A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect.

PURPOSE: Roberts syndrome (RBS) is a rare, recessively transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities, and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (DSB) repair. Here we characterize DNA damage responses (DDRs) for the first time in an RBS-affected family. METHODS AND MATERIALS: Lymphoblastoid cell lines were established from an RBS family, including the proband and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including cell survival, chromosome break, and apoptosis assays; checkpoint activation indicators; and measures of DNA breakage and repair. RESULTS: Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and mitomycin C-induced DNA damage. In this ESCO2 compound heterozygote, other DDRs were also defective, including enhanced IR-induced clastogenicity and apoptosis; increased DNA DSB induction; and a reduced capacity for repairing IR-induced DNA DSBs, as measured by γ-H2AX foci and the comet assay. CONCLUSIONS: In addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DDR-defective syndrome, which contributes to its cellular, molecular, and clinical phenotype.

LINE-1 DNA methylation: A potential forensic marker for discriminating monozygotic twins.

Discriminating individuals within a pair of monozygotic (MZ) twins using genetic markers remains unresolved. This inability causes problems in criminal or paternity cases involving MZ twins as suspects or alleged fathers. Our previous study showed DNA methylation differences in interspersed repeat sequences such as Alu and LINE-1 within pairs of newborn MZ twins. To further evaluate the possible value of LINE-1 DNA methylation for discriminating MZ twins, this study investigated the LINE-1 DNA methylation of a large number of twins. We collected blood samples and buccal cell samples from 119 pairs of MZ and 57 pairs of dizygotic (DZ) twins. Genomic DNA was extracted and LINE-1 methylation level was detected using bisulfite pyrosequencing. The mean methylation level of the three CpG sites in the blood sample among the 176 unrelated individuals was 76.60% and 70.08% in buccal samples. This difference was significant, indicating the tissue specificity of LINE-1 DNA methylation. Among 119 pairs of MZ twins, 15 pairs could be discriminated according to the difference of CpG methylation level between them, which accounted for 12.61% of total number of MZ pairs. As for DZ twins, 10 pairs had significant differences between two individuals, which accounted for 17.54% of the total 57 DZ pairs. In conclusion, there are global DNA methylation differences within some healthy concordant monozygotic (MZ) twin pairs. LINE-1 DNA methylation might be a potential marker for helping to discriminate individuals within MZ twin pairs, and the tissue specificity must be considered in practice.

Validation of DNA methylation biomarkers for diagnosis of acute lymphoblastic leukemia.

BACKGROUND: DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing. METHODS: We evaluated DNA methylation of promoter regions of TLX3 (T-cell leukemia homeobox) and FOXE3 (forkhead box E3) in bone marrow biopsies from 197 patients classified as leukemic (n = 95) or clear of the disease (n = 102) by MALDI-TOF. Using a single nucleotide extension assay (methylSABER), we tested 10 bone marrow biopsies collected throughout the course of patient chemotherapy. Using reference materials, diagnostic thresholds and limits of detection were characterized for both methods. RESULTS: Reliable detection of DNA methylation of TLX3 and FOXE3 segregated ALL from those clear of disease with minimal false-negative and false-positive results. The limit of detection with MALDI-TOF was 1000-5000 copies of methylated allele. For methylSABER, the limit of detection was 10 copies of methylated TLX3, which enabled monitoring of minimal residual disease in ALL patients. CONCLUSIONS: Mass spectrometry procedures can be used to regionally multiplex and detect rare DNA methylation events, establish DNA methylation loci as clinically applicable biomarkers for disease diagnosis, and track pediatric ALL.

Accuracy of cystatin C-based estimates of glomerular filtration rate in kidney transplant recipients: a systematic review.

BACKGROUND: As with creatinine, cystatin C can be incorporated into a formula to estimate the glomerular filtration rate (GFR). The overall performance of cystatin C-based equations in kidney transplantation is unclear with conflicting results between studies. METHODS: Systematic review of adult kidney transplant recipients. Studies that reported mean bias (mean difference between the measured and estimated GFRs) or accuracy of the cystatin C-based GFR estimation equation (e.g. percentage of estimates within 30% of the measured GFR) against the measured GFR using renal or plasma clearance of contrast agents, radioisotopes or inulin were included. RESULTS: The search identified 10 studies that examined 14 different cystatin C-based estimating equations (n = 5 equations evaluated in more than one study). The Le Bricon equation had the best performance with a bias that ranged from -6.4 to +2.8 mL/min/1.73 m(2); 85% (95% CI, 82-88) of estimates were within 30% of the measured GFR. For the other equations, 66-82% of estimates were within 30% of the measured GFR. For the modification of diet in renal disease (MDRD) equation, 68% (95% CI, 65-72) of estimates were within 30% of the measured GFR. CONCLUSIONS: The cystatin C-based Le Bricon equation was the most accurate, and most of the cystatin C-based equations showed improvements in 30% and 50% accuracy compared with the creatinine-based MDRD equation. Cystatin C-based equations may offer an advantage over the MDRD equation in kidney transplant recipients. Estimating equations re-expressed with standardized cystatin C have been developed and their accuracy needs to be tested in the kidney transplant population.