<i>In silico</i> prediction of the <i>CDH1</i> p.T560R variant.

<p>Prediction of the potential effects of the <i>CDH1 c</i>.<i>1679 C>G</i> (p.T560R) alteration on normal mRNA splicing. Top panel: reference sequence; bottom panel: mutated sequence. The text at the left of each panel represents the names of the tools used to predict splice site strength. The number ranges indicate the strength of each target sequence in each prediction tool with the upper range being the perfect match. The reference sequence and mutated sequences are shown at the bottom of each panel with nucleotide positions indicated above. Blue vertical bars indicate 5’ (donor) site. The height of each bar is proportional to the maximum possible score computed by the corresponding algorithm.</p

Patient pedigree, <i>CDH1</i> mutation and H&E image of diffuse gastric cancer.

<p>(A) The patient (indicated with the arrow head) is a 50 year old man who was diagnosed with gastric cancer at the age of 50. In his generation, three members were diagnosed with gastric cancers (one brother died of gastric cancer at 45, another brother was diagnosed with gastric cancer at 63). All three brothers affected with gastric cancer were determined to have the <i>CDH1 c</i>.<i>1679 C>G</i> p.T560R variant. (B) our patient’s germline <i>CDH1</i> sequencing demonstrating the <i>CDH1 c</i>.<i>1679C>G</i> variant. (C) Haematoxylin and eosin stain (H&E stain) of patient’s biopsy specimen showing infiltrating adenocarcinoma poorly differentiated with mucinous and signet ring cell features. The signet ring cells are characteristic of hereditary diffuse gastric cancer (HDGC). They contain large amount of mucin which pushes the nucleus to the cell periphery (see arrows).</p

<i>CDH1 c</i>.<i>1679 C>G</i> (p.T560R) mutant allele creates a novel splice site that results in a 32 nucleotide deletion of the 3’ end of exon 11.

<p>(A) Sequence adjacent to wild type allele, <i>CDH1 c</i>.<i>1679 C</i>, indicating normal splice site. Electropherogram data of normally spliced cDNA with wild type allele highlighted in blue. (B) Sequence adjacent to mutant allele, <i>CDH1 c</i>.<i>1679 G</i>, indicating aberrant splice site produced by mutant allele (solid lines) and location of normal splice site (dashed lines). Electropherogram data of aberrantly spliced cDNA with the mutant allele highlighted in light blue.</p

A Germline Variant on Chromosome 4q31.1 Associates with Susceptibility to Developing Colon Cancer Metastasis

<div><p>We tested for germline variants showing association to colon cancer metastasis using a genome-wide association study that compared Ashkenazi Jewish individuals with stage IV metastatic colon cancers versus those with stage I or II non-metastatic colon cancers. In a two-stage study design, we demonstrated significant association to developing metastatic disease for rs60745952, that in Ashkenazi discovery and validation cohorts, respectively, showed an odds ratio (OR) = 2.3 (P = 2.73E-06) and OR = 1.89 (P = 8.05E-04) (exceeding validation threshold of 0.0044). Significant association to metastatic colon cancer was further confirmed by a meta-analysis of rs60745952 in these datasets plus an additional Ashkenazi validation cohort (OR = 1.92; 95% CI: 1.28–2.87), and by a permutation test that demonstrated a significantly longer haplotype surrounding rs60745952 in the stage IV samples. rs60745952, located in an intergenic region on chromosome 4q31.1, and not previously associated with cancer, is, thus, a germline genetic marker for susceptibility to developing colon cancer metastases among Ashkenazi Jews.</p></div

Regional Plot of loci on chromosomal region 4 (4q31.1) associated with Stage IV vs. Stage I/II colon cancer in Discovery Dataset.

<p>The horizontal axis shows SNPs along the chromosomal region and the left vertical axis shows (-log₁₀ transformed) observed P-value. SNPs near the most significant SNP (rs60745952) are color coded to depict their LD with this SNP (derived as pairwise R2 values from HapMap CEU data). rs60745952, which is shown in the purple circle, is in strong LD with rs72737820, which is illustrated by the red circle. Estimated recombination rates from HapMap are plotted on the right vertical axis in cyan to reflect the local LD structure.</p

Summary of Association and Test of Homogeneity Results (a) and Forest Plots (b) for the Meta-Analysis evaluating the rs60745952 SNP and Stage IV vs. Stage I and II Colon Cancers in the Discovery and Validation #1 and Validation #2 data sets under a fixed effects and random effects model.

<p>Summary of Association and Test of Homogeneity Results (a) and Forest Plots (b) for the Meta-Analysis evaluating the rs60745952 SNP and Stage IV vs. Stage I and II Colon Cancers in the Discovery and Validation #1 and Validation #2 data sets under a fixed effects and random effects model.</p

Manhattan Plot of the Discovery Dataset for Associations Between SNPs and Stage IV vs. Stage I/II Colon Cancers.

<p>The vertical axis indicates the (-log₁₀ transformed) observed P-value and the horizontal axis indicates the chromosomal position of each SNP. Arrow denotes position of rs60745952 and rs72737810, which are not individually discernible at this scale of presentation.</p

Germline <i>ETV6</i> Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia

<div><p>Somatic mutations affecting <i>ETV6</i> often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline <i>ETV6</i> p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second <i>ETV6</i> p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type <i>ETV6</i> with retention of the <i>ETV6</i> p. N385fs. Enforced expression of the <i>ETV6</i> mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of <i>ETV6</i> target genes. Our findings highlight a novel role for <i>ETV6</i> in leukemia predisposition.</p></div

Clinical features of individuals in the study kindreds.

<p>RCC = renal cell carcinoma; ALL = acute lymphoblastic leukemia; MDS = myelodysplastic syndrome; AML = acute myeloid leukemia; MCV = mean corpuscular volume. ETV6 status, hematologic phenotypes, and additional clinical features of patients in two kindreds with segregating germline <i>ETV6</i> mutations.</p><p>Clinical features of individuals in the study kindreds.</p

Germline <i>ETV6</i> mutations impair localization of the ETV6 protein.

<p>Western blots of HeLa cell fractions probed for ETV6 protein show (a) presence of ETV6 within the cells transiently overexpressing ETV6 WT and the P214L, R369Q and R339C mutants within the nucleus. Both the L349P and N385fs mutant were not detected in the nuclear fraction. (b) Presence of ETV6 protein is abundant in the cytoplasmic fraction. The frameshift mutant N385fs showed a protein product that was smaller (45kDa) than the full-length protein (53kDa). (c) Densitometric analysis of the western blots shows that the WT localization is predominantly nuclear, while the L349P and N385fs are cytoplasmic. Other mutants P214L, R369Q and R339C show localization to a lesser extent in the nucleus.</p