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Supravalvular aortic stenosis (SVAS) is a localized or diffuse congenital narrowing of the ascending aorta which may occur sporadically, as a familial defect, or in association with Williams syndrome. Familial cases suggest an autosomal dominant gene defect but the underlying molecular basis of SVAS is unknown. In this study, we sought to localize the genetic defect in familial SVAS by linkage analysis in a large three generation family. A total of 44 polymorphic markers were examined for linkage, including 17 Southern blot-based RFLPs, 2 PCR-based RFLPs, and 25 microsatellites, primarily of the (CA)n repeat type. We report linkage of the disease phenotype to a highly informative (CA)n repeat marker, Mfd 50, at locus D7S440 which has been localized to chromosome arm 7q. Using a 100% penetrance model, which was more conservative than lower values of penetrance, a peak LOD score of 4.66 at a recombination frequency of 0.043 was found. A number of candidate genes have been localized to this region, including collagen 1A2, laminin B1, and elastin. Based on our preliminary linkage data, the abnormal microscopic appearance of aortic elastic fibers in SVAS, and analogous animal and human diseases associated with elastic fiber and vascular abnormalities, there is indirect evidence suggesting elastin as a possible candidate gene for this disorder.

The detection of defective mismatch repair (MMR), as assessed by the presence of tumor microsatellite instability (MSI) and/or loss of MMR protein expression by IHC, has been useful for risk assessment, prognosis, and prediction of treatment in patients with colorectal cancer. We analyzed tumors for the presence of defective MMR from 5927 Colorectal Cancer Family Registry patients recruited at six international consortium sites. We evaluated the appropriate percentage instability cutoff used to distinguish the three MSI phenotypes [ie, stable (MSS), low instability (MSI-L), and high instability (MSI-H)]; the sensitivity, specificity, and performance characteristics of individual markers; and the concordance between MSI and IHC phenotypes. Guided by the results of the IHC testing, our findings indicate that the distinction between an MSI-H phenotype from a low-instability or MSS phenotype can best be accomplished by using a cutoff of 30% or greater of the markers showing instability. The sensitivity and specificity of the mononucleotide markers were higher than those of the dinucleotide markers. Specifically, BAT26 and BAT25 had the highest sensitivity (94%) and specificity (98%), and the use of mononucleotide markers alone identified 97% of the MSI-H cases correctly. As expected, the presence of MSI-H correlated with an older age of diagnosis, the presence of tumor in the proximal colon, and female sex.

Background: We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability. Methodology/Principal Findings: We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promotermethylation status and MLH1 immunohisotchemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value =0.72 vs. 2.3610 24 when the SNP was examined alone). Conclusions/Significance: The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

Background & Aims Direct germline analysis could be used to screen high-risk patients for mutations in DNA mismatch repair genes associated with Lynch Syndrome. We examined the prevalence of mutations in MLH1, MSH2, and MSH6 in a population-based sample of patients with young-onset (age Methods Young-onset CRC cases were randomly selected from 3 Colon Cancer Family Registry sites. DNA was extracted from peripheral blood leukocytes; MLH1, MSH2, and MSH6 were sequenced, and duplication and deletion analyses was performed for MLH1 and MSH2. Results were reported as deleterious or suspected deleterious, likely neutral, variant of uncertain significance, or no alteration detected. Germline data were compared to Amsterdam II criteria (ACII) and immunohistochemistry results in secondary analyses. Results Among 195 subjects, 11 had deleterious/suspected deleterious mutations (5.6%; 95% confidence interval [CI], 2.8%–9.9%), 12 had likely neutral alterations (6.2%; 95% CI, 3.2%–10.5%), 14 had variants of uncertain significance (7.2%; 95% CI, 4.0%–11.8%), 2 had a likely neutral alteration and a variant of uncertain significance (1.0%; 95% CI, 0.1%–3.7%), and 156 had no alteration detected (80.0%; 95% CI, 73.7%–85.4%). Sensitivity, specificity, and positive and negative predictive values for detecting deleterious/suspected deleterious mutations, based on ACII, were 36.4% (4/11), 96.7% (178/184), 40.0% (4/10), and 96.2% (178/185), respectively; based on immunohistochemistry these values were 85.7% (6/7), 91.9% (136/148), 33.3% (6/18), and 99.3% (136/137), respectively. Conclusions In a population-based sample of young-onset CRC cases, germline mutations in MLH1, MSH, and/or MSH6 were more prevalent than reported for CRC patients overall. Because only about 5% of young-onset CRC cases had confirmed deleterious or suspected deleterious mutations, further comparative effectiveness research is needed to determine the most appropriate screening strategy for Lynch Syndrome in this high-risk group.

Background Cigarette smoking is an established risk factor for colorectal cancer. Because colorectal carcinogenesis is a heterogeneous process, we investigated whether cigarette smoking is differentially associated with molecularly defined subtypes of colorectal cancer. Methods We evaluated associations between smoking and incident colorectal cancer, overall and by microsatellite instability (MSI) phenotype (MSI-high vs MSI-low or microsatellite stable), CpG island methylator phenotype (CIMP positive or CIMP negative), and BRAF mutation status (BRAF mutation positive or BRAF mutation negative), among 37 399 participants in a population-based cohort study (the Iowa Women's Health Study). Cigarette smoking (and other exposures) was assessed by self-report at baseline in 1986, including smoking status (never and ever [former or current]), age at initiation, total duration, average number of cigarettes smoked per day, cumulative pack-years, and induction period. Vital status and state of residence were determined by mailed follow-up questionnaires in 1987, 1989, 1992, and 1997 and by linkage to Iowa death certificate records. Nonrespondents were checked via the National Death Index to identify descendants. Participants with newly diagnosed (ie, incident) colorectal cancer were identified through annual linkage with the Iowa Cancer Registry. Archived paraffin-embedded tumor tissue specimens were obtained for 555 patients with colorectal cancer who were diagnosed from January 1, 1986, through December 31, 2002, and MSI status, CIMP status, and BRAF status were determined. Multivariable Cox regression models were fit to estimate relative risks (RRs) and 95% confidence intervals (CIs). Results Ever-smokers were at moderately increased risk for incident colorectal cancer (RR = 1.19, 95% CI = 1.05 to 1.35) compared with never-smokers. Higher risk estimates were observed for current smokers with MSI-high tumors (RR = 1.99, 95% CI = 1.26 to 3.14), CIMP-positive tumors (RR = 1.88, 95% CI = 1.22 to 2.90), and BRAF mutation-positive tumors (RR = 1.92, 95% CI = 1.22 to 3.02). Other smoking-related variables (ie, age at initiation, total duration, average number of cigarettes smoked per day, cumulative pack-years, and induction period) were also associated with MSI-high, CIMP-positive, and BRAF mutation-positive tumor subtypes. Conversely, cigarette smoking status (ever vs never) was not associated with the MSI-low or microsatellite stable (RR = 1.00, 95% CI = 0.79 to 1.25), CIMP-negative (RR = 1.02, 95% CI = 0.81 to 1.30), or BRAF mutation-negative subtypes (RR = 1.00, 95% CI = 0.65 to 1.27). Conclusions In this prospective study of older women, cigarette smoking was associated with the MSI-high, CIMP-positive, and BRAF mutation-positive colorectal cancer subtypes, which indicates that epigenetic modification may be functionally involved in smoking-related colorectal carcinogenesis.

International audience; Multiple genome-wide scans for hereditary prostate cancer (HPC) have identified susceptibility loci on nearly every chromosome. However, few results have been replicated with statistical significance. One exception is chromosome 22q, for which five independent linkage studies yielded strong evidence for a susceptibility locus in HPC families. Previously, we refined this region to a 2.53 Mb interval, using recombination mapping in 42 linked pedigrees. We now refine this locus to a 15 kb interval, spanning Apolipoprotein L3 (APOL3), using family-based association analyses of 150 total prostate cancer (PC) cases from two independent family collections with 506 unrelated population controls. Analysis of the two independent sets of PC cases highlighted single nucleotide polymorphisms (SNPs) within the APOL3 locus showing the strongest associations with HPC risk, with the most robust results observed when all 150 cases were combined. Analysis of 15 tagSNPs across the 5' end of the locus identified six SNPs with P-values < or =2 × 10(-4). The two independent sets of HPC cases highlight the same 15 kb interval at the 5' end of the APOL3 gene and provide strong evidence that SNPs within this 15 kb interval, or in strong linkage disequilibrium with it, contribute to HPC risk. Further analyses of this locus in an independent population-based, case-control study revealed an association between an SNP within the APOL3 locus and PC risk, which was not confirmed in the Cancer Genetic Markers of Susceptibility data set. This study further characterizes the 22q locus in HPC risk and suggests that the role of this region in sporadic PC warrants additional studies.

Lynch syndrome is an autosomal dominant cancer predisposition syndrome characterized by loss of function of DNA mismatch repair enzyme MLH1, MSH2, MSH6, or PMS2. Mutations in MLH1 and MSH2 account for ∼80% of the inherited cases. However, in up to 20% of cases suspected of having a germline mutation in MSH2 due to loss of MSH2 expression, a germline mutation is not identified. Recent studies have shown that some Lynch syndrome cases are due to 3′ EPCAM/TACSTD1 deletions that subsequently lead to MSH2 promoter hypermethylation. In this study, we examined the frequency of this novel mechanism for MSH2 inactivation in cases recruited through the Colon Cancer Family Registry and from the Mayo Clinic Molecular Diagnostics Laboratory. From the combined cohort, 58 cases were selected in which immunohistochemical staining suggested a mutation in MSH2 or MSH6, but no mutations were identified on follow-up testing. Of these 58 cases, 11 demonstrated a deletion of EPCAM/TACSTD1. Of cases with a deletion, the methylation status of the MSH2 promoter was confirmed in tumor tissue using methylation-sensitive PCR primers. One case showed MSH2 promoter hypermethylation in the absence of a detectable EPCAM/TACSTD1 deletion. These results indicate that approximately 20% to 25% of cases suspected of having a mutation in MSH2 but in which a germline mutation is not detected, can be accounted for by germline deletions in EPCAM/TACSTD1. These data also suggest the presence of other alterations leading to MSH2 promoter hypermethylation.

Purpose: The recognition of breast cancer as a spectrum tumor in Lynch syndrome remains controversial. The aim of this study was to explore features of breast cancers arising in Lynch syndrome families. Experimental Design: This observational study involved 107 cases of breast cancer identified from the Colorectal Cancer Family Registry (Colon CFR) from 90 families in which (a) both breast and colon cancer co-occurred, (b) families met either modified Amsterdam criteria, or had at least one early-onset ( Results: Breast cancer arose in 35 mutation carriers, and of these, 18 (51%) showed immunohistochemical absence of MMR protein corresponding to the MMR gene mutation segregating the family. MMR-deficient breast cancers were more likely to be poorly differentiated (P = 0.005) with a high mitotic index (P = 0.002), steroid hormone receptor–negative (estrogen receptor, P = 0.031; progesterone receptor, P = 0.022), and to have peritumoral lymphocytes (P = 0.015), confluent necrosis (P = 0.002), and growth in solid sheets (P < 0.001) similar to their colorectal counterparts. No difference in age of onset was noted between the MMR-deficient and MMR-intact groups. Conclusions: MMR deficiency was identified in 51% of breast cancers arising in known mutation carriers. Breast cancer therefore may represent a valid tissue option for the detection of MMR deficiency in which spectrum tumors are lacking. Clin Cancer Res; 16(7); 2214–24. ©2010 AACR.

Prostate cancer (PC) is generally believed to have a strong inherited component, but the search for susceptibility genes has been hindered by the effects of genetic heterogeneity. The recently developed sumLINK and sumLOD statistics are powerful tools for linkage analysis in the presence of heterogeneity.We performed a secondary analysis of 1,233 PC pedigrees from the International Consortium for Prostate Cancer Genetics (ICPCG) using two novel statistics, the sumLINK and sumLOD. For both statistics, dominant and recessive genetic models were considered. False discovery rate (FDR) analysis was conducted to assess the effects of multiple testing.Our analysis identified significant linkage evidence at chromosome 22q12, confirming previous findings by the initial conventional analyses of the same ICPCG data. Twelve other regions were identified with genome-wide suggestive evidence for linkage. Seven regions (1q23, 5q11, 5q35, 6p21, 8q12, 11q13, 20p11-q11) are near loci previously identified in the initial ICPCG pooled data analysis or the subset of aggressive PC pedigrees. Three other regions (1p12, 8p23, 19q13) confirm loci reported by others, and two (2p24, 6q27) are novel susceptibility loci. FDR testing indicates that over 70% of these results are likely true positive findings. Statistical recombinant mapping narrowed regions to an average of 9 cM.Our results represent genomic regions with the greatest consistency of positive linkage evidence across a very large collection of high-risk PC pedigrees using new statistical tests that deal powerfully with heterogeneity. These regions are excellent candidates for further study to identify PC predisposition genes.

Genomic imprinting refers to a parent-of-origin specific effect on gene expression. At least 1% of genes in the human genome are modulated in this manner. We sought evidence for genomic imprinting in colorectal cancer by studying the ages at diagnosis in the offspring of 2,061 parent-child pairs in which both parent and child were affected by nonsyndromic colorectal cancer. Families were ascertained through the colon Cancer Family Registry [http://epi.grants.cancer.gov/CFR/] from both population-based and clinic-based sources. We found that the affected offspring of affected fathers were on average younger than offspring of affected mothers (55.8 vs. 53.7 years; p = 0.0003), but when divided into sons and daughters, this difference was driven entirely by younger age at diagnosis in daughters of affected fathers compared to sons (52.3 years vs. 55.1 years; p = 0.0004). A younger age at diagnosis in affected daughters of affected fathers was also observable in various subsets including families that met Amsterdam II Criteria, families that did not meet Amsterdam Criteria, and in families with documented normal DNA mismatch repair in tumors. Imprinting effects are not expected to be affected by the sex of the offspring. Possible explanations for these unexpected findings include: (i) an imprinted gene on the pseudoautosomal regions of the X chromosome; (ii) an imprinted autosomal gene that affects a sex-specific pathway; or (iii) an X-linked gene unmasked because of colonic tissue-specific preferential inactivation of the maternal X chromosome.

Background. Recent clinical trials establish a beneficial effect for adjuvant chemotherapy after surgical resection of the primary tumor (1) as single treatment for patients with colonic cancer and (2) combined with radiation therapy for patients with rectal cancer. Because adjuvant chemotherapy is not universally effective and is associated with toxicity and some degree of risk, it would be desirable to supplement standard pathologic staging criteria to define more precisely the subset of patients at high risk for tumor recurrence who would benefit most from adjuvant therapy. Tumor cell DNA content and cell proliferation measured by flow cytometry were identified as important and independent prognostic factors for patients undergoing curative resection of colorectal cancer

Complete and partial hydatidiform moles are genetically aberrant conceptuses. Usually, complete moles have 46 chromosomes (diploidy), all of paternal origin. Most partial moles have 69 chromosomes (triploidy), including 23 of maternal origin and 46 of paternal origin. Triploidy that involves 23 paternal chromosomes and 46 maternal chromosomes is not associated with molar placental changes and, rarely, can result in a live-born infant with multiple birth defects. Herein we review the mechanisms of fertilization that may produce these unbalanced sets of parental chromosomes and the role of genomic imprinting as a possible explanation for these clinical conditions.

Synthesis of renin by tubulocystic epithelium in autosomal-dominant polycystic kidney disease. Evidence suggests an important role for the renin-angiotensin system in the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). Therefore, we studied the presence of immunoreactive renin in renal biopsies and measured the concentrations of renin in cyst fluids. Normal kidneys and kidneys with renal artery stenosis were used for comparison. In ADPKD, immunoreactive renin was present in juxtaglomerular apparatus, associated arterioles, and in some cells within the connective tissue surrounding the cysts. Vascular immunoreactive renin was less prominent than in renal artery stenosis. Increased amounts of tubular immunoreactive renin were noted in polycystic kidneys, as compared to normal kidneys and kidneys with renal artery stenosis. Cyst fluids contained renin detected by Western analysis and enzymatic activity; concentrations were greater in gradient cysts than in nongradient cysts. Seventy-four percent of the renin in gradient cysts was active as compared to 23% in nongradient cysts and 15% in plasma. To determine whether cyst epithelial cells are capable of synthesizing renin, these cells were isolated in tissue culture. Enzymatic assay of extracts from these cells revealed the presence of renin-like enzymatic activity (1.3 ± 0.8ng AI/mg protein/hr). The synthesis of renin by tubulocystic epithelium was confirmed by [35S]-methionine radiolabeling of cyst-derived cells, followed by immunoprecipitation and SDS-PAGE and by detection of renin mRNA by the polymerase chain reaction. These results indicate that the tubulocystic epithelium has the potential to synthesize renin. Elevated levels of active renin in renal cysts may be linked to the pathogenesis of hypertension in ADPKD. The occurrence of renin in the lining epithelium of cyst walls raises the possibility that abnormal expression of the renin-angiotensin system may, by a paracrine or autocrine mechanism, regulate epithelial hyperplasia in growing renal cysts.

Mental retardation unassociated with the Fragile X syndrome accounts for up to 60% of patients with X-linked mental retardation. In this investigation, we report on a family with mild non-specific X-linked mental retardation (MRX) without other apparent phenotypic abnormalities. Linkage analysis on 27 relatives using 18 polymorphic markers spanning the X-chromosome demonstrated close linkage to DXYS1 with a peak LOD score of 2.14 at a theta of 0. Numerous families with various types of MRX have now been studied by other investigators using molecular genetic techniques. In addition to the family described in this report, a number of these have demonstrated linkage to the DXYS1 locus. These data suggest that a gene for mental retardation may exist in the region of DXYS1. Alternatively, this area of the X-chromosome may harbor multiple different but closely linked genes which cause the various types of MRX.

Prostate cancer, a complex disease, can be relatively harmless or extremely aggressive. To identify candidate genes involved in causal pathways of aggressive prostate cancer, we implemented a systems biology approach by combining differential expression analysis and coexpression network analysis to evaluate transcriptional profiles using lymphoblastoid cell lines from 62 prostate cancer patients with aggressive phenotype (Gleason grade ≥ 8) and 63 prostate cancer patients with nonaggressive phenotype (Gleason grade ≤ 5). From 13,935 mRNA genes and 273 microRNAs (miRNA) tested, we identified significant differences in 1,100 mRNAs and 7 miRNAs with a false discovery rate (FDR) of

Background Mutation of Wnt signal antagonists Apc or Axin activates beta-catenin signaling in many cancers including the majority of human colorectal adenocarcinomas. The phenotype of apc or axin mutation in the fruit fly Drosophila melanogaster is strikingly similar to that caused by mutation in the segment-polarity gene, naked cuticle (nkd). Nkd inhibits Wnt signaling by binding to the Dishevelled (Dsh/Dvl) family of scaffold proteins that link Wnt receptor activation to beta-catenin accumulation and TCF-dependent transcription, but human NKD genes have yet to be directly implicated in cancer. Methodology/principal findings We identify for the first time mutations in NKD1--one of two human nkd homologs--in a subset of DNA mismatch repair-deficient colorectal tumors that are not known to harbor mutations in other Wnt-pathway genes. The mutant Nkd1 proteins are defective at inhibiting Wnt signaling; in addition, the mutant Nkd1 proteins stabilize beta-catenin and promote cell proliferation, in part due to a reduced ability of each mutant Nkd1 protein to bind and destabilize Dvl proteins. Conclusions/significance Our data raise the hypothesis that specific NKD1 mutations promote Wnt-dependent tumorigenesis in a subset of DNA mismatch-repair-deficient colorectal adenocarcinomas and possibly other Wnt-signal driven human cancers.

Genetic instability, which leads to an accumulation of various genetic abnormalities, has been considered an essential component of the human neoplasic transformation process. However, the molecular basis of genomic instability during tumorigenesis remains incompletely understood. Growing evidence indicates that checkpoint with forkhead and ring finger domains (CHFR), a recently identified mitotic checkpoint protein, plays an important role in maintaining chromosome integrity and functions as a tumor suppressor. In this study, we used high-throughput technology to conduct gene expression profiling of human colon cancers and found that loss of CHFR expression frequently occurred in colon cancers with high microsatellite instability (MSI-H). Downregulation of CHFR expression was closely associated with overexpression of Aurora A, an important mitotic kinase. Mice with deficiencies in both Chfr and Mlh1 (the gene that encodes the DNA mismatch-repair protein Mlh1) displayed dramatically higher incidence of spontaneous tumors relative to mice deficient for only one of these genes. These results suggest that defects in both Chfr and Mlh1 synergistically increase predisposition to tumorigenesis.

We assessed mismatch repair by immunohistochemistry (IHC) and microsatellite instability (MSI) analysis in an early onset endometrial cancer and a sister’s colon cancer. We demonstrated high-level MSI and normal expression for MLH1, MSH2 and MSH6. PMS2 failed to stain in both tumors, strongly implicating a PMS2 defect. This family did not meet clinical criteria for Lynch syndrome. However, early onset endometrial cancers in the proband and her sister, a metachronous colorectal cancer in the sister as well as MSI in endometrial and colonic tumors suggested a heritable mismatch repair defect. PCR-based direct exonic sequencing and multiplex ligation-dependent probe amplification (MLPA) were undertaken to search for PMS2 mutations in the germline DNA from the proband and her sister. No mutation was identified in the PMS2 gene. However, PMS2 exons 3, 4, 13, 14, 15 were not evaluated by MLPA and as such, rearrangements involving those exons cannot be excluded. Clinical testing for MLH1 and MSH2 mutation revealed a germline deletion of MLH1 exons 14 and 15. This MLH1 germline deletion leads to an immunodetectable stable C-terminal truncated MLH1 protein which based on the IHC staining must abrogate PMS2 stabilization. To the best of our knowledge, loss of PMS2 in MLH1 truncating mutation carriers that express MLH1 in their tumors has not been previously reported. This family points to a potential limitation of IHC-directed gene testing for suspected Lynch syndrome and the need to consider comprehensive MLH1 testing for individuals whose tumors lack PMS2 but for whom PMS2 mutations are not identified.

Family history is a strong predictor of colorectal cancer risk; however, a diagnosis of colorectal cancer among first-degree relatives has not been systematically investigated as a function of the colorectal cancer molecular subtypes related to tumor microsatellite instability (MSI) status. We investigated whether the observable familial colorectal cancer risks differed according to tumor MSI subtypes, stratified as MSI-High (>30% instability), MSI-Low (

Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, some of which are causally involved in tumorigenesis (drivers) and others that have little functional impact (passengers). To help distinguish between these two classes of alterations, we used a transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in gastrointestinal tract epithelium. Most of the offspring developed intestinal lesions, including intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 16,000 transposon insertions identified 77 candidate CRC genes, 60 of which are mutated and/or dysregulated in human CRC and thus are most likely to drive tumorigenesis. These genes include APC, PTEN , and SMAD4 . The screen also identified 17 candidate genes that had not previously been implicated in CRC, including POLI, PTPRK , and RSPO2 .

1. Clarifying how to define the clinical disorder—Lynch syndrome. In this supplementary review, Lynch syndrome refers to individuals with a predisposition to CRC and certain other malignancies as a result of a germline mismatch repair (MMR) gene mutation—including those with an existing cancer and those who have not yet developed cancer. This definition allows planned analyses of clinical validity and utility to be more straightforward. Several recent editorials and publications recommend that the ambiguous term HNPCC be abandoned and that this clarified definition of Lynch syndrome should be used instead. 2. Removing family history from consideration as a preliminary test. A previous evidence review showed that screening performance of both the Amsterdam and the Bethesda criteria to identify individuals with Lynch syndrome were highly heterogeneous, possibly due to differences among the populations tested. In a general population, Amsterdam criteria are associated with relatively low sensitivity (28‐45%), but high specificity (99%), whereas Bethesda criteria are associated with higher sensitivity (73‐91%), but at the cost of lower specificity (82‐77%). Neither provides the necessary high sensitivity/specificity in a reliable and consistent manner. There are also gaps in knowledge relating to the time required to

Background Colon cancer arises from the accumulation of multiple genetic and epigenetic alterations to normal colonic tissue. microRNAs (miRNAs) are small, non-coding regulatory RNAs that post-transcriptionally regulate gene expression. Differential miRNA expression in cancer versus normal tissue is a common event and may be pivotal for tumor onset and progression. Methods To identify miRNAs that are differentially expressed in tumors and tumor subtypes, we carried out highly sensitive expression profiling of 735 miRNAs on samples obtained from a statistically powerful set of tumors (n = 80) and normal colon tissue (n = 28) and validated a subset of this data by qRT-PCR. Results Tumor specimens showed highly significant and large fold change differential expression of the levels of 39 miRNAs including miR-135b, miR-96, miR-182, miR-183, miR-1, and miR-133a, relative to normal colon tissue. Significant differences were also seen in 6 miRNAs including miR-31 and miR-592, in the direct comparison of tumors that were deficient or proficient for mismatch repair. Examination of the genomic regions containing differentially expressed miRNAs revealed that they were also differentially methylated in colon cancer at a far greater rate than would be expected by chance. A network of interactions between these miRNAs and genes associated with colon cancer provided evidence for the role of these miRNAs as oncogenes by attenuation of tumor suppressor genes. Conclusion Colon tumors show differential expression of miRNAs depending on mismatch repair status. miRNA expression in colon tumors has an epigenetic component and altered expression that may reflect a reversion to regulatory programs characteristic of undifferentiated proliferative developmental states.

T-cell receptor gene rearrangement analysis is a useful technique to detect clonality and determine lineage of lymphoid neoplasms. We examined 103 patients with mycosis fungoides, Sézary syndrome, peripheral T cell lymphoma, potentially malignant lymphoproliferative disorders including pre-Sézary syndrome, large plaque parapsoriasis, lymphomatoid papulosis and follicular mucinosis, and various benign inflammatory infiltrates. A clonal rearrangement was detected in skin samples in 20 of 24 patients with mycosis fungoides and in peripheral blood samples in 19 of 21 patients with Sézary syndrome. A clonal population was also detected in seven of eight cases classified as peripheral T cell lymphoma. The potentially malignant dermatoses tended to have clonal rearrangement, with the exception of large plaque parapsoriasis, and further follow-up is needed to correlate clonality with the disease course. These studies demonstrate the value of molecular genetics as an adjunct to morphology in the examination of patients with cutaneous lymphoproliferative disease.

The most common genetic cause of mental retardation after Down's syndrome, the fragile X syndrome, is associated with the occurrence of a fragile site at Xq27.3. This X-linked disease is intriguing because transmission can occur through phenotypically normal males. Theories to explain this unusual phenomenon include genomic rearrangements and methylation changes associated with a local block of reactivation of the X chromosome. Using microdisected markers close to the fragile site, we have been able to test these hypotheses. We present evidence for the association of methylation with the expression of the disease. However, there is no simple relationship between the degree of methylation and either the level of expression of the fragile site or the severity of the clinical phenotype.

The mitochondrion, conventionally thought to be an organelle specific to energy metabolism, is in fact multifunctional and implicated in many diseases, including cancer. To evaluate whether mitochondria-related genes are associated with increased risk for prostate cancer, we genotyped 24 single-nucleotide polymorphisms (SNP) within the mitochondrial genome and 376 tagSNPs localized to 78 nuclear-encoded mitochondrial genes. The tagSNPs were selected to achieve ≥80% coverage based on linkage disequilibrium. We compared allele and haplotype frequencies in ∼1,000 prostate cancer cases with ∼500 population controls. An association with prostate cancer was not detected for any of the SNPs within the mitochondrial genome individually or for 10 mitochondrial common haplotypes when evaluated using a global score statistic. For the nuclear-encoded genes, none of the tagSNPs were significantly associated with prostate cancer after adjusting for multiple testing. Nonetheless, we evaluated unadjusted P values by comparing our results with those from the Cancer Genetic Markers of Susceptibility (CGEMS) phase I data set. Seven tagSNPs had unadjusted P ≤ 0.05 in both our data and in CGEMS (two SNPs were identical and five were in strong linkage disequilibrium with CGEMS SNPs). These seven SNPs (rs17184211, rs4147684, rs4233367, rs2070902, rs3829037, rs7830235, and rs1203213) are located in genes MTRR, NDUFA9, NDUFS2, NDUFB9, and COX7A2, respectively. Five of the seven SNPs were further included in the CGEMS phase II study; however, none of the findings for these were replicated. Overall, these results suggest that polymorphisms in the mitochondrial genome and those in the nuclear-encoded mitochondrial genes evaluated are not substantial risk factors for prostate cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3558–66)

To determine the prevalence of Fragile X-associated Tremor Ataxia Syndrome (FXTAS) among men with undiagnosed ataxic disorders. PCR amplification of CGG repeats in the FMR1 gene and a chart review of clinical features were performed for 286 male subjects who had non diagnostic genetic testing for spinocerebellar ataxia between November 1998 and October 2002 prior to widespread clinical testing of FXTAS. Chart review showed that 55% of tested subjects manifested only one cardinal clinical feature of FXTAS (progressive intention tremor, ataxia, and cognitive decline), 20% had two of the three findings, and 4% had all three. Gait ataxia associated with clinical features not characteristic of FXTAS was reported in 47% of subjects. Molecular analysis yielded one FMR1 premutation (100 CGG repeats). Combining our data with that of comparable published studies shows 17/1,320 (1.3%) males tested for adult-onset ataxia had FMR1 premutations. FMR1 premutations are an uncommon cause of spinocerebellar ataxia. This study is in agreement with other similar studies and supports recommendations that testing be considered only if there are additional supporting clinical features indicating that a possible FMR1 premutation may be involved.

MUTYH adenomatous polyposis (MAP) can mimic both the familial adenomatous polyposis (FAP) and hereditary nonpolyposis colon cancer (HNPCC) phenotypes. As a result of MAP's phenotypic overlap with FAP, some DNA diagnostic laboratories perform MUTYH testing in conjunction with APC testing in patients with suspected FAP or attenuated FAP (AFAP). In addition to testing FAP/AFAP samples for MUTYH mutations, we were interested whether there would also be value in testing samples referred for HNPCC testing. To determine this, we tested a consecutive series of 229 samples referred for HNPCC testing for the two most common MUTYH mutations in the Caucasian population. To enrich our study population with MAP cases, we only included samples from patients with early onset colorectal cancer (CRC diagnosed

Inactivating mutations in several genes that encode components of the DNA repair machinery have been associated with an increased risk of breast cancer. To assess whether alterations in other DNA repair genes contribute to breast cancer and to further determine the relevance of these genes to pancreatic cancer, we performed mutational analysis of 32 DNA double-strand break repair genes in genomic DNA from 38 breast tumors, 48 pancreatic tumors, and 10 non-BRCA1/BRCA2 hereditary breast cancer patients. A total of 494 coding exons were screened by denatured high-performance liquid chromatography and direct DNA sequencing. Two inactivating mutations were identified in breast tumor samples, a germline single-nucleotide deletion in POLQ (c.3605delT) and a somatic nonsense change in PRKDC (c.2408C>A, p.Ser803X). Two germline-inactivating mutations in RAD50 (c.1875C>G, p.Tyr625X and IVS14+1G>A) were also detected in separate pancreatic tumor samples. In addition, 35 novel nonsynonymous amino acid substitutions, resulting from two in-frame deletions and 33 single nucleotide alterations, were identified. Seven of these were predicted to influence protein function. A separate analysis of the CLSPN c.3839C>T (rs35490896) variant that was observed more frequently in breast tumors than in pancreatic tumors or normal controls failed to detect a significant association with breast cancer risk in a Mayo Clinic breast cancer case-control study. In conclusion, this screen of DNA repair genes implicates PRKDC and POLQ as candidate tumor suppressor genes involved in breast cancer and suggests that inactivating mutations in RAD50 predispose to pancreatic cancer as well as breast cancer. [Cancer Res 2008;68(4):971–5]

Background & Aims It is not clear what proportion of synchronous or metachronous colorectal cancers (CRCs) are associated with DNA mismatch repair (MMR) alterations or unsuspected Lynch syndrome. On the basis of tissue analyses, the aims were to evaluate DNA MMR expression in metachronous, synchronous, and isolated sporadic CRCs and to assess within-patient concordance of MMR expression in metachronous and synchronous CRCs. Methods Tissue was evaluated from 34 patients with metachronous CRC, 34 matched solitary CRC patients, and 40 patients with synchronous CRCs. Subjects with known hereditary CRC were excluded. Immunohistochemical staining for MLH1 and MSH2 was performed on all tissues. Results Absent MLH1 or MSH2 staining of the initial metachronous tumor was observed in 27% compared with 21% of control CRCs, P = .58. The odds of metachronicity with absent immunostaining were 1.33 (95% confidence interval, 0.46–3.84). Loss of MMR expression was observed in at least one cancer in 30% of patients with synchronous CRC. MMR expression loss was discordant in 70% of metachronous CRCs and 50% of synchronous CRCs. Lynch syndrome was subsequently diagnosed in 2 patients with synchronous CRCs, both with concordant tumor MMR loss but in no patient with metachronous CRC. Conclusions Among those without known Lynch syndrome, development of multiple primary CRCs appears to be due largely to somatic events and often occurs via different molecular pathways within the same patient. Altered MMR expression in sporadic CRC has low predictive value for metachronicity. MMR expression analysis in cases of multiple primary CRC might identify a small number of patients with unsuspected Lynch syndrome.

Background & Aims: Although the clinical phenotype of Lynch syndrome (also known as hereditary nonpolyposis colorectal cancer) has been well described, little is known about disease in PMS2 mutation carriers. Now that mutation detection methods can discern mutations in PMS2 from mutations in its pseudogenes, more mutation carriers have been identified. Information about the clinical significance of PMS2 mutations is crucial for appropriate counseling. Here, we report the clinical characteristics of a large series of PMS2 mutation carriers. Methods: We performed PMS2 mutation analysis using long-range polymerase chain reaction and multiplex ligation-dependent probe amplification for 99 probands diagnosed with Lynch syndrome-associated tumors showing isolated loss of PMS2 by immunohistochemistry. Penetrance was calculated using a modified segregation analysis adjusting for ascertainment. Results: Germ-line PMS2 mutations were detected in 62% of probands (n = 55 monoallelic; 6 biallelic). Among families with monoallelic PMS2 mutations, 65.5% met revised Bethesda guidelines. Compared with the general population, in mutation carriers, the incidence of colorectal cancer was 5.2-fold higher, and the incidence of endometrial cancer was 7.5-fold higher. In North America, this translates to a cumulative cancer risk to age 70 years of 15%–20% for colorectal cancer, 15% for endometrial cancer, and 25%–32% for any Lynch syndrome-associated cancer. No elevated risk for non-Lynch syndrome-associated cancers was observed. Conclusions: PMS2 mutations contribute significantly to Lynch syndrome, but the penetrance for monoallelic mutation carriers appears to be lower than that for the other mismatch repair genes. Modified counseling and cancer surveillance guidelines for PMS2 mutation carriers are proposed.

The clinical and histologic findings in five young patients with mycosis fungoides are reviewed. The skin specimen from a 16-year-old boy had an infiltrate predominately Leu-1 + , Leu-4 + , Leu-3a + 3b + , OKT6 + , Leu-2a − , Leu-8 − , and Leu-9 − , and rearrangements were noted in T cell receptor gene ( β chain [constant region probe] and γ chain [J region probe] ); no rearrangements were found in a histologically normal lymph node or peripheral blood. The skin specimen from an 11-year-old girl contained predominately Leu-4 + and Leu-9 + lymphoid cells. No T cell receptor gene rearrangements were found in the skin or in an involved lymph node. The variations in clinical, histologic, and immunopathologic features suggest that mycosis fungoides in young patients represents a heterogeneous subgroup of cutaneous T cell lymphoma.

We used the X-linked restriction fragment length polymorphism (RFLP)- methylation strategy to study the clonal basis of the myelodysplastic syndrome (MDS) in seven patients. RFLP-methylation analysis was performed on cell populations from bone marrow (BM) aspirates and peripheral blood using probes specific for the hypoxanthine phosphoribosyltransferase (HPRT) or phosphoglycerate kinase (PGK) gene regions. Density gradient centrifugation methods were used to separate granulocytes and monocytes, and T lymphocytes were positively selected by CD2 (a pan-T marker) immunoconjugated magnetic beads. Cell populations from BM aspirates in 6 of the 7 patients with MDS showed a monoclonal pattern of X-inactivation. The neutrophilic and T- lymphocytic cell fractions were analyzed in 4 of the 6 patients, and the monocytic cell fraction in one of these, and all fractions analyzed showed a similar monoclonal pattern. In 2 of the latter 4 patients, both of whom had normal karyotypes, DNA from a skin biopsy showed a polyclonal pattern. Our data suggest that MDS is a clonal disorder, even in the absence of detectable cytogenetic abnormalities, and that the abnormal clone is capable of myeloid, monocytic, and lymphoid differentiation.

Recent publications have reported that common variants on 8q24 are associated with both prostate and colorectal cancers (CRC). In addition, one of these studies (the ARCTIC study) initially observed an association with a single nucleotide polymorphism (SNP) on 9p24 that was not confirmed in some of their validation data sets. In the research described here, we conducted a case-unaffected sibling analysis using population- and clinic-based discordant sibships (N = 1,567 sibships) from the Colon Cancer Family Registry (Colon CFR) to investigate the associations between common variants at 9p24 and 8q24 and risk of CRC. We also evaluated whether these associations differed by age, family history, and tumor characteristics, including microsatellite instability and tumor site. Associations were estimated using conditional logistic regression, treating sibship as the matching factor. Analyses were adjusted for age and sex, and stratified by ascertainment source (population versus clinic). We observed an association between a SNP on 9p24 (rs719725) and risk of CRC in the population-based series (AA versus CC: odds ratios, 1.46; 95% confidence interval, 1.06–2.02; AC versus CC: odds ratios, 1.50; 95% confidence interval, 1.14–1.98; P = 0.011 on 2 df). In the population-based series, we also detected statistically significant associations between two SNPs on 8q24, rs10505477 and rs6983267, and risk of CRC (P = 0.005 and P = 0.002, respectively). There was no evidence of statistically significant heterogeneity by age at diagnosis, family history of CRC, microsatellite instability, or tumor site at either locus and no evidence of interaction between SNPs on 8q24 and 9p24. These data suggest that common variants may play important roles in the risk of CRC. [Cancer Res 2007;67(23):11128–32]

The frequency of colorectal cancer (CRC) among the Alaska Native people is the highest of any ethnic group in the United States. In this study, CRC from 329 Alaska Native people (165 Eskimo, 111 Indians, and 53 Aleut) were evaluated for evidence of defective DNA mismatch repair (MMR) by testing tumors for altered protein expression (hMLH1, hMSH2, and hMSH6) and for the presence of microsatellite instability. Of the 329 samples tested, 46 (14%) showed both microsatellite instability and altered protein expression; 42 (91%) with a loss of hMLH1, 3 (7%) hMSH2, and 1 (2%) hMLH1/hMSH6. Tumors with loss of hMLH1 were further evaluated for hMLH1 promoter hypermethylation and for the presence of the BRAF-V600E mutation. Among cases tested, all 19 (100%) tumors among the Eskimo patients showed hMLH1 promoter hypermethylation, whereas this was the case for only 3 of the 7 (42%) tumors among the Aleut (P = 0.002) and 5 of the 10 (50%) tumors from the Indians (P = 0.002). The majority of hypermethylated cases (23 of 27) tested positive for the V600E alteration. Of the nine tumors from the Aleut and Indian patients that did not exhibit hMLH1 hypermethylation, eight tested negative for V600E. Overall, the frequency of defective MMR among the three groups was not statistically different (P = 0.75). However, the data suggest that the pathogenesis of CRC may differ between the three groups. The CRC with defective MMR among the Eskimo sample fit the typical profile for hMLH1-related cancer associated with sporadic CRC, whereas the pattern in the Aleut and Indian suggests the possibility that germ line hMLH1 mutations may be present. (Cancer Epidemiol Biomarkers Prev 2007;16(11):2344–50)

In response to certain types of DNA damage, ataxia telangiectasia and rad3-related (ATR) phosphorylates checkpoint kinase 1 (CHEK1) resulting in cell cycle arrest and subsequent DNA repair. ATR and CHEK1 contain mononucleotide microsatellite repeat regions, which are mutational targets in tumors with defective mismatch repair (MMR). This study examined the frequency of such mutations in colon cancers and their impact on biologic behavior. Screening for ATR mutations in 48 tumors was performed using denaturing high-performance liquid chromatography (DHPLC) and confirmed with sequencing analysis. The CHEK1 exon 7 A(9) region was sequenced in 20 of the 27 (74%) tumors with high frequency of microsatellite instability (MSI-H). Univariate and multivariate analyses were used to examine associations with clinical outcomes. Frequent mutations in MSI-H colon cancers were identified within the ATR (37%)/CHEK1(5%) damage response pathway. Stage and MSI status both independently predicted overall survival (OS) and disease-free survival (DFS). ATR status was not associated with stage, but was associated with a trend toward improved DFS: 0/9 cancers recurred in MSI-H cases harboring ATR mutations vs. 4/18 recurrences in MSI-H cases without ATR mutations. This suggests that ATR mutations may affect clinical behavior and response to therapy in MSI-H colon cancers.

The role of genes that influence the risk of developing pancreatic cancer (PC) has not been well studied. The mitochondrion, conventionally thought to be an organelle specific to energy metabolism, is in fact multifunctional and has been implicated in many diseases, including cancer. To evaluate whether single nucleotide polymorphisms in mitochondrial DNA (mtSNP) are associated with increased risk of PC, we screened Caucasian cases diagnosed or seen at the Mayo Clinic with primary pancreatic adenocarcinoma (n = 955), and healthy clinic-based Caucasian controls (n = 1,102). A total of 24 mtSNPs, including 10 of the most common tagSNPs, 7 non-tagSNPs in the coding region, and 7 common SNPs in the regulatory region were genotyped. For analysis, these samples were grouped into two phases, the “testing” set (474 cases and 615 controls), and the “validation” set (481 cases and 487 controls). In the testing set, one mtSNP (SNP11719) suggested an association in single SNP analysis, with an odds ratio of 1.34 (95% confidence intervals, 1.05-1.72; P = 0.020), but did not remain statistically significant after correction for multiple testing. In the validation set, none of the 24 variants indicated any association with PC. For haplogroup analysis, 10 core SNPs that form common haplogroups in Caucasians (1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, and 16391) were evaluated. No significant associations with PC were identified either by analyzing the two sets separately or combined (combined global P = 0.17). Overall, these results do not support a significant involvement of mitochondrial DNA variation in the risk of developing PC. Investigation of other mitochondrial genetic variations (i.e., nuclear-encoded mitochondrial proteins) would be necessary to elucidate any role of mitochondrial DNA variation in PC. (Cancer Epidemiol Biomarkers Prev 2007;16(7):1455–9)

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant colon cancer syndrome. The first description of a cancer-prone family with HNPCC dates back to the late 1800s.1 However, it was not until the work of Lynch in the 1970s that a more complete clinical picture of this disorder began to emerge.2,3 The diagnosis of HNPCC has, until recently, been based primarily on family history. As a result, reliably differentiating patients with HNPCC from those with sporadic cancer has been difficult. However, the constellation of several clinical characteristics, in addition to family history, may raise suspicion of HNPCC.

© 2015 Macmillan Publishers Limited All rights reserved. Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0, This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to ascertain the relevance of MEK5/ERK5 signalling in colon cancer. Expression of MEK5 and ERK5 was evaluated in 323 human colon cancer samples. To evaluate the role of MEK5/ERK5 signalling in colon cancer, we developed a stable cell line model with differential MEK5/ERK5 activation. Impact of differential MEK5/ERK5 signalling was evaluated on cell cycle progression by flow cytometry and cell migration was evaluated by wound healing and transwell migration assays. Finally, we used an orthotopic xenograft mouse model of colon cancer to assess tumour growth and progression. Our results demonstrated that MEK5 and ERK5 are overexpressed in human adenomas (P, This study was supported by Fundação para a Ciência e Tecnologia (HMSP-ICT/0018/2011, SFRH/BD/96517/2013, SFRH/BD/88619/2012 and SFRH/BD/79356/2011).

Importance Use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with lower risk of colorectal cancer. Objective To identify common genetic markers that may confer differential benefit from aspirin or NSAID chemoprevention, we tested gene × environment interactions between regular use of aspirin and/or NSAIDs and single-nucleotide polymorphisms (SNPs) in relation to risk of colorectal cancer. Design, Setting, and Participants Case-control study using data from 5 case-control and 5 cohort studies initiated between 1976 and 2003 across the United States, Canada, Australia, and Germany and including colorectal cancer cases (n=8634) and matched controls (n=8553) ascertained between 1976 and 2011. Participants were all of European descent. Exposures Genome-wide SNP data and information on regular use of aspirin and/or NSAIDs and other risk factors. Main Outcomes and Measures Colorectal cancer. Results Regular use of aspirin and/or NSAIDs was associated with lower risk of colorectal cancer (prevalence, 28% vs 38%; odds ratio [OR], 0.69 [95% CI, 0.64-0.74]; P = 6.2 × 10 −28 ) compared with nonregular use. In the conventional logistic regression analysis, the SNP rs2965667 at chromosome 12p12.3 near the MGST1 gene showed a genome-wide significant interaction with aspirin and/or NSAID use ( P = 4.6 × 10 −9 for interaction). Aspirin and/or NSAID use was associated with a lower risk of colorectal cancer among individuals with rs2965667-TT genotype (prevalence, 28% vs 38%; OR, 0.66 [95% CI, 0.61-0.70]; P = 7.7 × 10 −33 ) but with a higher risk among those with rare (4%) TA or AA genotypes (prevalence, 35% vs 29%; OR, 1.89 [95% CI, 1.27-2.81]; P = .002). In case-only interaction analysis, the SNP rs16973225 at chromosome 15q25.2 near the IL16 gene showed a genome-wide significant interaction with use of aspirin and/or NSAIDs ( P = 8.2 × 10 −9 for interaction). Regular use was associated with a lower risk of colorectal cancer among individuals with rs16973225-AA genotype (prevalence, 28% vs 38%; OR, 0.66 [95% CI, 0.62-0.71]; P = 1.9 × 10 −30 ) but was not associated with risk of colorectal cancer among those with less common (9%) AC or CC genotypes (prevalence, 36% vs 39%; OR, 0.97 [95% CI, 0.78-1.20]; P = .76). Conclusions and Relevance In this genome-wide investigation of gene × environment interactions, use of aspirin and/or NSAIDs was associated with lower risk of colorectal cancer, and this association differed according to genetic variation at 2 SNPs at chromosomes 12 and 15. Validation of these findings in additional populations may facilitate targeted colorectal cancer prevention strategies.

To test the hypothesis that actin dysfunction leads to heart failure, patients with hereditary idiopathic dilated cardiomyopathy (IDC) were examined for mutations in the cardiac actin gene ( ACTC ). Missense mutations in ACTC that cosegregate with IDC were identified in two unrelated families. Both mutations affect universally conserved amino acids in domains of actin that attach to Z bands and intercalated discs. Coupled with previous data showing that dystrophin mutations also cause dilated cardiomyopathy, these results raise the possibility that defective transmission of force in cardiac myocytes is a mechanism underlying heart failure.

SULT1A1, which catalyzes the sulfate conjugation of a wide variety of natural and synthetic compounds, is genetically polymorphic. Biochemical and pharmacogenetic studies have demonstrated that individual variation in the level of enzyme activity is inherited. Common single-nucleotide polymorphisms (SNPs) located in the open reading frame and in the 5'-flanking region (5'-FR) may account for a portion of this individual variation. In this study, we demonstrate the presence of SULT1A1 gene deletions and duplications, representing an additional source of variability in the metabolic activity of this enzyme. A quantitative multiplex PCR assay was used to measure the extent of copy number differences and the frequency of these events in different populations. An analysis of DNA from 362 Caucasian-American and 99 African-American showed the presence of 1 to approximately 5 copies of SULT1A1 in individual samples: 5% of Caucasian subjects contained a single copy of the gene and 26% had three or more copies, while 63% of African-American subjects had three or more copies. Analysis of the genomic region surrounding the SULT1A1 gene in three separate cases with a deletion demonstrated that the entire SULT1A1 gene was affected. Reporter assays, constructed for each of the various 5'-FR SNP haplotypes, suggest that these may also play a role in SULT1A1 activity. However, the variability in the level of enzyme activity among 23 human platelet and 267 human liver samples was best explained by gene copy number differences when all sources of genetic variability were considered (P < 0.0001). Overall, these observations have obvious implications for the effectiveness of SULT1A1 as a drug and hormone metabolizing enzyme and its potential role as a risk factor for disease.

Background/Aims: Genetic linkage analysis of common diseases is complicated by the heterogeneity of genetic and environmental factors that increase disease risk, and possibly interactions among them. Most linkage methods that account for covariates are restricted to sib pairs, with the exception of the conditional logistic regression model [1] implemented in LODPAL in the S.A.G.E. software [2]. Although this model can be applied to arbitrary pedigrees, at times it can be difficult to maximize the likelihood due to model constraints, and it does not account for the dependence among the different types of relative pairs in a pedigree. Methods: To overcome these limitations, we developed a new approach based on score statistics for quasi- likelihoods, implemented as weighted least squares. Our methods can be used to test three different hypotheses: (1) a test for linkage without covariates; (2) a test for linkage with covariates, and (3) a test for effects of covariates on identity by descent sharing (i.e., heterogeneity). Furthermore, our methods are robust because they account for the dependence among different relative pairs within a pedigree. Results and Conclusion: Although application of our methods to a prostate cancer linkage study did not find any critical covariates in our data, the results illustrate the utility and interpretation of our methods, and suggest, nonetheless, that our methods will be useful for a broad range of genetic linkage heterogeneity analyses.

Purpose: The genomic instability in colon cancer can be divided into at least two major types, microsatellite instability (MSI) or chromosomal instability (CIN). Although initially felt to be mutually exclusive, recent evidence suggests that there may be overlap between the two. The aim of this study was to identify chromosomal alterations at high resolution in sporadic colon cancers with high-level microsatellite instability (MSI-H) and to compare them to those present in a set of matched microsatellite stable (MSS) tumors. Experimental Design: Array-based comparative genomic hybridization was used to analyze a set of 23 sporadic MSI-H and 23 MSS colon cancers matched for location, gender, stage, and age. The arrays consisted of 2,464 bacterial artificial chromosome clones. Results: MSI and MSS colon cancers differed significantly with respect to frequency and type of chromosomal alterations. The median fraction of genome altered was lower among MSI-H tumors than MSS tumors (2.8% versus 30.7%, P = 0.00006). However, the MSI-H tumors displayed a range of genomic alterations, from the absence of detectable alterations to extensive alterations. Frequent alterations in MSI-H tumors included gains of chromosomes 8, 12, and 13, and loss of 15q14. In contrast, the most frequent alterations in MSS tumors were gains of 7, 13, 8q, and 20, and losses of 8p, 17p, and 18. A small, previously uncharacterized, genomic deletion on 16p13.2, found in 35% of MSI-H and 21% of MSS tumors, was confirmed by fluorescence in situ hybridization. Conclusion: MSI and CIN are not mutually exclusive forms of genomic instability in sporadic colon cancer, with MSI tumors also showing varying degrees of CIN.

Germ line mutations in several genes (BRCA1, BRCA2, and CHEK2) whose products are involved in the DNA damage–signaling pathway have been implicated in prostate cancer risk. To identify additional genes in this pathway that might confer susceptibility to this cancer, we analyzed a recently identified DNA damage–response gene, p53AIP1 (a gene encoding for p53-regulated apoptosis-inducing protein 1), for genetic variants in prostate cancer. Five novel germ line variants were identified. The two truncating variants (Ser32Stop and Arg21insG) were found in 3% (4 of 132) of unselected prostate tumor samples. Genotyping of the two variants in an additional 393 men with sporadic prostate cancer showed a frequency of 3.1% (12 of 393) in contrast to 0.6% (2 of 327) in 327 unaffected men (Fisher's exact test, P = 0.018), with an odds ratio (OR) of 5.1 [95% confidence interval (95% CI), 1.1-23.0]. In addition, two of six tumors carrying the truncating variants were associated with loss of heterozygosity of the wild-type alleles, suggesting that p53AIP1 may act as a tumor suppressor. We also showed that the truncated p53AIP1 was unable to induce apoptosis and suppress cell growth in HeLa and COS-7 cells. These results suggest that loss-of-function variants in p53AIP1 associated with the risk of sporadic prostate cancer and further support the concept that the genetic defects in the DNA damage–response genes play an important role in the development of prostate cancer. (Cancer Res 2006; 66(21): 10302-7)

Background: Laboratory testing in suspected α-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency.Methods: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis.Results: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result.Conclusions: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

Colon cancers with high frequency microsatellite instability (MSI-H) are preferentially located in the proximal colon. Given that 15-20% of sporadic colon cancers are MSI-H, we determined whether tumor site-specific differences in clinicopathological variables, biomarkers, and prognosis are due to inclusion of MSI-H cases.TNM stage II and III primary colon carcinomas (N = 528) from patients enrolled in 5-fluorouracil-based adjuvant trials were analyzed for MSI using 11 microsatellite markers. Immunostaining for DNA mismatch repair (hMLH1, hMSH2, hMSH6) and p53 proteins was performed. DNA ploidy (diploid vs aneuploid/tetraploid) and proliferative indices (PI: S-phase + G(2)M) were analyzed by flow cytometry.MSI-H was found in 95 (18%) colon cancers. Proximal tumors (N = 286) were associated with MSI-H, older age (65 yr), poor differentiation, and diploid DNA content compared with distal tumors (all Por = 0.016). Nuclear p53 staining was more frequent in distal tumors (P= 0.002); PI was unrelated to tumor site. When MSI-H tumors were excluded, no tumor site-related differences were found except for age, which remained associated with proximal cancers (P= 0.030). Proximal site was associated with improved disease-free survival in all patients (P= 0.042), but not when MSI-H cases were excluded (P= 0.236). MSI-H status or loss of mismatch repair proteins, diploidy, and lower PI were associated with improved survival rates.Tumor site-related differences in clinicopathological variables, biomarkers, and prognosis of sporadic colon cancers can be explained by the inclusion of MSI-H cases. Older age, however, is associated with proximal tumor site independent of MSI status.

Background & Aims: Genomic instability in colon cancers is a consequence of chromosomal instability characterized by aneuploidy or defective DNA mismatch repair (MMR) indicated by microsatellite instability (MSI). Given that high-frequency MSI (MSI-H) and diploidy are correlated, we determined whether they are independent prognostic variables. Methods: Astler–Coller stage B2 and C colon cancers (N = 528) from patients treated in 5-fluorouracil–based adjuvant therapy trials were analyzed for MSI using 11 microsatellite markers. Immunostaining for hMLH1, hMSH2, and p53 proteins was performed. DNA ploidy was analyzed by flow cytometry. Associations with disease-free and overall survival were determined. Results: MSI-H was detected in 95 tumors (18%), and 70 (74%) of these were diploid. Tumors showing MSI-H (hazard ratio, 0.65; 95% confidence interval, 0.44–0.96; P = .023) or loss of MMR proteins ( P = .024) were associated with better overall survival. Improved disease-free and overall survival were found for diploid versus aneuploid/tetraploid tumors (overall survival: hazard ratio, 0.59; 95% confidence interval, 0.43–0.79; P = .0003). In the subgroups of MSI-H and microsatellite stable (MSS)/low-frequency MSI (MSI-L) tumors, diploidy was associated with better survival. The prognostic impact of ploidy was similar in stage B2 and C tumors. Ploidy did not predict the benefit of 5-fluorouracil–based treatment. When ploidy, MSI, and MMR proteins were analyzed in the same multivariate model, only ploidy remained significant. Conclusions: DNA ploidy and MSI-H status were independent prognostic variables, yet ploidy was the strongest marker. Diploidy was associated with better survival in MSI-H and in MSS/MSI-L patient subgroups.