Noninvasive prediction of axillary lymph node status in breast cancer using promoter profiling of circulating cell-free DNA.

BACKGROUND: Lymph node metastasis (LNM) is one of the most important factors affecting the prognosis of breast cancer. The accurate evaluation of lymph node status is useful to predict the outcomes of patients and guide the choice of cancer treatment. However, there is still lack of a low-cost non-invasive method to assess the status of axillary lymph node (ALN). Gene expression signature has been used to assess lymph node metastasis status of breast cancer. In addition, nucleosome footprint of cell-free DNA (cfDNA) carries gene expression information of its original tissues, so it may be used to evaluate the axillary lymph node status in breast cancer. METHODS: In this study, we found that the cfDNA nucleosome footprints between the ALN-positive patients and ALN-negative patients showed different patterns by implementing whole-genome sequencing (WGS) to detect 15 ALN-positive and 15 ALN-negative patients. In order to further evaluate its potential for assessing ALN status, we developed a classifier with multiple machine learning models by using 330 WGS data of cfDNA from 162 ALN-positive and 168 ALN-negative samples to distinguish these two types of patients. RESULTS: We found that the promoter profiling between the ALN-positive patients and ALN-negative patients showed distinct patterns. In addition, we observed 1071 genes with differential promoter coverage and their functions were closely related to tumorigenesis. We found that the predictive classifier based on promoter profiling with a support vector machine model, named PPCNM, produced the largest area under the curve of 0.897 (95% confidence interval 0.86-0.93). CONCLUSIONS: These results indicate that promoter profiling can be used to distinguish ALN-positive patients from ALN-negative patients, which may be helpful to guide the choice of cancer treatment.

Noninvasive inferring expressed genes and in vivo monitoring of the physiology and pathology of pregnancy using cell-free DNA.

BACKGROUND: Noninvasive monitoring of fetal development and the early detection of pregnancy-associated complications is challenging, largely because of the lack of information about the molecular spectrum during pregnancy. Recently, cell-free DNA in plasma was found to reflect the global nucleosome footprint and status of gene expression and showed potential for noninvasive health monitoring during pregnancy. OBJECTIVE: We aimed to test the relationships between plasma cell-free DNA profiles and pregnancy biology and evaluate the use of a cell-free DNA profile as a noninvasive method for physiological and pathologic status monitoring during pregnancy. STUDY DESIGN: We used genome cell-free DNA sequencing data generated from noninvasive prenatal testing in a total of 2937 pregnant women. For each physiological and pathologic condition, features of the cell-free DNA profile were identified using the discovery cohort, and support vector machine classifiers were built and evaluated using independent training and validation cohorts. RESULTS: We established nucleosome occupancy profiles at transcription start sites in different gestational trimesters, demonstrated the relationships between gene expression and cell-free DNA coverage at transcription start sites, and showed that the cell-free DNA profiles at transcription start sites represented the biological processes of pregnancy. In addition, using cell-free DNA data, nucleosome profiles of transcription factor binding sites were identified to reflect the transcription factor footprint, which may help to reveal the molecular mechanisms underlying pregnancy. Finally, by using machine-learning models on low-coverage noninvasive prenatal testing data, we evaluated the use of cell-free DNA nucleosome profiles for distinguishing gestational trimesters, fetal sex, and fetal trisomy 21 and highlighted its potential utility for predicting physiological and pathologic fetal conditions by using low-coverage noninvasive prenatal testing data. CONCLUSION: Our analyses profiled nucleosome footprints and regulatory networks during pregnancy and established a noninvasive proof-of-principle methodology for health monitoring during pregnancy.

Characterization of a Novel 71.8 kb α0-Thalassemia Deletion and Subsequent Summary of a Practical Procedure for Thalassemia Molecular Diagnosis.

Thalassemia is the most common monogenic disorder around the world. Based on the principle of genotype-phenotype correlation, identification of thalassemia mutations is the essential prerequisite for clinical diagnosis and management. Because only common mutations are routinely detected, the identification of rare or undetermined mutations is a challenge for clinical laboratories. Herein, a proband presenting with inconsistent phenotype-genotype correlation after routine molecular screening was investigated by multiplex ligation-dependent probe amplification (MLPA), targeted-next generation sequencing (targeted-NGS), gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. Eventually, a novel 71.8 kb deletion (- -71.8) was identified and characterized, which included HBZ (ζ), HBA2 (α2), and HBA1 (α1) genes and was causing α0-thalassemia (α0-thal). Furthermore, we summarized a practical procedure based on accumulated experience in studies and clinical practice, which can be a guide for molecular screening and clinical diagnosis of thalassemia, especially for identification of undetermined or novel mutations.

Semiconductor Sequencing Analysis of Chromosomal Copy Number Variations in Spontaneous Miscarriage.

BACKGROUND Array CGH is the criterion standard for identifying copy number variations (CNV), but the restrictive requirement of DNA quality and relatively high cost prevent the use of this method as a general assay in hospitals in developing countries. Our principal objective was to determine whether the semiconductor sequencing platform (SSP) could be an alternative method in CNV detection for spontaneous miscarriage. MATERIAL AND METHODS A total of 443 spontaneous miscarriage samples were collected and subjected to low-coverage (0.1X) whole-genome analysis by SSP. These samples were verified by array CGH and 8 low-quality DNA samples were analyzed by SSP and validated by MLPA. RESULTS SSP detected 195 chromosomal numerical abnormalities, 74 CNVs, and 9 mosaicisms among the 435 samples. Among 74 CNV abnormalities, SSP detected an equal number (56) of CNVs 56 >1 Mb with array CGH. However, SSP missed more 6 cases CNVs <1 Mb than array CGH (12 vs. 18). SSP detected more mosaicisms than array CGH (9 vs. 7, p=0.5). Interestingly, SSP detected the mosaicism which had only 8% X monosomy, which was much lower than the minimal percentage of monosomy that was detected by array CGH. CONCLUSIONS SSP is of equivalent efficacy as array CGH in detecting CNVs >1 Mb, and performs better in identifying mosaicism. With the merits of low cost and less demand of input DNA, SSP is a good alternative for use in genetic diagnosis.

Genetic polymorphisms in estrogen-related genes and the risk of breast cancer among Han Chinese women.

Exposure to high levels of estrogen is considered an important risk factor for susceptibility to breast cancer. Common polymorphisms in genes that affect estrogen levels may be associated with breast cancer risk, but no comprehensive study has been performed among Han Chinese women. In the present study, 32 single-nucleotide polymorphisms (SNPs) in estrogen-related genes were genotyped using the MassARRAY IPLEX platform in 1076 Han Chinese women. Genotypic and allelic frequencies were compared between case and control groups. Unconditional logistic regression was used to assess the effects of SNPs on breast cancer risk. Associations were also evaluated for breast cancer subtypes stratified by estrogen receptor (ER) and progesterone receptor (PR) status. Case-control analysis showed a significant relation between heterozygous genotypes of rs700519 and rs2069522 and breast cancer risk (OR = 0.723, 95% CI = 0.541-0.965, p = 0.028 and OR = 1.500, 95% CI = 1.078-2.087, p = 0.016, respectively). Subgroup comparisons revealed that rs2446405 and rs17268974 were related to ER status, and rs130021 was associated with PR status. Our findings suggest that rs700519 and rs2069522 are associated with susceptibility to breast cancer among the Han Chinese population and have a cumulative effect with three other identified SNPs. Further genetic and functional studies are needed to identify additional SNPs, and to elucidate the underlying molecular mechanisms.

CHRNA3 polymorphism modifies lung adenocarcinoma risk in the Chinese Han population.

Recent genome-wide association studies (GWASs) have identified 15q25.1 as a lung cancer susceptibility locus. Here, we sought to explore the direct carcinogenic effects of genetic variants in this region on the risk of developing lung adenocarcinoma (ADC). Five common SNPs (rs8034191, rs16969968, rs1051730, rs938682, and rs8042374) spanning the 15q25.1 locus were assayed in a case-control study examining a cohort of 301 lung ADCs and 318 healthy controls. Stratification analysis by gender, smoking status, and tumor, node, metastasis (TNM) classification, was performed. In addition, sections from ADC tissue and normal tissue adjacent to tumors were stained with an anti-CHRNA3 (cholinergic receptor nicotinic α3) antibody by immunohistochemistry in 81 cases. Our results demonstrate that rs8042374, a variant of the CHRNA3 gene, is associated with an increased risk of ADC with an OR of 1.76 (95% CI: 1.17-2.65, p=0.024). This variant was linked to a greater risk of ADC in female nonsmokers (OR (95% CI): 1.81 (1.05-3.12), p=0.032) and female stage I+II cases (OR (95% CI): 1.92 (1.03-3.57), p=0.039). Although located within the same gene, rs938682 showed protective effects for smokers, stage III+IV cases, and male stage III+IV cases. Additionally, the CHRNA3 protein level in ADC tissue was slightly higher than in the surrounding normal lung tissue, based on immunohistochemical analysis. Our results suggest that the CHRNA3 polymorphism functions as a genetic modifier of the risk of developing lung ADC in the Chinese population, particularly in nonsmoking females.

A functional variant of NEDD4L is associated with obesity and related phenotypes in a Han population of Southern China.

NEDD4L is a candidate gene for hypertension, both functionally and genetically. Recently, studies showed evidence for the association of NEDD4L with obesity, a key intermediate phenotype in hypertension. To further investigate the relationship between NEDD4L and body mass-related phenotypes, we genotyped three common variants (rs2288774, rs3865418 and rs4149601) in a population-based study of 892 unrelated Han Cantonese using the Sequenom MALDI-TOF-MS platform. Allele frequencies and genotype distribution were calculated in lean controls and overweight/obese cases and analyzed for association by the Chi-squared test and Logistic regression. Linear regression analysis was used to analyze the effect of individual genotypes on quantitative traits. Multivariate analyses demonstrated that the minor allele of rs4149601(A = 20.9%) was associated with a 2.60 kg, 2.78 cm and 0.97 kg/m2 decrease per allele copy in weight, waist and BMI, respectively. Carriers of this allele also had a significant lower risk of overweight/obesity (p < 0.0001, OR = 0.52, 95% CI: 0.37-0.74) as compared to non-carriers. However, no significant association between genotypes at rs2288774 and rs3865418 and covariate-adjusted overweight/obesity or any related phenotypes was observed. These results suggested that the functional variant of NEDD4L, rs4149601, may be associated with obesity and related phenotypes, and further genetic and functional studies are required to understand its role in the manifestation of obesity.

Neurogenic locus notch homolog protein 4 and brain-derived neurotrophic factor variants combined effect on schizophrenia susceptibility.

OBJECTIVES: To investigate the relationships between single-nucleotide polymorphisms (SNPs) in NOTCH4 and brain-derived neurotrophic factor (BDNF) with schizophrenia among Han Chinese in Southern China. METHODS: Two NOTCH4 SNPs (rs520688 and rs415929) and two BDNF SNPs (rs2030324 and rs12273539) were examined in 464 schizophrenics and 464 healthy controls from Hunan province in South China, using the Sequenom MassARRAY® iPLEX System. RESULTS: In the study population, rs520688 and rs2030324 were significantly associated with schizophrenia. A decreased risk of schizophrenia was associated with the rs520688 GA genotype (p = 0.035), whereas an increased risk of schizophrenia was associated with the rs2030324 CC/CT genotype (p = 0.044). The genotype distributions of rs415929 in NOTCH4 and rs12273539 in BDNF did not differ significantly between the case and control groups. Although no allele-allele interactions were detected between rs520688 and rs2030324, recombination analysis revealed a combined effect of the two on the susceptibility to schizophrenia, with GA-TT decreasing and CT/CC-GG/GA increasing the risk of schizophrenia. CONCLUSION: In conclusion, rs520688 in NOTCH4 and rs2030324 in BDNF are significantly associated with schizophrenia among Han Chinese in Southern China. The two had a combined effect on the susceptibility to schizophrenia among Han Chinese in Southern China, but this may not be caused by an allele-allele interaction.

[DAZL gene polymorphisms and astheno-teratozoospermia].

OBJECTIVE: To investigate the association between single nucleotide polymorphism (SNP) of the DZAL gene in infertile Han Chinese males with astheno-teratozoospermia. METHODS: We collected semen samples from 173 infertile Han Chinese men with astheno-teratozoospermia (case group) and 175 age-matched normal male volunteers (control group) for semen routine and morphological analyses. We obtained genomic DNA, genotyped the polymorphisms of the DAZL gene A260G and A386G via the Sequenom MassARRAY system, and compared the frequencies of the genotypes between the case and control groups. RESULTS: The AA nucleotide variant was found in the A260G and A386G polymorphisms of the DZAL gene in both the cases and controls, but the heterozygous AG variant in neither. CONCLUSION: The A260G and A386G polymorphisms of the DAZL gene are not correlated with astheno-teratozoospermia-induced male infertility in the Han Chinese population, and therefore could not be considered as molecular markers of male infertility.

Association of 10q23 with colorectal cancer in a Chinese population.

Recently, a genome-wide association study of gastric cancer (GC) reported the significant association of seven genetic variants (rs4072037 and rs4460629 on 1q22; rs753724, rs11187842, rs3765524, rs2274223, and rs3781264 on 10q23) with GC in a Chinese population. These findings were confirmed in a subsequent independent study. However, it remains unknown whether these loci are associated with an increased risk of colorectal cancer (CRC). This study was to test whether the seven single nucleotide polymorphisms (SNPs) associated with GC were also associated with CRC in a Chinese population. The seven SNPs were genotyped using MassARRAY system. Allelic, genotypic, and haplotypic associations of the SNPs with CRC were investigated using χ(2) tests and logistic regression analysis. The SNPs rs3765524 and rs2274223, located on 10q23, were found to have significant protective effects against CRC, with equal odds ratios per allele. The two SNPs located on 1q22 (rs4072037 and rs4460629) showed a weak association with CRC. No significant association was identified with CRC for the remaining three SNPs located on 10q23 (rs753724, rs11187842, and rs3781264). These results suggest that rs3765524 and rs2274223 on 10q23 are associated with a protective effect against CRC in a Chinese population.

Risk-association of DNA methyltransferases polymorphisms with gastric cancer in the Southern Chinese population.

DNA hypomethylation and/or hypermethylation are presumed to be early events in carcinogenesis, and one or more DNA methyltransferases (DNMTs) have been suggested to play roles in carcinogenesis of gastric cancer (GC). However, there have been no systematic studies regarding the association between DNMT gene polymorphisms and GC risk. Here, we examined the associations of 16 single nucleotide polymorphisms (SNPs) from DNMT1 (rs2114724, rs2228611, rs2228612, rs8101866, rs16999593), DNMT2 (rs11695471, rs11254413), DNMT3A (rs1550117, rs11887120, rs13420827, rs13428812, rs6733301), DNMT3B (rs2424908, rs2424913, rs6087990) and DNMT3L (rs113593938) with GC in the Southern Chinese population. We assessed the associations of these 16 SNPs with GC in a case-control study that consisted of 242 GC cases and 294 controls, using the Sequenom MALDI-TOF-MS platform. Association analyses based on the χ(2) test and binary logistic regression were performed to determine the odds ratio (OR) and 95% confidence interval (95%CI) for each SNP. We found that rs16999593 in DNMT1, rs11254413 in DNMT2 and rs13420827 in DNMT3A were significantly associated with GC susceptibility (OR 1.45, 0.15, 0.66, respectively; 95% CI 1.00-2.11, p = 0.047; 0.08-0.27, p < 0.01; 0.45-0.97, p = 0.034, respectively, overdominant model). These results suggested that DNMT1, DNMT2 and DNMT3A may play important roles in GC carcinogenesis. However, further studies are required to elucidate the mechanism.

[Association of PRM1-190C- > A polymorphism with teratozoospermia].

OBJECTIVE: To investigate the association of single nucleotide polymorphism (SNP) of the Protamine 1 (PRM1) gene in infertile men with teratozoospermia. METHODS: We collected semen samples from 157 infertile men with teratozoospermia (case group) and 37 age-matched male volunteers (control group), and subjected them to morphological analysis. We extracted genome DNA, genotyped the polymorphism of the PRM1-190C- > A SNP (rs2301365) using the Sequenom MassARRAY system, compared the genotype frequencies between the case and control groups, and analyzed the sperm morphological parameters of different genotypes in the infertile males with teratozoospermia. RESULTS: The frequencies of the genotypes CC, CA and AA were 38.9% (61), 44.6% (70) and 16.6% (26) in the case group, as compared with 45.9% (17), 51.4% (19) and 2.7% (1) in the control, with that of AA significantly higher in the patients than in the volunteers (P<0.05). The frequencies of the alleles C and A were 57.6% and 42.4% in the former, with no significant differences from 71.6% and 28.4% in the latter (P>0.05). Nor were any statistically significant differences observed in sperm morphology parameters between the genotype CC and CA, AA and CA + AA in the male patients (P>0.05). CONCLUSION: The SNP of PRM1-190C- > A might be associated with teratozoospermia-induced male infertility in the Han Chinese. Although this SNP may attribute to abnormal sperm morphology, the targeted part of sperm remains unclear.

Single-nucleotide polymorphism associations for colorectal cancer in southern chinese population.

OBJECTIVE: Genome-wide association studies (GWAS) have identified 11 loci that influence the risk of developing colorectal cancer (CRC). Given that these studies were conducted in European Caucasian populations, it is not clear whether the results are relevant for populations with different ethnicities. The aim of this study was to examine these associations in a southern Chinese population. METHODS: Eleven single-nucleotide polymorphisms (SNPs), rs12701937, rs16892766, rs7014346, rs6983267, rs719725, rs10795668, rs3802842, rs4444235, rs9929218, rs10411210, and rs961253, were genotyped in 229 CRC patients and 267 controls using the MassArray SNP genotyping system. RESULTS: Evidence of an association with CRC was found for four of the 11 loci. The strongest associations were with rs4444235 and rs961253, with significant odds ratios close to those reported in previous GWAS. Among these four loci, rs719725 and rs4444235 were significantly associated with female gender, rs3802842, rs961253, and rs4444235 with early disease onset, and rs3802842 with later disease onset. However, no associations with CRC risk were detected for six other loci (rs9929218, rs10411210, rs12701937, rs7014346, rs6983267, and rs10795668), and one SNP, rs16892766, was not polymorphic in any of the study participants. CONCLUSION: The rs4444235 and rs961253 loci are strongly associated with the risk of CRC in southern Chinese.

A risk-associated single nucleotide polymorphism of SMAD7 is common to colorectal, gastric, and lung cancers in a Han Chinese population.

SMAD7 has been demonstrated to antagonize TGF-ß-mediated fibrosis, carcinogenesis, and inflammation. Two previous genome-wide association studies identified three single nucleotide polymorphisms (SNPs) (rs4939827, rs12953717 and rs4464148) in SMAD7 to be associated with colorectal cancer in a Western population. We conducted the first case-control study in a Han Chinese population to explore the associations between these three SNPs and colorectal, gastric, and lung cancers. Of the three SNPs, only rs12953717 was strongly associated with the three types of cancer, fitting the overdominant model. Compared with the CC/TT (CC combined with TT) genotype, the adjusted odds ratios for the CT genotype were 2.002 (95% CI, 1.250-3.207, P = 0.004), 1.678 (95% CI, 1.048-2.689, P = 0.031), 3.825 (95% CI, 2.310-6.335, P < 1 × 10(-4)), and 2.294 (95% CI, 1.537-3.343, P < 1 × 10(-4)), respectively, for colorectal, gastric, lung, and combined cancers. These outcomes suggest that rs12953717 is a common risk marker of these three types of cancer in the Han Chinese.

Aberrant CpG island hypermethylation and down-regulation of Oct-6 mRNA expression in human hepatocellular carcinoma.

BACKGROUND: Aberrant CpG island hypermethylation is a major epigenetic mechanism that can inactivate the transcription of cancer-related genes. PURPOSE: This study aimed to investigate whether Oct-6 transcription was regulated by CpG island methylation in hepatocellular carcinoma (HCC). METHODS: Quantitative real-time PCR and the MassARRAY platform (Sequenom) were employed in 38 HCC tissues samples and four cell lines. RESULTS: The levels of Oct-6 mRNA were decreased by more than twofold in 31 of 38 tumor tissues compared to that of adjacent non-cancerous tissues. Among the 31 tumor tissues with lower levels of Oct-6 mRNA, 17 tumor tissues also had higher methylation levels in Oct-6 CpG island. Based on these results, we hypothesized that CpG island hypermethylation may down-regulate Oct-6 mRNA expression in HCC. To confirm this hypothesis, we also analyzed the changes in Oct-6 mRNA expression and CpG island methylation in four HCC cell lines (Huh7, Bel-7402, HepG2 and SMMC-7721) after treatment with 0.1, 0.5 and 2.5 µM 5-Aza-2-deoxycytidine (5-Aza-CdR), a demethylating agent. The results demonstrated that the CpG island methylation levels decreased and Oct-6 mRNA levels increased in a dose-dependent manner in both Huh7 and Bel7402 cells, but there were only slight changes in HepG2 cell. Interestingly, there were no significant alterations of Oct-6 mRNA levels observed in SMMC7721 cell; although lower levels of CpG island methylation were detected after treatment with 5-Aza-CdR. CONCLUSIONS: Our study shows that CpG island hypermethylation contributes to down-regulation of Oct-6 mRNA expression in HCC.

Association of TGF-beta1, IL-4 and IL-13 gene polymerphisms with asthma in a Chinese population.

BACKGROUND: Asthma is a common respiratory disease caused by genetic and environmental factors. It has been suggested that TGF-beta1, IL-4 and IL-13 play important roles in asthma. OBJECTIVES: We attempted to confirm the roles of TGF-beta1, IL-4 and IL-13 polymorphisms in asthma in a Chinese population. METHODS: Five SNPs (rs1800469, rs2241712, rs2070874, rs20541 and rs1800925) in TGF-beta1, IL-4 and IL-13 were genotyped using the MassArray SNP genotyping system. Allelic and genotypic associations between these SNPs and asthma were evaluated using logistic regression analysis. RESULTS: The CT genotype of rs1800469 and T allele of rs20541 were significantly associated with asthma. Among atopic subjects, the CT genotype of rs1800469 and GA genotype of rs2241712 decreased the risk of asthma, while the CC genotype of rs2070874 showed a decreasing trend of asthma risk with a borderline significance. No significant association was found between rs1800925 and asthma. CONCLUSION: In the present study, we confirmed the association of rs1800469 in TGF-beta1 and rs20541 in IL-13 with asthma and found a trend toward association between rs2241712 in TGF-beta1 and rs2070874 in IL-4 with asthma among atopic subjects, suggesting TGF-beta1, IL-4 and IL-13 may be associated with the susceptibility and development of asthma in this Chinese population.

[Association of the C3 gene polymorphisms with susceptibility to adult asthma].

OBJECTIVE: To investigate the association between single nucleotide polymorphisms (SNPs) of the complement component 3 gene (C3) and adult asthma of Hans in southern China. METHODS: A case-control study was performed. Four hundred and eighty-four adult asthma patients diagnosed in Nanfang Hospital and Affiliated Hospital of Guangdong Medical College, and 553 healthy subjects were collected from 2006 to 2010 for the study. MassARRAY-IPLEX and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) techniques was used to determine the genotypes of the rs10402876 and rs366510 loci of C3 gene. RESULTS: Genotypes GG, GT and TT in the rs366510 locus, and genotypes GG, GT and TT in the rs10402876 locus were detected. A total of 98.94 percent of samples were genotyped. There were no significant differences in genotype frequencies (chi-square =0.346,P=0.841) and allele frequencies (chi-square =0.101,P=0.751) of rs10402876 between the two groups. However, genotype and allele frequencies of the rs366510 locus were significantly different (chi-square =9.759, P=0.008, Bonferroni correction, P=0.016; chi-square =5.294,P=0.021, Bonferroni correction, P=0.042, respectively). Compared with genotypes GG+GT, genotype TT of rs366510 significantly increased the risk of asthma, with the odds ratio of 1.471 (95% confidence interval 1.125-1.923). CONCLUSION: These results suggest that C3 gene could be associated with adult asthma of Han population in southern China.

[Association of single nucleotide polymorphisms of MD-1 gene with asthma in adults of Han Nationality in Southern China].

OBJECTIVE: To investigate the correlation between MD-1 gene single nucleotide polymorphism (SNP) and bronchial asthma in adults of the Han nationality in Southern China as well as their lung functions. METHODS: From 2006 to 2010, 332 adult asthmatic patients of the Han nationality diagnosed in Nanfang Hospital, Southern Medical University, and 276 healthy volunteers were recruited for the study. The SNPs of MD-1 gene loci rs2233128 and rs7740529 were genotyped by using MassARRAY-IPLEX technology and matrix-assisted laser desorption ionization time of flight mass spectrometry platform (MALDI-TOF-MS). In addition, the lung function of 105 cases initially diagnosed with asthma was measured. RESULTS: There was no significant difference in sex (χ(2) = 0.50, P = 0.824) and age (t = 0.930, P = 0.357) between the asthma and the healthy groups. Genotypes AG and GG were detected in MD-1 gene at rs2233128 locus and genotypes CC, CT and TT at rs7740529 locus in this Han population at Southern China. Genotyping success rate was 94.74%. The rs2233128 genotype distribution (χ(2) = 0.030, P = 0.863) and allele (χ(2) = 0.029, P = 0.865) showed no significant difference between the asthma and the control groups. However, the rs7740529 genotype distribution (χ(2) = 8.681, P = 0.013) and allele (χ(2) = 8.005, P = 0.005) were significantly different in the asthma group as compared to the control group. Compared with the TT genotype, the rs7740529 of the CC and CC + CT genotypes could dramatically increase the risk of asthma, the odds ratio (95% confidence interval) being 2.451 (1.299 - 4.9626) and 2.172 (1.173 - 4.020), respectively. In the 105 patients initially diagnosed with asthma, FVC% and FEV(1)% were significantly different among the 3 different genotypes (CC, CT and TT) of the rs7740529 locus (F = 18.405, P = 0.000 and F = 25.700, P = 0.000). The percentages of predicted value of FVC and FEV(1) were decreased [(65 ± 12)%, (64 ± 11)%] in the CC genotype as compared to the TT genotypes [(86 ± 9)%, (88 ± 8)%], P < 0.05. There was no significant difference of genotype distribution of rs7740529 in different grades of severity of newly diagnosed asthma (χ(2) = 5.017, P = 0.081). CONCLUSIONS: MD-1 may be a susceptible gene for adult asthma in a Southern Han population in China. Genotype distribution of rs7740529 locus in newly diagnosed patients with asthma may be related to their lung function changes.

Genetic Variants in MMP9 and TCF2 Contribute to Susceptibility to Lung Cancer.

OBJECTIVE: The Wnt signaling pathway is crucial for pulmonary development and differentiation; dysregulation of the Wnt signaling pathway may impair lung function. Indeed, single nucleotide polymorphisms (SNPs) of Wnt pathway-related genes have been suggested as risk factors for certain types of cancers. In this study, we aimed to evaluate the influence of SNPs in Wnt-related genes (TCF2, MMP9) on susceptibility to lung cancer. METHODS: Polymorphisms of TCF2 rs4430796, MMP9 rs2250889, and MMP9 rs17576 were studied in Han Chinese subjects, including 135 patients with lung cancer and 176 controls, using the Sequenom MassARRAY platform. The association of genotypes with susceptibility to lung cancer was analyzed using odds ratio (OR), with 95% confidence interval (95% CI) and χ(2). RESULTS: The three SNPs (rs4430796, rs2250889, and rs17576) were found to be significantly associated with an increased risk of lung cancer. The AA genotype and AG+AA genotype of rs4430796 showed a significantly increased susceptibility to lung cancer compared with the GG genotype (adjusted OR=6.03, 95% CI: 1.30-28.09, P=0.022; 5.55, 95% CI: 1.20-25.58, P=0.028). Compared with the rs17576 GG genotype, the AG and AG+AA genotypes were also associated with a significant risk (adjusted OR=2.65, 95% CI: 1.60-4.37, P≤0.001; 2.57, 95% CI: 1.59-4.19, P≤0.001) whereas the rs2250889 CG and CG+GG genotypes had 2.97-fold (95% CI: 1.81-4.85; P≤0.001) and 2.80-fold increased associations with lung cancer (95% CI: 1.73-4.54; P≤0.001), respectively, compared with the rs2250889 CC genotype. Furthermore, the association of rs4430796 with lung cancer became insignificant (P>0.05) after adjusting for gender and rs2250889. CONCLUSION: The three SNPs may play a role in the predisposition of members of the Han Chinese population to lung cancer.

Targeted Next-Generation Sequencing of Circulating Tumor DNA Mutations among Metastatic Breast Cancer Patients.

Liquid biopsy through the detection of circulating tumor DNA (ctDNA) has potential advantages in cancer monitoring and prediction. However, most previous studies in this area were performed with a few hotspot genes, single time point detection, or insufficient sequencing depth. In this study, we performed targeted next-generation sequencing (NGS) with a customized panel in metastatic breast cancer (MBC) patients. Fifty-four plasma samples were taken before chemotherapy and after the third course of treatment for detection and analysis. Paired lymphocytes were also included to eliminate clonal hematopoiesis (CH)-related alternatives. A total of 1182 nonsynonymous mutations in 419 genes were identified. More ctDNA mutations were detected in patients with tumors > 3 cm (p = 0.035) and HER2(-) patients (p = 0.029). For a single gene, the distribution of ctDNA mutations was also correlated with clinical characteristics. Multivariate regression analysis revealed that HER2 status was significantly associated with mutation burden (OR 0.02, 95% CI 0-0.62, p = 0.025). The profiles of ctDNA mutations exhibited marked discrepancies between two time points, and baseline ctDNA was more sensitive and specific than that after chemotherapy. Finally, elevated ctDNA mutation level was positively correlated with poor survival (p < 0.001). Mutations in ctDNA could serve as a potential biomarker for the evaluation, prediction, and clinical management guidance of MBC patients with chemotherapy.