (20–29 repeats) and were unmethylated. The remaining 368 cases were negative (< 20 repeats). C9orf72 repeat expansion was absent in patients with HD and was identified in a small subset (1.2%) of patients with negative HD genetic testing. Conclusion: These findings suggest that C9orf72 repeat expansion does not coexist with HTT repeat expansion and that C9orf72 repeat expansion testing is unnecessary for patients with HD. In addition, C9orf72 evaluation may be considered for individuals negative for HD genetic testing. Similar to in previous studies, methylation of C9orf72 repeat expansion was limited to large expansions.