Mitochondrial haplogroups and cognitive progression in Parkinson's disease.

Mitochondria are a culprit in the onset of Parkinson's disease, but their role during disease progression is unclear. Here we used Cox proportional hazards models to exam the effect of variation in the mitochondrial genome on longitudinal cognitive and motor progression over time in 4064 patients with Parkinson's disease. Mitochondrial macro-haplogroup was associated with reduced risk of cognitive disease progression in the discovery and replication population. In the combined analysis, patients with the super macro-haplogroup J, T, U# had a 41% lower risk of cognitive progression with P = 2.42 × 10-6 compared to those with macro-haplogroup H. Exploratory analysis indicated that the common mitochondrial DNA variant, m.2706A>G, was associated with slower cognitive decline with a hazard ratio of 0.68 (95% confidence interval 0.56-0.81) and P = 2.46 × 10-5. Mitochondrial haplogroups were not appreciably linked to motor progression. This initial genetic survival study of the mitochondrial genome suggests that mitochondrial haplogroups may be associated with the pace of cognitive progression in Parkinson's disease over time.

Major-effect quantitative trait locus qLKR4.1 encodes a phospholipase Dδ protein associated with low-K+ stress tolerance by promoting root length.

KEY MESSAGE: qLKR4.1, controlling low K+ resistance in tomato, was fine-mapped to an interval of 67.5 kb on chromosome A04, and one gene encoding phospholipase Dδ was identified as a candidate gene. In plants, changes in root length are an important morphological response to low K+ (LK) stress; however, the underlying genetics in tomato remain unclear. Here, we combined bulked segregant analysis-based whole-genome sequencing, single-nucleotide polymorphism haplotyping, and fine genetic mapping to identify a candidate gene as a major-effect quantitative trait loci (QTL), i.e., qLKR4.1, which was associated with LK tolerance due to increased root elongation in the tomato line JZ34. Through multiple analyses, we found that Solyc04g082000 is the most likely candidate for qLKR4.1, which encodes phospholipase Dδ (PLDδ). Increased root elongation under LK in JZ34 may be attributed to a non-synonymous single-nucleotide polymorphism in the Ca2+-binding domain region of this gene. Solyc04g082000 increases root length through its PLDδ activity. Silencing of Solyc04g082000Arg in JZ34 led to a significant decrease in root length compared with silencing of Solyc04g082000His allele in JZ18 under LK conditions. Mutation of a Solyc04g082000 homologue in Arabidopsis, pldδ, resulted in decreased primary root lengths under LK conditions, compared to the wild type. Transgenic tomato expressing the qLKR4.1Arg allele from JZ34 exhibited a significant increase in root length compared with the wild type expressing the allele from JZ18 under LK conditions. Taken together, our results confirm that the PLDδ gene Solyc04g082000 exerts important functions in increasing tomato root length and LK tolerance.

Blocker displacement amplification-based genetic diagnosis for autosomal dominant polycystic kidney disease and the clinical outcomes of preimplantation genetic testing.

OBJECTIVE: Given that the molecular diagnosis of autosomal dominant polycystic kidney disease (ADPKD) is complicated, we aim to apply blocker displacement amplification (BDA) on the mutational screening of PKD1 and PKD2. METHODS: A total of 35 unrelated families with ADPKD were recruited from the Center for Reproductive Medicine, Women and Children's Hospital of Chongqing Medical University (Chongqing, China), from October 2018 to October 2021. Long-range PCR followed by next-generation sequencing were applied for resequencing of PKD1 and PKD2, and the putatively disease-causative variants were verified with BDA. The effects of ADPKD on male and female infertility and the factors influencing the clinical outcomes of preimplantation genetic testing (PGT) for ADPKD were investigated. RESULTS: A total of 26 PKD1 variants and 5 PKD2 variants were identified, of which 13 were newly discovered. The BDA system worked effectively for eliminating the interference of pseudogenes in genetic testing of PKD1 (1-33 exons) with different concentrations of genome DNA. The females with ADPKD have no specific infertility factors, while 68.2% of the affected men were with abnormal sperm concentration and/or motility with an indefinite genotype-phenotype relationship. As for PGT, the fertilization rate of couples with the male partner having ADPKD was relatively lower compared to those with the female partner being affected. The ADPKD patients receiving PGT usually achieved high rates of live births. CONCLUSION: These findings expanded the variant spectrum of PKD genes and emphasized the application prospect of blocker displacement amplification on PKD1-related genetic diagnosis.

Joint Modeling Study Identifies Blood-Based Transcripts Link to Cognitive Decline in Parkinson's Disease.

BACKGROUND: Cognitive decline in Parkinson's disease (PD) is prevalent, insidious, and burdensome during the progression of the disease. OBJECTIVES: We aimed to find transcriptome-wide biomarkers in blood to predict cognitive decline and identify patients at high risk with cognitive impairment in PD. METHODS: We carried out joint modeling analysis to characterize transcriptome-wide longitudinal gene expression and its association with the progression of mild cognitive impairment (MCI) in PD patients. The average time-dependent area under the curves (AUCs) were used for evaluating the accuracy of the significant joint models. A cognitive survival score (CogSs) derived from joint model was leveraged to predict the occurrence of MCI. All predicting models were built in a discovery cohort with 272 patients and replicated in an independent cohort with 177 patients. RESULTS: We identified five longitudinal varied expression of transcripts that were significantly associated with MCI progression in patients with PD. The most significant transcript IGLC1 joint model accurately predicted the progression of MCI in PD patients in the discovery and replication cohorts (average time-dependent AUCs >0.82). The CogSs derived from the optimal IGLC1 joint model had a high accuracy at early study stage in both cohorts (AUC ≥0.91). CONCLUSIONS: Our transcriptome-wide joint modeling analysis uncovered five blood-based transcripts related to cognitive decline in PD. The joint models will serve as a useful resource for clinicians and researchers to screen PD patients with high risk of development of cognitive impairment and pave the path for Parkinson's personalized medicine. © 2022 International Parkinson and Movement Disorder Society.

Hemodiafiltration improves red blood cell lifespan in patients with end-stage renal disease.

INTRODUCTION: Uremic toxin-induced shortening of red blood cell (RBC) lifespan is an important mechanism of anemia in end-stage renal disease (ESRD). Conventional hemodialysis does not improve RBC lifespan; the efficacy of hemodiafiltration (HDF) for alleviating RBC lifespan has not yet been evaluated in patients with ESRD. METHODS: Twenty-three patients with ESRD in maintenance hemodialysis were enrolled. Baseline data for sex, age, dialysis vintage, pre-dialysis hemoglobin (Hb), blood urea nitrogen (BUN), intact parathyroid hormone (iPTH), single pool Kt/V (spKt/V), and plasma indophenol sulfate (IS) were collected. RBC lifespans before and after one session of HDF were compared. The resultant differences were subjected to correlational analyses with baseline data. RESULTS: RBC lifespan increased from 73 (66, 89) days at baseline to 77 (71, 102) days after a single HDF treatment (p = 0.034). Meanwhile, plasma IS concentration decreased from 113.05 (80.67, 133.05) mg/L to 83.87 (62.98, 96.78) mg/L (p < 0.001). RBC lifespan increases correlated negatively with Hb levels. CONCLUSIONS: A single HDF treatment improved RBC lifespan in ESRD patients on maintenance hemodialysis, with more severe pre-HDF anemia at baseline being associated with greater increases in RBC lifespan.

A Validation Study of a Locally Adapted Brussels Infant and Toddler Stool Scale of the Chinese Version.

The Brussels Infant and Toddler Stool Scale was developed to improve the reliability of constipation diagnosis in non-toilet-trained children. The aim of this study was to evaluate the validity of simplified Chinese versions of the Brussels Infant and Toddler Stool Scale when used by parents, community doctors, pediatricians, and nurses. Photographs of the Scale were categorized into four categories (hard stools, formed stools, loose stools, and watery stools) and subjects assigned each photograph to a category. The study included two stages. In the first stage (n = 237 observers), percent correct allocations of the seven photographs ranged from 68.4% to 93.2%. We observed poorer recognition of the three hard stool items (77.4%, 85.8%, and 74.0%) than had been reported in the original Brussels Infant and Toddler Stool Scale validity study (95.9%, 93.4%, and 96.2%). Because hard stool items were commonly miscategorized as formed stools (21.6%, 9.5%, and 26.0%), we modified the descriptors "hard stools" and "formed stools" into "dry/hard stools" and "formed loose stools," respectively, and examined the performance of the modified Chinese Brussels Infant and Toddler Stool Scale in stage 2 of our study. The proportions of correct allocations of the three "hard stool" items in the modified Chinese Brussels Infant and Toddler Stool Scale increased to 94.7%, 90.4%, and 84.6%, values that were statistically similar to those reported previously in the original Brussels Infant and Toddler Stool Scale publisher. Renaming these categories to remove ambiguity in Chinese improved the identifiability of these items. The resultant Chinese Brussels Infant and Toddler Stool Scale was found to be valid for use with Chinese observers.

Comparison of Levitt's CO breath test and the 15 N-glycine labeling technique for measuring the lifespan of human red blood cells.

The red blood cell (RBC) lifespan is an important physiological indicator of clear significance in clinical research, used for the differential diagnosis of various diseases such as anemia, compensatory phase hemolysis, and polycythemia. The 15 N-glycine labeling technique is the gold standard method for determining RBC lifespans. However, the usefulness of this technique in clinical settings is seriously hindered by the several weeks required to complete the analyses. Levitt's CO breath test is another reliable technique for determining RBC lifespans, with a simpler protocol giving much faster results, making it more useful in clinical applications. We compared the CO breath test and 15 N-glycine labeling technique for measuring the human RBC lifespan. We investigated human RBC lifespans where each subject undertook both the 15 N-glycine labeling technique and the CO breath test. The correlation between the results from these two methods was analyzed. Eight of the ten subjects successfully completed the study. The RBC lifespan values obtained by Levitt's CO breath test were lower than those obtained by the 15 N-glycine labeling technique. The RBC lifespan values determined from the 15 N-glycine labeling technique and the CO breath test were significantly correlated, with a Pearson correlation coefficient of R = 0.98 (p < 0.05), while the R2 of the linear regression equation was 0.96. The CO breath test exhibits as good performance as the 15 N-glycine labelling technique in distinguishing healthy subjects from subjects with hemolysis. The result suggests that the CO breath test is a reliable method for quickly determining human RBC lifespans in clinical applications.

Red Blood Cell Lifespan Shortening in Patients with Early-Stage Chronic Kidney Disease.

BACKGROUND: Although reduced red blood cell (RBC) lifespan has been reported to be a contributory factor to anemia in patients with end-stage chronic kidney disease (CKD), there are limited data regarding RBC lifespan in early-stage CKD. Serum erythropoietin (EPO) is considered a primary causative factor of renal anemia. The aims of this study were to compare the RBC lifespan, serum EPO levels, and other renal anemia indicators across CKD-stage groups of patients and to analyze the impacts of etiological factors on renal anemia. METHODS: A cohort of 74 non-smoking patients with CKD were enrolled, including 15 in stage 1, 18 in stage 2, 15 in stage 3, 15 in stage 4, and 11 in stage 5. RBC lifespan was determined by CO breath tests. Potential correlations of hemoglobin (Hb) concentration with RBC lifespan, reticulocyte count (Ret), and levels of EPO, ferritin, folic acid, and vitamin B12 were analyzed. RESULTS: CKD progression was associated with decreases in (Hb) and RBC lifespan. RBC lifespan durations in CKD stages 1-5 were 122 ± 50, 112 ± 26, 90 ± 32, 88 ± 28, and 60 ± 24 days, respectively. RBC lifespan means for the stage 3, 4 and 5 groups were significantly shorter than those for the stage 1 and 2 groups. Serum EPO did not differ significantly between the CKD stage groups. (Hb) correlated directly with RBC lifespan (r = 0.372, p = 0.002) and Ret (r = 0.308, p = 0.011), but did not correlate with serum EPO, ferritin, folic acid, or vitamin B12 levels. CONCLUSIONS: Reduced RBC lifespan in early-stage CKD, demonstrated in this study, suggests that increased RBC destruction may play a more important etiological role in renal anemia than other indicators in patients with CKD.

Ferroelectric Nanorods as a Polymer Interface Additive for High-Performance Garnet-Based Solid-State Batteries.

Sandwiching polymer interlayers between the electrode and solid electrolyte is considered promising in solving the interfacial issues arising from solid-solid contact in garnet-based solid-state batteries, but drawbacks including low ionic conductivity, inferior Li+ transference number, and unsatisfying mechanical property of the polymer hindered the practical application of such strategy. To solve the mentioned shortcomings of the polymer interlayer simultaneously, we introduce the ferroelectric material, BaTi2O5 (BT) nanorods, into the polymer matrix in this work. By taking full advantage of the plasticization effect and intrinsic spontaneous polarization of the introduced ferroelectric, the polymer's ionic conductivity and Li+ transference number have been significantly enhanced. The built-in electric field BT introduced also benefits the modulation of CEI components formed on the cathode particles, further enhancing the battery performance by decreasing cathode degradation. Besides, the BT nanorods' particular high aspect ratio also helps increase the mechanical property of the obtained polymer film, making it more resistant to lithium dendrite growth across the interface. Benefitting from the merits mentioned above, the assembled lithium symmetric cells using garnet SE with the BT-modified polymer interlayer exhibit stable cycling performance (no short circuit after 1000 h under RT) with low polarization voltage. The full battery employing LiFePO4 as a cathode also presents superior capacity retentions (94.6% after 200 cycles at 0.1 C and 93.4% after 400 cycles at 0.2 C). This work highlights the importance of ferroelectric materials with specific morphology in enhancing the electrochemical performance of polymer-based electrolytes, promoting the practical application of solid-state batteries.

Structure-function relationships of three kinds of immobilized DNA probes in the discrimination abilities of single nucleotide variation.

We have compared here the nucleic acid hybridization abilities of three kinds of immobilized probes with different structures. Under the conventional structural design, we found that the share-stem hairpin structure probe (SHP) was more easily hybridized with the target than the linear probe and conventional hairpin shaped probe (HP). The HP probe had the lowest hybridization ability. However, it was shown a contrary result in the single-nucleotide mismatch discrimination ratio. Subsequently, we increased the Tm of the two hairpin-shaped probes and found both of the two probes were improved on the hybridization specificities even though the hybridization abilities were decreased. The result of the hybridization temperature optimization experiment showed that at 45 degrees C, the Dr Ratio (mismatch discrimination ratio) of HP-28T and SHP-32T were both as high as 8.5 while the ratios of linear probe were -0.3-0.7. The results suggested that the immobilized shared stem hairpin shaped probes also had the ability to discriminate single-nucleotide variation under proper design.

High-Throughput Variant Detection Using a Color-Mixing Strategy.

Many diseases are related to multiple genetic alterations within a single gene. Probing for highly multiple (>10) variants in a single quantitative PCR tube is impossible because of a limited number of fluorescence channels and the limited ability to test one variant per channel, increasing the need for tubes. Herein, a novel color-mixing strategy was experimentally validated that uses fluorescence combinations as digital color codes to probe multiple variants simultaneously. The color-mixing strategy relies on a simple intratube assay that can probe for 15 variants as part of an intertube assay that can probe for an exponentially increased number of variants. This strategy is achieved by using multiplex double-stranded toehold probes modified with fluorophores and quenchers; the probes are designed to be quenched or remain luminous after binding to wild-type or variant templates. The color-mixing strategy was used to probe for 21 pathogenic variants in thalassemia and to distinguish between heterozygous and homozygous variants in six tubes, with a specificity of 99% and a sensitivity of 94%. To support tuberculosis diagnosis, the same strategy was applied to simultaneously probe in Mycobacterium tuberculosis for rifampicin-resistance mutations occurring within one 81-bp region and one 48-bp region in the rpoB gene, plus five isoniazid-resistance mutations in the inhA and katG genes.

Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE).

One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.

Validation of a highly sensitive Sanger sequencing in detecting EGFR mutations from circulating tumor DNA in patients with lung cancers.

BACKGROUND: The novel method, named blocker displacement amplification (BDA) Sanger, was applied to detect low variant allele frequency mutations in the circulating tumor DNA (ctDNA). This study aimed to evaluate the performance of the BDA Sanger method for the EGFR mutation detection in the ctDNA from lung cancer patients. METHODS: A total of 195 plasma samples of lung cancer patients were included. The EGFR mutation status in the ctDNA was detected by the BDA Sanger and Super-ARMS assays. Next-generation sequencing (NGS) was further used to verify the mutant of EGFR with inconsistencies. RESULTS: BDA Sanger assay was capable of detecting EGFR mutations with a 0.20% VAF from plasma samples. Among treatment-naive patients with paired tissue and plasma samples, the EGFR positive percent agreement (PPA) was 79% by BDA sanger. EGFR mutation was detected in 34.4% (67/195) ctDNA samples by the Super-ARMS and in 41.0% (80/195) ctDNA samples by the BDA Sanger assay. The overall concordance rate between the BDA Sanger and Super-ARMS assays was 82% (160/195). The BDA Sanger also enabled the detection of rare EGFR mutations, which were not discovered by the Super-ARMS. CONCLUSION: The results supported the validity and efficiency of the BDA Sanger method for EGFR detection in patients with lung cancer, indicating that BDA Sanger has a great potential for application in detecting mutations in the ctDNA.

Levitt's CO breath test in the differential diagnosis of chronic isolated hyperbilirubinemia.

A key component of the differential diagnosis of isolated hyperbilirubinemia (HB) is distinguishing between hemolytic and non-hemolytic types. Routine hemolysis screening markers have unsatisfactory sensitivity and specificity. Erythrocyte (RBC) lifespan shortening, the gold standard marker of hemolysis, is seldomly measured due to the cumbersome and protracted nature of standard methods. A new Levitt's CO breath test method may enable simple, rapid RBC lifespan measurement. In this pilot prospective diagnostic study, Levitt's CO breath test was evaluated to discriminate hemolytic from non-hemolytic HB in adults. One hundred and thirty eligible non-smoking adult patients who were aged 18 or older, referred for chronic (>6 months) isolated HB or had a known diagnosis of isolated HB of a rare cause, were recruited, including 77 with non-hemolytic HB and 53 with hemolytic HB. ROC curve analysis was applied to determine the optimal cutoff for discriminating between hemolytic and non-hemolytic HB, and the performance was calculated. Results showed that the mean RBC lifespan in non-hemolytic HB (93 ± 26 d) was reduced (p= 0.001 vs. normal reference value of 126 d), but longer than that in hemolytic HB (36 ± 17 d;p= 0.001). RBC lifespans did not differ significantly between 26 patients with simple hemolytic HB (32 ± 14 d) and 27 patients with a Gilbert syndrome comorbidity (40 ± 18 d). ROC curve analysis revealed an optimal lifespan cutoff for discriminating between hemolytic and non-hemolytic HB of 60 d (AUC = 0.982), with a diagnostic accuracy of 95.4%, 94.3% sensitivity and 96.1% specificity respectively. These results indicate that Levitt's CO breath test seems to be very sensitive and specific for detecting hemolysis in adult patients with chronic isolated HB, and could enable simple, rapid, and reliable differential diagnosis of isolated HB. A large-scale validation study of the method is warranted.

Detection method for methylation density on microarray using methyl-CpG-binding domain protein.

A method for determining methylation density of target CpG islands has been established. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite-converted sample DNAs. The PCR products on the microarray were treated by SssI methyltransferase and labeled with TAMRA fluorescence. A recombinant, antibody-like methyl-CpG-binding protein labeled with Cy5 fluorescence was used to identify symmetrical methyl-CpG dinucleotide of the PCR products on the microarray. By use of a standard curve with control mixtures, the ratio of two fluorescence signals can be converted into percentage values to assess methylation density of targeted fragments. We obtained the methylation density of six CpG islands on the two tumor suppressor genes of CDK2A and CDK2B from seven cancer cell line samples and two normal blood samples. The validity of this method was tested by bisulfite sequencing. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples.

Targeted regulation by ROCK2 on bladder carcinoma via Wnt signaling under hypoxia.

Bladder cancer is frequently occurred in urinary system and has complicated pathogenesis factors including both genetics and environmental factors that have not been fully illustrated. Hypoxia can further induce tumor progression. ROCK2 has abnormal expression in various tumors but its expression or functional role in bladder cancer have not been illustrated. In vitro cultured bladder cancer cell line T24 was randomly assigned into control group, hypoxia group (prepared under hypoxic culture), and ROCK2 siRNA group (transfected with ROCK2 siRNA after hypoxia treatment). Real-time PCR and Western bot measured ROCK2 expression. MTT assay tested cell proliferation, and cell migration was quantified. Cell apoptosis was measured by caspase3 activity assay kit and Transwell chamber measured cell migration. Western blot quantified expressional change of HIF-1α and E-cadherin, and Wnt signal pathway proteins including Wnt4, and ß-catenin. ROCK2 is up-regulated in bladder cancer T24 cells under hypoxia, and can facilitate cell proliferation, migration and invasion, inhibited Caspase3 activity, enhanced HIF-1α expression, decreased E-cadherin expression, and up-regulated Wnt4 and ß-catenin (p< 0.05 comparing to hypoxia group). Under hypoxia conditions, ROCK2 can facilitate apoptosis of bladder cancer cells via modulating Wnt signal pathway, inhibit cell proliferation, migration, invasion or formation of epithelial mesenchymal transition (EMT).

Effect of Hemodialysis on the Red Blood Cell Life Span in Patients with End-Stage Kidney Disease.

The aim of this study was to use a CO breath test to investigate hemodialysis effects on red blood cell lifespan in patients with chronic kidney disease. A cohort of 17 non-smoking men with end-stage kidney disease undergoing hemodialysis via a polysulfone dialysis membrane (as opposed to a traditional cellulose acetate membrane) were subjected to a repeated Levitt's CO breath test to compare red blood cell lifespan before vs. after dialysis. None of the patients showed significant fluctuations in endogenous CO concentration during the dialysis procedure. The mean red blood cell lifespan was 66.0 ± 31.0 days before dialysis and 72.0 ± 26.0 days after dialysis, with no significant difference between the assessment time points (P > 0.05). In conclusion, dialysis using a polysulfone membrane did not appear to disrupt red blood cells or reduce their lifespan in patients with end-stage kidney disease.

A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray.

BACKGROUND: DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing. RESULTS: We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients. CONCLUSION: This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

Microarray-based Ms-SNuPE: near-quantitative analysis for a high-throughput DNA methylation.

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In the present study, a microarray-based methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for parallel detecting changes of DNA methylation in cancer was developed. After modification by sodium sulfite, the unmethylated cytosine in the genomic DNA is converted to uracil while leaving the 5-methylcytosine unchanged, which can be detected by bifunctional primer carrying a unique sequence tag in addition to a locus-specific sequence. Because each locus has a distinct tag, the detecting reactions can be performed in a highly multiplexed fashion and the resulting product then be hybridized to the reverse complements of the sequence tags arrayed on a glass slide for methylation analysis. The calibration curves with the correlation coefficient >0.97 were established, which suggested that the method could be used in near-quantitative DNA methylation analysis. Two breast tumor-related genes (E-cad and p16) are successfully analyzed by two group primers (22 primers total), and the results are compatible with that of methylation-specific PCR (MSP). Our research proved that the method is simple and inexpensive, and could be applied as a high-throughput tool to quantitatively determine methylation status of the investigated genes.

Detailed methylation patterns and protein expression profiles of MGMT in colorectal carcinoma surgical margins.

OBJECTIVES: Molecular margin analysis is considered more sensitive in detecting preneoplastic lesions and residual cancer cells than conventional histological margin examination. Hence, we examined MGMT expression profile and methylation status in histologically negative margins of colorectal cancer patients. DESIGN AND METHODS: This study included 24 colorectal tumor tissues and corresponding negative surgical margin tissues. MGMT promoter methylation patterns were analyzed by using methylation-specific oligonucleotide microarray. In addition, MGMT protein expression was analyzed by immunohistochemistry. MGMT clinical significance was evaluated together with other well-known clinicopathological factors. RESULTS: Extensive MGMT promoter methylation was observed in tumor tissues; a moderate methylation level was found in surgical margin tissues and little or no methylation was observed in the normal control. There was a trend towards longer overall survival for those patients with negative MGMT immunostaining in surgical margins. CONCLUSIONS: MGMT expression negative in surgical margin tissues indicates longer overall survival for colorectal tumor patients.