BackgroundThere is an increasing demand for accurate biomarkers for early non-invasive colorectal cancer detection. We employed a genome-scale marker discovery method to identify and verify candidate DNA methylation biomarkers for blood-based detection of colorectal cancer.Methodology/Principal FindingsWe used DNA methylation data from 711 colorectal tumors, 53 matched adjacent-normal colonic tissue samples, 286 healthy blood samples and 4,201 tumor samples of 15 different cancer types. DNA methylation data were generated by the Illumina Infinium HumanMethylation27 and the HumanMethylation450 platforms, which determine the methylation status of 27,578 and 482,421 CpG sites respectively. We first performed a multistep marker selection to identify candidate markers with high methylation across all colorectal tumors while harboring low methylation in healthy samples and other cancer types. We then used pre-therapeutic plasma and serum samples from 107 colorectal cancer patients and 98 controls without colorectal cancer, confirmed by colonoscopy, to verify candidate markers. We selected two markers for further evaluation: methylated THBD (THBD-M) and methylated C9orf50 (C9orf50-M). When tested on clinical plasma and serum samples these markers outperformed carcinoembryonic antigen (CEA) serum measurement and resulted in a high sensitive and specific test performance for early colorectal cancer detection.Conclusions/SignificanceOur systematic marker discovery and verification study for blood-based DNA methylation markers resulted in two novel colorectal cancer biomarkers, THBD-M and C9orf50-M. THBD-M in particular showed promising performance in clinical samples, justifying its further optimization and clinical testing.

Age, fasting period and blood glucose levels did not influence physiological uptake. However, there seemed to be a patient-specific pattern for stomach and bowel uptake.

This pilot study shows that (1) as predicted the prevalence of risk-related HbP and of alpha(+)-thalassemia is high in the immigrant population. (2) The compliance with carrier analysis in both native Dutch and immigrants is virtually total and (3) carrier diagnosis in early pregnancy and partner analysis in Hospital Laboratories is possible and is an effective tool for primary prevention of HbP in The Netherlands.

Alpha-Thalassemia (thal) is generally considered to be an expression defect caused mostly by deletions silencing one or more alpha-globin genes. Although nondeletional alpha-thalassemia is considered rare, in our laboratory we frequently observe alpha-thal phenotypes induced by point mutations. We report a new point mutation generating an abnormal hemoglobin (Hb) associated with a mild alpha-thal phenotype in two members of a Moroccan family, who presented with mild but persistent microcytic hypochromic parameters and a balanced beta/alpha synthetic ratio. All attempts to separate an abnormal native or denatured fraction were unsuccessful using electrophoresis, isoelectrofocusing (IEE), ion exchange and reversed phase high performance liquid chromatography (HPLC), denaturing polyacrylamide gel electrophoresis (PAGE), and electrospray mass spectrometry (ES/MS). The anomalous protein was only predictable by DNA analysis. The mutated gene product, not separable with any of the techniques used, could be a monomer unsuitable for tetramer formation, which is proteolyzed at an early stage. Alternatively, this mutation could perhaps lead to an abnormal splicing. The CCTCT sequence generated by the mutant, not found in the translated region of the gene, but normally present at the end of the IVS-II, could induce a possible exon skipping. This mutant could generate a mild or a critical Hb H disease in combination with one of the common alpha0-thal deletion defects.

We report a new structural defect of the alpha2-globin chain, not detectable on high performance liquid chromatography (HPLC) or electrophoresis, characterized in a 12-year-old boy of Surinamese-Hindustani origin. The child was suspected to be a carrier of alpha-thalassemia (thal) because of microcytic hypochromic parameters in the absence of iron depletion. Gap-polymerase chain reaction (gap-PCR) revealed only normal fragments in the proband, and the pattern of a -alpha4.2 (leftward) deletion in his father and sister. Direct sequencing of the alpha-globin genes revealed an ACC-->AAC transversion at codon 108 of the alpha2-globin gene in the proband, in his mother and in a younger sister. The new mutation predicts a Thr -->Asn amino acid substitution at the corresponding residue. Threonine, a covalent binder with an R-active OH group, situated in the G helix of the alpha-globin chain, is involved in alpha1beta1 contacts. Asparagine, being an equally covalent binder but with a different R-active H2N-C=O group, could make the mutated chain less suitable for tetramer cooperation. Alternatively, an absent or reduced interaction with the alpha hemoglobin (Hb) stabilizing protein (AHSP) could lead to loss of alpha chains. Hb Bleuland is the first mutation described at codon 108 and is therefore interesting in regard to the possible effects and genetic risk. The nearest variant, Hb Suan-Dok [alpha109(G16)Leu -->Arg, CTG-->CGG (alpha2)] was originally observed in a Thai patient affected with Hb H, in combination with an alpha0-thal allele. The same Hb Suan-Dok mutation, recently described in our laboratory in a carrier of African ancestry, was also not detectable as a protein and presented with an alpha-thal phenotype similar to Hb Bleuland.