Objective
To evaluate whether targeted sequencing and relative mutation dosage can be used to diagnose correctly inheritance of maternal β‐thalassaemia mutations in cell‐free DNA.
Design
Feasibility ...study using samples collected in a prenatal clinic.
Setting
South East Asia.
Population
Couples where both partners were known to be carriers of one of four common β‐thalassaemia mutations or an HbE mutation, and therefore at risk of carrying a fetus affected with β‐thalassaemia.
Methods
49 samples previously identified as having inherited a paternal β‐thalassaemia mutation were amplified using nested polymerase chain reaction (PCR), and then sequencing. Relative mutation dosage was used to classify the fetus as having inherited the wild‐type or mutant maternal allele.
Main outcome measures
Classification of the fetus as ‘unaffected’ (if the maternal wild‐type allele was inherited) or ‘affected’ with β‐thalassaemia (if the maternal mutant allele was inherited).
Results
A classification for inheritance of maternal allele was obtained in 48/49 samples (98.0%). A concordant call was made in 44/48 cases (91.7%): one false‐positive and three false‐negatives were obtained. Thus, we had an overall sensitivity of 87.5% 95% confidence interval (CI) 67.6–97.3% and a specificity of 95.8% (95% CI 78.9–99.9%) for inheritance of maternal genotype.
Conclusions
RMD for detection of inheritance of maternal β‐thalassaemia mutations has potential for clinical use. Our sequential approach could be applied to other single‐gene disorders.
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NIPT for β‐thalassaemia achieved using nested‐PCR followed by relative mutation dosage.
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NIPT for β‐thalassaemia achieved using nested‐PCR followed by relative mutation dosage.
MR imaging can detect nasopharyngeal carcinoma that is hidden from endoscopic view, but for accurate detection carcinoma confined within the nasopharynx (stage T1) must be distinguished from benign ...hyperplasia of the nasopharynx. This study aimed to document the MR imaging features of stage T1 nasopharyngeal carcinoma and to attempt to identify features distinguishing it from benign hyperplasia.
MR images of 189 patients with nasopharyngeal carcinoma confined to the nasopharynx and those of 144 patients with benign hyperplasia were reviewed and compared in this retrospective study. The center, volume, size asymmetry (maximum percentage difference in area between the right and left nasopharyngeal halves), signal intensity asymmetry, deep mucosal white line (greater contrast enhancement along the deep tumor margin), and absence/distortion of the adenoidal septa were evaluated. Differences were assessed with logistic regression and the χ
test.
The nasopharyngeal carcinoma center was lateral, central, or diffuse in 134/189 (70.9%), 25/189 (13.2%), and 30/189 (15.9%) cases, respectively. Nasopharyngeal carcinomas involving the walls showed that a deep mucosal white line was present in 180/183 (98.4%), with a focal loss of this line in 153/180 (85%) cases. Adenoidal septa were absent or distorted in 111/111 (100%) nasopharyngeal carcinomas involving the adenoid. Compared with benign hyperplasia, nasopharyngeal carcinoma had a significantly greater volume, size asymmetry, signal asymmetry, focal loss of the deep mucosal white line, and absence/distortion of the adenoidal septa (
< .001). Although size asymmetry was the most accurate criterion (89.5%) for nasopharyngeal carcinoma detection, use of this parameter alone would have missed 11.9% of early-stage T1 nasopharyngeal carcinomas.
MR imaging features can help distinguish stage T1 nasopharyngeal carcinoma from benign hyperplasia in most cases.
Our previous nasopharyngeal carcinoma detection study, comparing MR imaging, endoscopy, and endoscopic biopsy, showed that MR imaging is a highly sensitive test that identifies nasopharyngeal ...carcinomas missed by endoscopy. However, at the close of that study, patients without biopsy-proved nasopharyngeal carcinoma nevertheless had shown suspicious abnormalities on endoscopy and/or MR imaging. The aim of this study was to determine whether there were any patients with undiagnosed nasopharyngeal carcinoma by obtaining long-term follow-up and to use these data to re-evaluate the diagnostic performance of MR imaging.
In the previous study, 246 patients referred to a hospital ear, nose, and throat clinic with suspected nasopharyngeal carcinoma, based on a wide range of clinical indications, had undergone MR imaging, endoscopy, and endoscopic biopsy, and 77 had biopsy-proved nasopharyngeal carcinoma. One hundred twenty-six of 169 patients without biopsy-proved nasopharyngeal carcinoma underwent re-examination of the nasopharynx after a minimum of 3 years, including 17 patients in whom a previous examination (MR imaging = 11; endoscopy = 7) had been positive for nasopharyngeal carcinoma, but the biopsy had been negative for it. Patients with nasopharyngeal carcinoma were identified by biopsy obtained in the previous and this follow-up study; patients without nasopharyngeal carcinoma were identified by the absence of a tumor on re-examination of the nasopharynx. The sensitivity and specificity of the previous investigations were updated and compared by using the Fisher exact test.
One patient with a previous positive MR imaging finding was subsequently proved to have nasopharyngeal carcinoma. Nasopharyngeal carcinomas were not found in the remaining 125 patients at follow-up, and the previous positive findings for nasopharyngeal carcinoma on MR imaging and endoscopy were attributed to benign lymphoid hyperplasia. The diagnostic performances for the previous MR imaging, endoscopy, and endoscopic biopsy were 100%, 88%, and 94%, respectively, for sensitivity, and 92%, 94%, and 100%, respectively, for specificity; the differences between MR imaging and endoscopy were significant for sensitivity (P = .003) but not specificity (P = .617).
MR imaging detected the 12% of nasopharyngeal carcinomas that were endoscopically invisible, including 1 cancer that remained endoscopically occult for several years. Lymphoid hyperplasia reduced the specificity of MR imaging.
The effect of corticosteroid treatment on the viral load of Severe Acute Respiratory Syndrome (SARS) patients is unknown.
To compare the plasma SARS-CoV RNA concentrations in ribavirin-treated ...patients who received early hydrocortisone therapy with those who received placebo.
Serial plasma SARS-CoV RNA concentrations measured in the setting of a prospective, randomized double-blinded, placebo-controlled trial designed to assess the efficacy of “early” (<7 days of illness) hydrocortisone use in previously healthy SARS patients were analyzed. SARS-CoV RNA was quantified using a one-step real-time RT-PCR assay targeting the nucleocapsid gene.
Among 16 non-ICU cases, SARS-CoV RNA was detected in plasma since day 3–4 after fever onset; viral concentration peaked in the first week, which then rapidly declined in the second week of illness. On days 8, 12, 16, and 20, the cumulative proportion of patients with undetectable virus in plasma was 31%, 69%, 92%, and 100%, respectively. Plasma SARS-CoV RNA concentrations in the second and third week of illness were significantly higher in patients who received initial hydrocortisone treatment (
n = 9), as compared to those who received placebo (
n = 7)(AUC; Mann–Whitney,
P = 0.023). The median time for SARS-CoV to become undetectable in plasma was 12 days (11–20 days) versus 8 days (8–15 days), respectively.
Our findings suggested “early” corticosteroid treatment was associated with a higher subsequent plasma viral load.
The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive prenatal diagnosis. Despite the rapid expansion in clinical applications, the molecular characteristics of ...plasma DNA in pregnant women remain unclear.
We investigated the size distribution of plasma DNA in 34 nonpregnant women and 31 pregnant women, using a panel of quantitative PCR assays with different amplicon sizes targeting the leptin gene. We also determined the size distribution of fetal DNA in maternal plasma by targeting the SRY gene.
The median percentages of plasma DNA with size >201 bp were 57% and 14% for pregnant and nonpregnant women, respectively (P <0.001, Mann-Whitney test). The median percentages of fetal-derived DNA with sizes >193 bp and >313 bp were 20% and 0%, respectively, in maternal plasma.
Plasma DNA molecules are mainly short DNA fragments. The DNA fragments in the plasma of pregnant women are significantly longer than those in the plasma of nonpregnant women, and the maternal-derived DNA molecules are longer than the fetal-derived ones.
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.
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.
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.
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.
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.
Tumour-associated changes have been observed in the circulating nucleic acids of cancer patients and have been proposed to be useful for the detection and monitoring of cancers. In this review, ...different approaches for detecting tumour-associated nucleic acids in the circulation and their potential applications as tumour markers are discussed.
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