Sandra Mammarella

Increased Variance in Germline Allele-Specific Expression of APC Associates With Colorectal Cancer

BACKGROUND & AIMS Germline variations in allele-specific expression (ASE) are associated with highly penetrant familial cancers, but their role in common sporadic cancers is unclear. ASE of adenomatous polyposis coli (APC) is associated with pathogenesis of familial adenomatous polyposis. We investigated whether moderate variations in ASE of APC contribute to common forms of colorectal cancer (CRC). METHODS Denaturing high-performance liquid chromatography was used to analyze germline ASE of APC in blood samples from patients with CRC (cases, n = 53) and controls (n = 68). Means, medians, and variances of ASE were compared. Variants in the APC gene region also were analyzed. RESULTS The distribution of ASE differed significantly between groups; cases had significantly larger amounts of variance than controls (P = .0004). Risk for CRC increased proportionally with the degree of deviation from the mean. The odds ratio for individuals with levels of ASE that deviated more than 1 standard deviation from the mean was 3.97 (95% confidence interval, 1.71-9.24; P = .001); for those with levels greater than 1.645 standard deviations, the odds ratio was 13.46 (95% confidence interval, 1.76-609.40; P = .005). Sequence analysis revealed that a patient with a high level of ASE who did not have a family history of CRC carried a nonsense mutation in APC (p.Arg216X). Genotype analysis of APC associated multiple single-nucleotide polymorphisms with ASE values and/or variance among cases, but not controls. Cis variants, therefore, might account for some of the variance in ASE of APC. CONCLUSIONS Patients with CRC have a larger variance in germline levels of ASE in APC than controls; large distances from the mean ASE were associated with risk for common forms of CRC.

Original ResearchClinical—Alimentary TractIncreased Variance in Germline Allele-Specific Expression of APC Associates With Colorectal Cancer

BACKGROUND & AIMS Germline variations in allele-specific expression (ASE) are associated with highly penetrant familial cancers, but their role in common sporadic cancers is unclear. ASE of adenomatous polyposis coli (APC) is associated with pathogenesis of familial adenomatous polyposis. We investigated whether moderate variations in ASE of APC contribute to common forms of colorectal cancer (CRC). METHODS Denaturing high-performance liquid chromatography was used to analyze germline ASE of APC in blood samples from patients with CRC (cases, n = 53) and controls (n = 68). Means, medians, and variances of ASE were compared. Variants in the APC gene region also were analyzed. RESULTS The distribution of ASE differed significantly between groups; cases had significantly larger amounts of variance than controls (P = .0004). Risk for CRC increased proportionally with the degree of deviation from the mean. The odds ratio for individuals with levels of ASE that deviated more than 1 standard deviation from the mean was 3.97 (95% confidence interval, 1.71-9.24; P = .001); for those with levels greater than 1.645 standard deviations, the odds ratio was 13.46 (95% confidence interval, 1.76-609.40; P = .005). Sequence analysis revealed that a patient with a high level of ASE who did not have a family history of CRC carried a nonsense mutation in APC (p.Arg216X). Genotype analysis of APC associated multiple single-nucleotide polymorphisms with ASE values and/or variance among cases, but not controls. Cis variants, therefore, might account for some of the variance in ASE of APC. CONCLUSIONS Patients with CRC have a larger variance in germline levels of ASE in APC than controls; large distances from the mean ASE were associated with risk for common forms of CRC.

Integrative Analysis of Hereditary Nonpolyposis Colorectal Cancer: the Contribution of Allele-Specific Expression and Other Assays to Diagnostic Algorithms

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

ANALYSIS OF GENE COPY NUMBER VARIATIONS USING A METHOD BASED ON LAB-ON-A-CHIP TECHNOLOGY

AIMS AND BACKGROUND Copy number variations (CNVs) contribute to genome variability and their pathogenic role is becoming evident in an increasing number of human disorders. Commercial assays for routine diagnosis of CNVs are available only for a fraction of known genomic rearrangements. Thus, it is important to develop flexible and cost-effective methods that can be adapted to the detection of CNVs of interest, both in research and clinical settings. METHODS We describe a new multiplex PCR-based method for CNV analysis that exploits automated microfluidic capillary electrophoresis through lab-on-a-chip technology (LOC-CNV). We tested the reproducibility of the method and compared the results obtained by LOC-CNV with those obtained using previously validated semiquantitative assays such as multiplex ligation-dependent probe amplification (MLPA) and nonfluorescent multiplex PCR coupled to HPLC (NFMP-HPLC). RESULTS The results obtained by LOC-CNV in control individuals and carriers of pathogenic MLH1 or BRCA1 genomic rearrangements (losses or gains) were concordant with those obtained by previously validated methods, indicating that LOC-CNV is a reliable method for the detection of genomic rearrangements. CONCLUSION Because of its advantages with respect to time, costs, easy adaptation of previously developed multiplex assays and flexibility in novel assay design, LOC-CNV may represent a practical option to evaluate relative copy number changes in genomic targets of interest, including those identified in genome-wide analyses.

Novel insulin receptor substrate 1 and 2 variants in breast and colorectal cancer

The insulin/insulin-like growth factor pathway is involved in breast and colorectal cancer (CRC) development. In the present study, we analyzed the coding region and short intron-exon borders of the insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) genes in 12 cell lines derived from breast cancer (BC), 14 cell lines derived from CRC and 33 primary CRCs. The nucleotide variants identified in BC were 3 in IRS-1, 1 of which (p.Arg267Cys) was novel and with a pathogenic potential as predicted by in silico analysis and 6 in IRS-2. Twenty-one variants in IRS-1 and 18 in IRS-2 were identified in the CRC samples. These included 11 novel IRS-1 variants detected exclusively in CRCs, which included 5 missense (p.Pro559Leu, p.Gln655His, p.Asp1014Gly, p.Asp1181His and pPro1203Ser) with a pathogenic potential as predicted by in silico analysis, 2 frameshifts predicted to generate a truncated protein, 1 splice-site mutation and 3 silent variants. In the CRC samples we also identified 7 novel IRS-2 variants, including 4 missense variants, which included 2 (p.Asp782Asn and p.Gly1230Ser) with a pathogenic potential as predicted by in silico analysis, 2 frame insertion mutations and 1 silent variant. Most of the novel IRS-1 and IRS-2 variants may be involved in the modulation of IRS-1 or IRS-2 functions and could be relevant to breast and colorectal tumorigenesis.

Association between rs12970134 Near MC4R and Adiposity Indexes in a Homogenous Population of Caucasian Schoolchildren

Background: To assess whether previously identified obesity-susceptibility loci were associated with overweight/obesity risk in a homogeneous population of Caucasian schoolchildren and whether these associations varied with age. Methods: Seven hundred and forty-five schoolchildren (353 boys, mean age: 8.3 ± 1.4 years) underwent anthropometric assessments. A saliva sample was collected for DNA extraction and assessment of 19 single-nucleotide polymorphisms previously associated with obesity. Results: Only the rs12970134 in the MC4R gene was significantly associated with overweight/obesity risk, with a higher frequency of the AA risk genotype in children with a BMI >85th (8.3%) than in those with a BMI <85th percentile (3.0%), p = 0.001; odds ratio (95% CI) of 1.544 (1.192-1.998), p = 0.001, after adjusting for age, sex and pubertal stage. BMI standard deviation scores (SDS) and waist-to-height ratio (W/Hr) progressively increased across the rs12970134 genotypes (GG vs. AG vs. AA): BMI SDS, p = 0.004; W/Hr, p = 0.009. When dividing the study population into two groups based on the median age of participants (8.3 years), the differences in BMI SDS and W/Hr across the MC4R genotypes persisted only in children older than 8.3 years. Conclusions: In a population of Caucasian schoolchildren, the rs12970134 MC4R variant was significantly associated with excess body weight, particularly in children older than 8 years of age.