Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing.

Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.

Systematic search for placental DNA-methylation markers on chromosome 21: toward a maternal plasma-based epigenetic test for fetal trisomy 21.

BACKGROUND: The presence of fetal DNA in maternal plasma represents a source of fetal genetic material for noninvasive prenatal diagnosis; however, the coexisting background maternal DNA complicates the analysis of aneuploidy in such fetal DNA. Recently, the SERPINB5 gene on chromosome 18 was shown to exhibit different DNA-methylation patterns in the placenta and maternal blood cells, and the allelic ratio for placenta-derived hypomethylated SERPINB5 in maternal plasma was further shown to be useful for noninvasive detection of fetal trisomy 18. METHODS: To develop a similar method for the noninvasive detection of trisomy 21, we used methylation-sensitive single nucleotide primer extension and/or bisulfite sequencing to systematically search 114 CpG islands (CGIs)-76% of the 149 CGIs on chromosome 21 identified by bioinformatic criteria-for differentially methylated DNA patterns. The methylation index (MI) of a CpG site was estimated as the proportion of molecules methylated at that site. RESULTS: We identified 22 CGIs which were shown to contain CpG sites that were either completely unmethylated (MI = 0.00) in maternal blood cells and methylated in the placenta (MI range, 0.22-0.65), or completely methylated (MI = 1.00) in maternal blood cells and hypomethylated in the placenta (MI range, 0.00-0.75). We detected, for the first time, placental DNA-methylation patterns on chromosome 21 in maternal plasma during pregnancy and observed their postpartum clearance. CONCLUSION: Twenty-two (19%) of the 114 studied CGIs on chromosome 21 showed epigenetic differences between samples of placenta and maternal blood cells; these CGIs may provide a rich source of markers for noninvasive prenatal diagnosis.

Hypermethylation of RASSF1A in human and rhesus placentas.

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.

Plasma Epstein-Barr viral deoxyribonucleic acid quantitation complements tumor-node-metastasis staging prognostication in nasopharyngeal carcinoma.

PURPOSE: To evaluate the effect of combining circulating Epstein-Barr viral (EBV) DNA load data with TNM staging data in pretherapy prognostication of nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS: Three hundred seventy-six patients with all stages of NPC were studied. Pretreatment plasma/serum EBV DNA concentrations were quantified by a polymerase chain reaction assay. Determinants of overall survival were assessed by multivariate analysis. Survival probabilities of patient groups, segregated by clinical stage (I, II, III, or IV) alone and also according to EBV DNA load (low or high), were compared. RESULTS: Pretherapy circulating EBV DNA load is an independent prognostic factor for overall survival in NPC. Patients with early-stage disease were segregated by EBV DNA levels into a poor-risk subgroup with survival similar to that of stage III disease and a good-risk subgroup with survival similar to stage I disease. CONCLUSION: Pretherapy circulating EBV DNA load is an independent prognostic factor to International Union Against Cancer (UICC) staging in NPC. Combined interpretation of EBV DNA data with UICC staging data leads to alteration of risk definition of patient subsets, with improved risk discrimination in early-stage disease. Validation studies are awaited.

Reduced plasma RNA integrity in nasopharyngeal carcinoma patients.

PURPOSE: Recent research has shown the feasibility of detecting cell-free RNA markers in human subjects. As elevated RNase activity has previously been described in the circulation of cancer patients, we hypothesized that cancer patients may have reduced plasma RNA integrity. In this study, we used nasopharyngeal carcinoma (NPC) as a model system to test this hypothesis. EXPERIMENTAL DESIGN: Plasma RNA integrity was determined using the ratio of the concentrations of transcript sequences corresponding to the 3' to those from the 5' end of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Transcript concentrations were measured using real-time quantitative reverse transcription-PCR assays targeting the 5' and 3' regions. We analyzed the plasma RNA integrity in 49 untreated NPC patients and 53 healthy controls. We also assessed the plasma samples from 19 NPC patients before and after radiotherapy to further show the clinical potential of this marker. RESULTS: The 3' to 5' GAPDH ratio was significantly lower in the plasma of untreated NPC patients when compared with healthy controls (0.0252 versus 0.0485, P = 0.024). Statistical analysis showed that plasma GAPDH ratio was correlated with tumor stage but not with sex and age. Moreover, 14 of 19 NPC patients (74%) showed significant increase in the plasma GAPDH ratio following radiotherapy (P = 0.003). All of these patients were in clinical remission after treatment. CONCLUSIONS: Our findings suggest that NPC is associated with disturbances in the integrity of cell-free circulating RNA, raising the possibility that measurement of plasma RNA integrity may serve as a useful marker for the diagnosis and monitoring of malignant diseases.

Plasma cell-free DNA as an indicator of severity of injury in burn patients.

BACKGROUND: Raised levels of plasma cell-free DNA have been detected in various patient groups, including trauma patients. We hypothesized that plasma DNA is increased in burn patients and may represent an objective indicator of burn severity and have predictive as well as prognostic significance. METHODS: This was a prospective clinical study with full ethical approval. With informed consent, blood samples were collected from 28 burn patients within 24 h of injury and from 12 control subjects. Plasma cell-free DNA was measured by real-time quantitative polymerase chain reaction (PCR) assay for the beta-globin gene. Descriptive analysis, non-parametric data comparison tests (Mann-Whitney) and correlation tests (Spearman rank) were performed on the data. RESULTS: Samples were taken at a mean time of 5.7 h after injury from 13 patients with flame/flash burns and 15 patients with scalds. Median plasma DNA levels in the control, scald and flame/flash burn patient groups were 287, 648 and 2685 kilogenome-equivalents/L, respectively. Plasma DNA levels correlated with the length of hospital stay, but not with admission to the intensive care unit (ICU) nor the length of ICU stay. DNA levels correlated with the burn surface area (Spearman rank r = 0.54, p = 0.04) and the number of operations needed (Spearman rank r = 0.55, p = 0.03) for scalds, but not for flame/flash burns. CONCLUSIONS: Plasma DNA is increased after burn injury and is significantly correlated with some outcome measures, including the length of hospital stay. DNA levels are higher in flame/flash patients than in scald patients; the difference may provide an objective indication of burn depth and inhalation injury.

Detection of the placental epigenetic signature of the maspin gene in maternal plasma.

The discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.

Improved accuracy of detection of nasopharyngeal carcinoma by combined application of circulating Epstein-Barr virus DNA and anti-Epstein-Barr viral capsid antigen IgA antibody.

BACKGROUND: Circulating Epstein-Barr viral (EBV) DNA and anti-EBV capsid antigen IgA (IgA VCA) represent two of the most sensitive peripheral blood markers of nasopharyngeal carcinoma (NPC), but direct comparative studies of these two markers are lacking. METHODS: The sensitivities and specificities of IgA-VCA and EBV DNA for diagnosis of NPC were determined in 139 new cases of NPC and 178 healthy individuals, respectively. EBV DNA was also assessed in 36 healthy family members identified as having false-positive IgA-VCA results at a screening clinic. EBV DNA was measured by a real-time quantitative PCR assay with a detection limit of 60 copies/mL. IgA-VCA was measured by semiquantitative indirect immunofluorescent method; a titer > or =1/10 was taken as positive. RESULTS: The sensitivities of EBV DNA and IgA-VCA for diagnosis of NPC were 95% (95% confidence interval, 91-98%) and 81% (73-87%), respectively. The combined marker panel had an overall sensitivity (positive result by either marker) of 99%. The concentrations of both markers showed dependence on cancer stage. The specificities of EBV DNA and IgA-VCA were 98% (96-99%) and 96% (91-98%), respectively. Among 36 healthy family members with false-positive IgA-VCA results, three-fourths had undetectable EBV DNA, whereas the others had increased EBV DNA concentrations that were significantly lower than in NPC patients. CONCLUSIONS: For diagnosis of NPC, EBV DNA identifies almost all false-negative IgA-VCA cases and gives a 99% diagnostic sensitivity when combined with IgA-VCA. In the screening setting, EBV DNA identifies three-fourths of false-positive IgA-VCA cases. The selective application of EBV DNA in an IgA-VCA-based screening protocol could improve screening accuracy with only moderate increases in cost.

Rapid clearance of plasma Epstein-Barr virus DNA after surgical treatment of nasopharyngeal carcinoma.

PURPOSE: Circulating EBV DNA analysis has been shown to be valuable in the detection, prognostication, and monitoring of nasopharyngeal carcinoma (NPC) patients. A previous study has shown that, after radiotherapy, plasma EBV DNA levels of NPC patients would decline exponentially with a median half-life of 3.8 days. We postulate that this decline in plasma EBV DNA reflects the decrease in cancer cell population and, therefore, the rate of decline reflects the radiosensitivity of the tumor. However, this postulation would hold true only if EBV DNA is rapidly eliminated from the circulation. In this study, we determined the in vivo elimination rate of plasma EBV DNA in NPC patients. EXPERIMENTAL DESIGN: We monitored the level of plasma EBV DNA in NPC patients during and after surgical resection of NPC. The half-life of plasma EBV DNA was then calculated by plotting the natural logarithm of EBV DNA concentrations against time. RESULTS: The median half-life of plasma EBV DNA after surgical resection of NPC was 139 min. After a median follow-up of 6.7 days, EBV DNA was undetectable in 8 of 11 patients. One of 8 patients with undectable EBV DNA and all of the patients with detectable EBV DNA developed clinical relapse. CONCLUSIONS: The in vivo elimination of EBV DNA is very rapid after surgical resection of NPC. The failure of complete and rapid elimination of EBV DNA from the circulation predicts disease recurrence.

Disparity of sensitivities in detection of radiation-naïve and postirradiation recurrent nasopharyngeal carcinoma of the undifferentiated type by quantitative analysis of circulating Epstein-Barr virus DNA1,2.

PURPOSE: The purpose of this research was to compare the sensitivities of plasma EBV DNA in detection of postirradiation locally recurrent nasopharyngeal carcinoma (NPC), postirradiation distant metastatic NPC, and radiation-naïve NPC. EXPERIMENTAL DESIGN: Twenty-four patients with postirradiation local recurrence of NPC were assessed for plasma EBV DNA levels by a real-time quantitative PCR system. The results were compared with those of a cohort of 140 patients with newly diagnosed NPC and with those of 25 patients with distant metastatic relapse. EBV-encoded RNA positivity was also assessed in locally recurrent tumors and newly diagnosed tumors with undetectable plasma EBV DNA levels. RESULTS: Postirradiation locally recurrent tumors were associated with a significantly lower rate of detectable plasma EBV DNA compared with radiation-naïve tumors of comparable stage [stage I-II tumors: 5 of 12 (42%) versus 47 of 51 (92%), P = 0.0002; stage III-IV tumors: 10 of 12 (83%) versus 88 of 89 (99%), P = 0.01; Fisher's exact test], and compared with distant metastatic recurrences [15 of 24 (63%) versus 24 of 25 (96%), P < 0.02; Fisher's exact test]. The median EBV DNA level in patients with detectable EBV DNA was also significantly lower in locally recurrent tumors than in radiation-naïve tumors. All of the tissue samples of tumors associated with undetectable EBV DNA levels, where available, were EBV-encoded RNA positive. CONCLUSIONS: The sensitivity of EBV DNA in the detection of tumors regrowing from an irradiated site is much lower than that from a radiation-naïve site. Although plasma EBV DNA is very effective in detecting distant metastatic relapse of NPC, it cannot be relied on as the sole surveillance tool for detection of local relapse.

[Mutation-function analysis in the lipoprotein lipase gene of Chinese patients with hypertriglyceridemic type 2 diabetes].

OBJECTIVE: To investigate the role of lipoprotein lipase (LPL) gene on Chinese patients with hypertriglyceridemic type 2 diabetes. METHODS: Three subject groups, including hypertriglyceridemic group, normalipidemic type 2 diabetes group and healthy controls, were recruited and screened for sequence changes in LPL gene with PCR, SSCP, restriction analysis and direct DNA sequencing. LPL mass and activity in post-heparin plasma and in in vitro expression were investigated. Comparative modeling was performed via Swiss-PDB Viewer to provide the potential 2-D structures of wildtype and mutant proteins. RESULTS: Four missense mutations, Ala71Thr, Val18Ile, Gly188Glu and Glu242Lys, were identified in patients with hypertriglyceridemic type 2 diabetes, and not in both normalipidemic diabetes and the control subjects. The four missense mutations were located in the highly conserved amino acid sites, which are involved in highly conserved exon 3, 5, or 6 regions. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed the differences to a great extent between the mutant and wide-type molecules. CONCLUSION: These results indicated that the 4 missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. The LPL deficiency predispose a progressive diabetic pathway to those affected individuals. LPL gene is one of susceptibility gene for hypertriglyceridemic type 2 diabetes.

Time course of early and late changes in plasma DNA in trauma patients.

BACKGROUND: Cell-free DNA concentrations increase in the circulation of patients after trauma and may have prognostic potential, but little is know concerning the temporal changes or clearance of the DNA or its relationships with posttraumatic complications. We investigated temporal changes in plasma DNA concentrations in patients after trauma with use of real-time quantitative PCR. METHODS: Serial plasma samples were taken from two trauma populations. In the first study, samples were collected every 20 min from 25 patients within the first 3 h of trauma. In the second study, samples were collected every day from 36 other trauma patients admitted to the intensive care unit (ICU). RESULTS: In the first study, plasma DNA was increased within 20 min of injury and was significantly higher in patients with severe injury and in patients who went on to develop organ failure. In patients with less severe injuries, plasma DNA concentrations decreased toward reference values within 3 h. In the second study, plasma DNA concentrations were higher in patients who developed multiple organ dysfunction syndrome between the second and fourth days of admission than in patients who did not develop the syndrome. In patients who remained in the ICU with continuing organ dysfunction, plasma DNA remained higher than in healthy controls even at 28 days after injury. Most survivors with multiple organ dysfunction syndrome showed an initial very high peak followed by a prolonged smaller increase. CONCLUSIONS: Plasma DNA concentrations increase early after injury and are higher in patients with severe injuries and in those who develop organ failure. Increased plasma DNA persists for days after injuries, especially in patients with multiple organ dysfunction syndrome.

Pretherapy quantitative measurement of circulating Epstein-Barr virus DNA is predictive of posttherapy distant failure in patients with early-stage nasopharyngeal carcinoma of undifferentiated type.

BACKGROUND: Patients with International Union Against Cancer (UICC) Stage I-II nasopharyngeal carcinoma (NPC) appear to have a relatively favorable prognosis and generally are excluded from trials of combined modality treatment. More recently, plasma/serum cell-free Epstein-Barr virus (EBV) DNA has been shown to be measurable in the majority of NPC patients at the time of diagnosis, and appears to have prognostic significance. However, within Stage I-II disease, in which failure events are infrequent, the prognostic impact of the pretreatment EBV DNA level has not been addressed to our knowledge. This issue has management implications because different therapeutic strategies currently are employed for patients with good-risk and those with poor-risk NPC. METHODS: A cohort of 90 patients with UICC Stage I-II NPC (World Health Organization Grade 2/3 histology) had their pretherapy plasma/serum EBV DNA levels determined by a quantitative polymerase chain reaction assay and correlated with the probability of posttherapy failure. All patients received radiation therapy only, except for three patients who also received concurrent chemotherapy. Kaplan-Meier plots of the probability of locoregional failure, distant failure, and cancer-specific survival were compared with reference to clinical stage and EBV DNA levels. RESULTS: With a median follow-up time of 45 months, 12 patients and 7 patients, respectively, had developed locoregional and distant failures, including 2 patients with both local and distant failures. Patients with distant failure had significantly higher pretherapy EBV DNA levels than those without failure (a median of 13,219 copies/mL [interquatile-range, 274,635 copies/mL] vs. a median of 423 copies/mL [interquatile-range, 2753 copies/mL]). The probability of distant failure was significantly higher in patients with high (>4000 copies/mL plasma) compared with low EBV DNA levels (P=0.0001, log-rank test) and for Stage IIB disease compared with Stage I and Stage IIA disease combined (P=0.0149, log-rank test), but was not significantly different between patients with Stage II and those with Stage I disease. The risks of locoregional failure were not significantly different between patients with high and those with low EBV DNA levels, and also was not significantly different between clinical substages. Approximately 35% of patients with Stage IIB disease were in the at-risk group for distant failure, as identified by high EBV DNA levels. CONCLUSIONS: Within a group of patients with UICC Stage I-II NPC, the pretherapy plasma EBV DNA level was found to identify a poor-risk group with a probability of distant failure similar to that of patients with advanced stage disease. This group of patients may warrant management considerations currently applicable only to cases of Stage III-IV disease. The prognostic significance of designating Stage IIB disease as per the 1997 UICC staging was confirmed, although the pretherapy EBV DNA level appears to be a more powerful prognostic discriminator in patients with early-stage NPC.

Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients.

Despite the increasing clinical applications of circulating EBV DNA analysis as a tumor marker, the molecular nature of these EBV DNA molecules remains unclear. We subjected plasma/serum samples of nasopharyngeal carcinoma and lymphoma patients to DNase digestion and ultracentrifugation and showed that circulating EBV DNA molecules are "naked" DNA fragments instead of being contained inside virions. We further showed that these EBV DNA fragments were relatively short, and 87% of them were shorter than 181 bp. These results provide fundamental information that may improve our understanding of the release of tumor-derived nucleic acids into the blood of cancer patients.

Quantitative analysis of circulating mitochondrial DNA in plasma.

BACKGROUND: Recent studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum, but the concentrations and physical characteristics of circulating mitochondrial DNA are unknown. The aim of this study was to develop an assay to quantify mitochondrial DNA in the plasma of healthy individuals. METHODS: We adopted a real-time quantitative PCR approach and evaluated the specificity of the assay for detecting mitochondrial DNA with a cell line (rho(0)) devoid of mitochondria. The concentrations and physical characteristics of circulating mitochondrial DNA were investigated by experiments conducted in three modules. In module 1, we evaluated the concentrations of mitochondrial DNA in plasma aliquots derived from four blood-processing protocols. In module 2, we investigated the existence of both particle-associated and free forms of mitochondrial DNA in plasma by subjecting plasma to filtration and ultracentrifugation. In module 3, we used filters with different pore sizes to investigate the size characteristics of the particle-associated fraction of circulating mitochondrial DNA. RESULTS: The mitochondrial DNA-specific, real-time quantitative PCR had a dynamic range of five orders of magnitude and a sensitivity that enabled detection of one copy of mitochondrial DNA in plasma. In module 1, we found significant differences in the amounts of circulating mitochondrial DNA among plasma aliquots processed by different methods. Data from module 2 revealed that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable by ultracentrifugation. Module 3 demonstrated that filters with different pore sizes removed mitochondrial DNA from plasma to different degrees. CONCLUSIONS: Both particle-associated and free mitochondrial DNA are present in plasma, and their respective concentrations are affected by the process used to harvest plasma from whole blood. These results may have implications in the design of future studies on circulating mitochondrial DNA measured in different disease conditions.

Quantitative analysis of pleural fluid cell-free DNA as a tool for the classification of pleural effusions.

BACKGROUND: Recently, much interest has been focused on the quantification of DNA in miscellaneous body fluids. In this study, the application is extended to classifying pleural effusions by measuring cell-free DNA in pleural fluid. METHODS: We recruited 50 consecutive patients with pleural effusions with informed consent. Pleural fluids were centrifuged at 13000 g, with supernatants aliquoted for extraction and analysis of beta-globin DNA sequence by quantitative real-time PCR. Serum and pleural fluid biochemistries were performed to classify pleural effusions using the modified criteria of Light et al. (Ann Intern Med 1972;77:507-13). The ROC curve was plotted to determine the cutoff DNA concentration for classifying pleural fluids as transudates or exudates. Indicators of diagnostic accuracy were calculated for both pleural fluid DNA and modified criteria of Light et al., using the discharge, microbiologic, and histologic diagnoses as the reference standard. RESULTS: The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.84-0.99]. At 509 genome-equivalents/mL, pleural fluid DNA alone correctly classified 46 of 50 pleural effusions with 91% sensitivity (95% CI, 76-98%), 88% specificity (95% CI, 64-98%), and positive and negative likelihood ratios of 7.7 (95% CI, 3.1-19.5) and 0.10 (95% CI, 0.04-0.27), respectively. With the modified criteria of Light et al., 43 of 50 pleural effusions were correctly classified with 97% sensitivity (95% CI, 91-100%) and 67% specificity (95% CI, 45-89%). There were significant correlations between cell-free DNA and both lactate dehydrogenase and total protein in pleural fluid, suggesting their common origin. CONCLUSIONS: Pleural fluid DNA concentrations are markedly increased in exudative effusions, making it a potential new tool to evaluate the etiologic causes of pleural effusions.

Pathogenic mutations of the lipoprotein lipase gene in Chinese patients with hypertriglyceridemic type 2 diabetes.

Elevated plasma triglyceride and nonesterified fatty acid concentrations may cause insulin resistance. Lipoprotein lipase (LPL) is a rate-determining enzyme in lipid metabolism. To investigate the role of the LPL gene in Chinese patients with hypertriglyceridemic type 2 diabetes, 277 patients with type 2 diabetes and 241 healthy control subjects were recruited and screened for sequence changes in the LPL gene by PCR, SSCP, restriction analysis and direct DNA sequencing. Ten mutations were identified: four missense mutations, Ala71Thr, Val181Ile, Gly188Glu and Glu242Lys; one nonsense mutation Ser447Ter; and five silent mutations. Ser447Ter was found in both patients and controls with no significant difference in frequency. The four missense mutations were located in the highly conserved exon 3, 5, and 6 regions and in highly conserved amino acid sites. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed major differences between the mutant and wildtype molecules. These results indicated that the four missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. Based on our study and published data, a putative pathogenic pathway was suggested: LPL enzyme deficiency causes elevated plasma triglyceride level and subsequent insulin resistance; both increased free fatty acids and insulin resistance promote gluconeogenesis and hyperglycaemia, a vicious circle leading to type 2 diabetes.