Zhihua Kang

Molecular Analysis of RNF213 Gene for Moyamoya Disease in the Chinese Han Population

Background Moyamoya disease (MMD) is an uncommon cerebrovascular disorder characterized by progressive occlusion of the internal carotid artery causing cerebral ischemia and hemorrhage. Genetic factors in the etiology and pathogenesis of MMD are being increasingly recognized. Previous studies have shown that the RNF213 gene was related to MMD susceptibility in the Japanese population. However, there is no large scale study of the association between this gene and MMD in the Chinese Han population. Thus we designed this case-control study to validate the R4810K mutation and to define the further spectrum of RNF213 mutations in Han Chinese. Methodology/Principal Findings Genotyping of the R4810K mutation in the RNF213 gene was performed in 170 MMD cases and 507 controls from a Chinese Han population. The R4810K mutation was identified in 22 of 170 MMD cases (13%), including 21 heterozygotes and a single familial homozygote. Two of the 507 controls (0.4%) were heterozygous R4810K carriers. The R4810K mutation greatly increased the risk for MMD (OR = 36.7, 95% CI: 8.6∼156.6, P = 6.1 E-15). The allele frequency of R4810K was significantly different between patients with ischemia and hemorrhage (OR = 5.4, 95% CI: 1.8∼16.1, P = 0.001). Genomic sequencing covering RNF213 exon 40 to exon 68 also identified eight other non-R4810K variants; P4007R, Q4367L, A4399T, T4586P, L4631V, E4950D, A5021V and M5136I. Among them A4399T polymorphism was found in 28/170 cases (16.5%) and 45/507 controls (8.9%) and was associated with MMD (OR = 2.0, 95% CI: 1.2∼3.3, P = 0.004), especially with hemorrhage (OR = 2.8, 95% CI: 1.2∼6.5, P = 0.014). Conclusions RNF213 mutations are associated with MMD susceptibility in Han Chinese. The ischemic type MMD is particularly related to the R4810K mutation. However, A4399T is also a susceptible variant for MMD, primarily associated with hemorrhage. Identification of novel variants in the RNF213 gene further highlights the genetic heterogeneity of MMD.

The mutation profile of JAK2 and CALR in Chinese Han patients with Philadelphia chromosome-negative myeloproliferative neoplasms

Mutations in JAK2, MPL and CALR are highly relevant to the Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs). We performed high resolution melting analysis and Sanger sequencing together with T-A cloning to elucidate the unique mutation profile of these genes, in Chinese patients with MPNs. Peripheral blood DNA samples were obtained from 80 patients with polycythemia vera (PV), 80 patients with essential thrombocytosis (ET) and 50 patients with primary myelofibrosis (PMF). Ten PV patients were identified with diverse JAK2 exon 12 mutations. Five novel JAK2 Exon 12 mutation patterns (M532V/E543G, N533D, M535I/H538Y/K549I, E543G and D544N) were described. JAK2 V617F was detected in 140 samples (66 PV, 45 ET and 29 PMF). JAK2 Exon 12 mutations were prevalent (13%) and variable in the Chinese patients. Compared with PV patients with JAK2 V617F mutations, PV patients with JAK2 exon 12 mutations had an earlier median onset of disease (P = 0.0013). MPL W515L/K mutations were discerned in 4 ET and 3 PMF patients. Two kinds of CALR mutation, c. 1179_1230del and c. 1234_1235insTTGTC were detected in 20 ET and 16 PMF patients. A novel CALR mutation pattern (c. 1173_1223del/c. 1179_1230del) was identified in 2 PMF samples. In addition, 17 scattered point mutations in CALR c.1153 to c.1255 were also detected in 13 cases with CALR frame-shifting variations and 2 cases without CALR frame-shifting variations. Female patients showed a predisposition to CALR mutations (P = 0.0035). Chinese Ph-negative MPN patients have a unique mutation landscape in the common molecular markers of MPN diagnosis. Validation of the molecular diagnostic pipeline should be emphasized since there is a considerable ethnical diversity in the molecular profiles of Ph-negative MPNs.

Downregulation of EphA5 by promoter methylation in human prostate cancer

BackgroundEphA5 is a member of the Eph/ephrin family and plays a critical role in the regulation of carcinogenesis. A significant reduction of EphA5 transcripts in high-grade prostate cancer tissue was shown using a transcriptomic analysis, compared to the low-grade prostate cancer tissue. As less is known about the mechanism of EphA5 downregulation and the function of EphA5, here we investigated the expression and an epigenetic change of EphA5 in prostate cancer and determined if these findings were correlated with clinicopathologic characteristics of prostate cancer.MethodsSeven prostate cell lines (RWPE-1, LNCap, LNCap-LN3, CWR22rv-1, PC-3, PC-3M-LN4, and DU145), thirty-nine BPH, twenty-two primary prostate carcinomas, twenty-three paired noncancerous and cancerous prostate tissues were examined via qRT-PCR, methylation-specific PCR, bisulfite sequencing, immunohistochemistry and western blotting. The role of EphA5 in prostate cancer cell migration and invasion was examined by wound healing and transwell assay.ResultsDownregulation or loss of EphA5 mRNA or protein expression was detected in 28 of 45 (62.2%) prostate carcinomas, 2 of 39 (5.1%) hyperplasias, and all 6 prostate cancer cell lines. Methylation of the EphA5 promoter region was present in 32 of 45 (71.1%) carcinoma samples, 3 of 39 (7.7%) hyperplasias, and the 6 prostate cancer cell lines. Among 23 paired prostate carcinoma tissues, 16 tumor samples exhibited the hypermethylation of EphA5, and 15 of these 16 specimens (93.8%) shown the downregulation of EphA5 expression than that of their respectively matched noncancerous samples. Immunostaining analysis demonstrated that the EphA5 protein was absent or down-regulated in 10 of 13 (76.9%) available carcinoma samples, and 8 of these 10 samples (80.0%) exhibited hypermethylation. The frequency of EphA5 methylation was higher in cancer patients with an elevated Gleason score or T3-T4 staging. Following the treatment of 6 prostate cancer cell lines with 5-aza-2′-deoxycytidine, the levels of EphA5 mRNA were significantly increased. Prostate cancer cells invasion and migration were significantly suppressed by ectopic expression of EphA5 in vitro.ConclusionOur study provides evidence that EphA5 is a potential target for epigenetic silencing in primary prostate cancer and is a potentially valuable prognosis predictor and thereapeutic marker for prostate cancer.

Oncogenic mutations in extramammary Paget's disease and their clinical relevance

Extramammary Pagets disease (EMPD) is a rare cutaneous malignant neoplasm. The genetic alterations underlying its pathogenesis have less been described. Therefore, we analyzed the possible mutations in the KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, PIK3CA, AKT1, CTNNB1 and APC genes as well as methylation and expression of CDH1 in 144 EMPD cases and 42 matched normal skin tissues. A distinct mutation profile was identified in EMPDs with 27 (19%) cases mutant for RAS and RAF genes and 50 (35%) cases harboring oncogenic mutations in PIK3CA and AKT1. Moreover, a mutually exclusive pattern was observed in the genetic variants in these two signaling pathways. No mutation was detected in CTNNB1 and APC genes. High prevalence of low expression and hypermethylation of CDH1 gene was detected in 33 and 48% of the EMPD cases, respectively. Furthermore, PIK3CA and AKT1 mutations were significantly correlated with CDH1 hypermethylation which could explain why the majority of EMPD cases with mutant PIK3CA and AKT1 were invasive. Our study demonstrates that genetic variants associated with constitutive activation of RAS/RAF and PI3K/AKT pathways are involved in the pathogenesis of EMPD. This may represent novel therapeutic targets for this skin cancer.

Downregulation of DLC-1 Gene by Promoter Methylation during Primary Colorectal Cancer Progression

Purpose. DLC-1 is a tumor suppressor gene frequently silenced in human cancers. However, the pathogenicity of DLC-1 epigenetic silencing in the mucosa-adenoma-carcinoma transformation process of colorectal cancer (CRC) has not been studied. Methods. Promoter methylation status of DLC-1 was evaluated in 4 human CRC cell lines, 48 normal mucosa, 57 adenomas, and 80 CRC tissues with methylation-sensitive high-resolution melting analysis (MS-HRMA), while the mRNA expression was examined by qPCR. HRMA was utilized to detect the KRAS codon 12, 13 and BRAF V600Emutations. Results. Partial (1%–10%) and extensive (10%–100%) DLC-1 promoter methylations were observed in 10% and 0% of normal mucosa, 46% and 14% of adenomas, and 60% and 36% of CRCs, respectively. The promoter methylation of DLC-1 was related with the reduction of gene expression and the advanced Dukes stages (Stage C and D). DLC-1 promoter methylation and KRAS mutations are common concurrent pathological alternations. Conclusions. Epigenetic alternation plays a key role in the transcriptional silencing of DLC-1. It is also an independent risk factor related to the carcinogenesis of colorectal tumors and spans over its pathogenesis process. Therefore, DLC-1 promoter methylation quantitation may have a promising significance in the evaluation and management of CRC patients.

Correlation of DLC1 gene methylation with oncogenic PIK3CA mutations in extramammary Paget's disease

Extramammary Paget’s disease is a rare cutaneous malignant neoplasm. The genetic and epigenetic mechanisms underlying its pathology remain unknown. In this study, we investigated the expression levels, and mutation and methylation status of a common tumor suppressor gene, deleted in liver cancer 1 (DLC1), and an oncogene, PIK3CA, in tumor (n=132) and normal tissues (n=20) from unrelated patients. The presence of epigenetic and genetic lesions was then correlated to the patient pathology data to determine the potential role of these genes in extramammary Paget’s disease etiology and progression. The DLC1 gene was found to be downregulated in 43 (33%) tumors, as compared with immunohistochemistry results from normal tissues. Methylation-sensitive, high-resolution melting analysis indicated that the DLC1 promoter was hypermethylated in 51 (39%) extramammary Paget’s disease tumors. This hypermethylation was associated with significantly decreased DLC1 levels (P=0.011), and had a strong positive correlation with advanced age (P=0.002). PIK3CA mutations were detected by direct sequencing in 32 (24%) tumors, the majority of which were invasive. Furthermore, PIK3CA mutations significantly correlated with DLC1 hypermethylation. Thus, aberrant DLC1 methylation and PIK3CA mutations may have important roles in extramammary Paget’s disease pathogenesis, and may represent potential molecular targets for therapy.

Effects of Ambient Fine Particles PM2.5 on Human HaCaT Cells

The current study was conducted to observe the effects of fine particulate matter (PM2.5) on human keratinocyte cell line (HaCaT) cells. The potential mechanism linking PM2.5 and skin was explored. HaCaT cells were cultured and then accessed in plate with PM2.5. Cell viability was tested by Cell Counting Kit-8. The mRNA and protein expression of Filaggrin, Loricrin, Involucrin, and Repetin were analyzed. The levels of Granulocyte-macrophage Colony Stimulating Factor, Thymic Stromal Lymphopoietin, Tumor Necrosis Factor-α, Interleukin-1α, and Interleukin-8 were detected in the supernatant of the HaCaT cell with enzyme-linked immunosorbent assay kits. Cell viability decreased with the increase in PM2.5. Compared with the control group, the protein expression of Filaggrin, Repetin, Involucrin, and Loricrin showed different expression patterns in PM2.5 treatment groups. The level of Tumor Necrosis Factor-α, Thymic Stromal Lymphopoietin, Interleukin-1α, and Interleukin-8 significantly increased in the cells treated with PM2.5. Ambient PM2.5 may increase the risk of eczema and other skin diseases. The relative mechanism may be associated with the impairment of the skin barrier and the elevation of inflammatory responses.

Toward point-of-care testing for JAK2 V617F mutation on a microchip.

Molecular genetics now plays a crucial role in diagnosis, the identification of prognostic markers, and monitoring of hematological malignancies. Demonstration of acquired changes such as the JAK2 V617F mutation within myeloproliferative neoplasms (MPN) has quickly moved from a research setting to the diagnostic laboratory. Microfluidics-based assays can reduce the assay time and sample/reagent consumption and enhance the reaction efficiency; however, no current assay has integrated isothermal amplification for point-of-care MPN JAK2 V617F mutation testing with a microchip. In this report, an integrated microchip that performs the whole human blood genomic DNA extraction, loop-mediated isothermal nucleic acid amplification (LAMP) and visual detection for point-of-care genetic mutation testing is demonstrated. This method was validated on DNA from cell lines as well as on whole blood from patients with MPN. The results were compared with those obtained by unlabeled probe melting curve analysis. This chip enjoys a high accuracy, operability, and cost/time efficiency within 1h. All these benefits provide the chip with a potency toward a point-of-care genetic analysis. All samples identified as positive by unlabeled probe melting curve analysis (n=27) proved positive when tested by microchip assay. None of the 30 negative controls gave false positive results. In addition, a patient with polycythemia vera diagnosed as being JAK2 V617F-negative by unlabeled probe melting curve analysis was found to be positive by the microchip. This microchip would possibly be very attractive in developing a point-of-care platform for quick preliminary diagnosis of MPN or other severe illness in resource-limited settings.

Genetic Analysis of Mismatch Repair Genes Alterations in Extramammary Paget Disease

Extramammary Paget disease (EMPD) is a rare cutaneous malignant neoplasm. The familial occurrence of EMPD and the high risk of concomitant secondary tumors in EMPD patients have gained much attention. These findings highlight the importance of genetic alterations in the tumorigenesis of this skin cancer. Genetic tests and functional analysis of mismatch repair (MMR) genes were performed in EMPD. The results showed that 8 of 20 cases with germline MMR genes mutations and 5 of them exhibited microsatellite instability (MSI). Immunohistochemical staining showed that the tumor tissues from 20 patients had the normal expression of MLH1 but 5 cases had the reduced expression of MSH2. There is a nearly significant correlation between MSI and germline mutations. In 172 cases, rates of germline and somatic mutations were 34.3% and 13.4%, respectively. The mutations of MLH1 V384D (15.7%), R217C (4.1%), and I219V (5.2%) were common in this cancer. In addition, the yeast 2-hybrid and immunoprecipitation assays exhibited reduced interaction between MLH1 and PMS2 in MLH1 V384D and R217C but not I219V. Moreover, MLH1 V384D and R217C had impaired MMR activity compared with the wild-type and I219V mutation by an in vitro MMR assay. The germline mutations in MMR genes are involved in the pathogenesis of EMPD and partially explain the genetic abnormalities for this disease.

Correlation of KIF3A and OVOL1, but not ACTL9, with atopic dermatitis in Chinese pediatric patients

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in Chinese pediatric patients. To date, the genetic susceptibility to AD in this population has not been fully clarified. Three single nucleotide polymorphisms have previously been associated with AD in Europeans, rs2897442 (KIF3A), rs479844 (OVOL1) and rs2164983 (ACTL9). To verify the correlation between AD and these three SNPs in the Chinese pediatric population, we conducted a case-control study including 235 pediatric patients with AD and 200 health controls. We confirmed the correlation between rs2897442 and rs479844 and AD in this population at both the genotype and allele levels. Statistical analysis showed that the C allele of rs2897442 is associated with an increased risk of developing AD, while the A allele of rs479844 is associated with a reduced risk. No correlation between rs2164983 and AD was identified. Our study indicates that KIF3A and OVOL1 are involved in the development of AD in the Chinese pediatric population.