Diagnostic Accuracy of Prenatal Fetal Ultrasound to Detect Cleft Palate in High-Risk Fetuses: A Systematic Review and Meta-Analysis.

OBJECTIVES: This systematic review and meta-analysis investigated the accuracy of prenatal fetal ultrasound (US) to detect cleft palate during the second and third trimester (12-36 weeks) of pregnancy in high-risk fetuses. METHODS: Pubmed and Embase databases were searched for studies that performed prenatal fetal US (comparator) and postnatal examination (reference standard) in fetuses at high risk for orofacial clefts. Risk of bias among included studies was assessed using the QUADAS-2. Area under the summary receiver operating characteristic (SROC) curve and pooled sensitivity and specificity were calculated. RESULTS: This meta-analysis included 7 studies involving 663 high-risk fetuses. The individual studies showed that prenatal fetal US accurately predicted the possibility of cleft palate in these fetuses. Pooled sensitivity was 87% (95% CI 71%-95%), pooled specificity was 98% (95%CI 90%-100%), and the area under the SROC curve was 0.98 (95% CI 0.97-0.99). CONCLUSION: Second and third trimester fetal US has excellent sensitivity and specificity for the detection of cleft palate in high-risk pregnancies.

Molecular diagnosis of sex chromosome mosaics in fetal amniotic cells.

ABSTRACT: Mosaicism can be observed in karyotype analyses of amniotic fluid cells. Differentiating between true mosaicism and pseudomosaicism and determining mosaic proportions can help avoid misjudgments by doctors and effectively reduce mental and physical harm to pregnant women. However, the detection of mosaicism and mosaic proportions via karyotype analysis and fluorescence in situ hybridization (FISH) is extremely complex. We have developed a novel approach, "segmental duplication quantitative fluorescent PCR" (SD-QF-PCR), to detect mosaicism and mosaic proportions.In this study, twenty control samples and fourteen mosaic samples were tested by first-line karyotype analysis; by second-line karyotype analysis, SD-QF-PCR and FISH were used to reassess fetal sex chromosome mosaicism and mosaic proportions.Detection of the 20 control samples by second-line karyotype analysis via FISH and SD-QF-PCR showed normal and consistent results. Among the 14 mosaic samples, the numbers of samples showing true mosaicism and pseudomosaicism detected by the three methods were 6 and 8, respectively.Our study demonstrates that SD-QF-PCR can be used as a complementary method to traditional cytogenetic analysis of amniotic fluid mosaics and has clinical application value.

Serum multiple cytokines for the prediction of spontaneous preterm birth in asymptomatic women: A nested case-control study.

BACKGROUND: Current biomarkers such as fetal fibronectin and cervical length are accurate predictors of spontaneous preterm birth (sPTB) in women with clinically suspected preterm risk; however, these are not effective for predicting the risk of sPTB in asymptomatic women. Therefore, we performed this study with the objective of determining whether the combinations of specific serum cytokines could accurately predict the sPTB risk in asymptomatic women. METHODS: We conducted a nested case-control study with 129 incident sPTB cases and 258 individually matched controls who participated in an ongoing birth cohort study. The maternal serum levels of the selected 35 cytokines were measured. We evaluated the relationship between the multiple cytokines and sPTB risk using conditional logistic regression and elastic net model. RESULTS: A panel of cytokines was significantly associated with an increased risk of sPTB. The odds ratio (OR) of sPTB per standard deviation (SD) increase of the predictive model score was 1.57 (95% CI 1.25-1.97) for the cytokines model. The combination of the selected serum cytokines was substantially more effective in predicting the risk for sPTB, as the receiver-operator characteristic curve (AUC) values were 0.546 and 0.559 in the single cytokine model and it improved to 0.642 in the multiple cytokines model (PAUC difference = 0.02 for TNF-α vs. multiple cytokines; PAUC difference = 0.05 for TRAIL vs. multiple cytokines). Moreover, the prediction was more accurate in overweight pregnant women, with an AUC = 0.879. CONCLUSIONS: The current study suggested that the combination of selected serum cytokines can more effectively predict the risk of sPTB in asymptomatic women compared with the use of single cytokine.

STARD-rapid screening for the 6 most common G6PD gene mutations in the Chinese population using the amplification refractory mutation system combined with melting curve analysis.

Dot-blot hybridization and high-resolution melting curve methods are used to detect G6PD gene mutations; however, the performance and throughput limitations of these methods hinder their use for screening large populations. For simple screening, we developed a novel approach called "Amplification Refractory Mutation System combined with Melting Curve Analysis (ARMS-MC)," which enables rapid and batch-based detection of the 6 most common G6PD mutations.In this method, we established 4 PCR reaction systems that can be used to detect the 6 most common G6PD mutations (c.95A>G, c.392G>T, c.871G>A, c.1024C>T, c.1376G>T, and c.1388G>A) in the Chinese population.The ARMS-MC method was evaluated with 174 cases of clinical G6PD-deficient samples, and the results were verified by direct sequencing at G6PD gene exons. The results showed that 170 samples had ≥1 of the 6 mutations, which accounted for 97.70% of all mutations. These results were consistent with the results of direct sequencing with 100% accuracy and specificity. Sequencing validation revealed other mutations in the 4 samples in which no mutation was detected by the ARMS-MC method.ARMS-MC provides a rapid, simple, inexpensive, and accurate screening method for detecting the most common G6PD mutations in Chinese people.

Identification of Three Types of α-Thalassemia Deletion, -α21.9, -α2.4, and - -THAI, and Their Frequencies, in One Family in the Population of Southern Guangxi Zhuang Autonomous Region, People's Republic of China.

Different types of deletional α-thalassemia (α-thal) have been reported by researchers in China. This study describes one family carrying -α21.9 (NG_000006.1: g.14373_36299delinsGGGAAGGGTGGGTGGGAATAACAGCTTTT), -α2.4 (NG_000006.1: g.36860_39251del) and - -THAI (Thailand) (NG_000006.1: g.10664_44164del) alleles in Guangxi Zhuang Autonomous Region, People's Republic of China (PRC), and reports the frequencies of these types in the population of this region. The proband was a 4-year-old girl, who screened positive for thalassemia, although the thalassemia genotype results were normal when screened using the routine kits. Samples of the proband's parents were also collected to perform further analyses. Two real-time gap-polymerase chain reaction (gap-PCR) systems were designed for separate detection of - -THAI and screening for -α21.9 and -α2.4. The genotype of the proband was -α21.9/-α2.4, and the two variants were inherited from her parents. In the frequency study, five - -THAI, four -α21.9 and 11 -α2.4 positive individuals were detected in the 3410 random samples. Thus, allele frequencies of -α21.9, - -THAI and -α2.4 in the population of southern Guangxi were determined as 0.059, 0.073 and 0.161%, respectively. This is the first report of an individual carrying the -α21.9/-α2.4 genotype, and the first report of the detection of -α21.9, -α2.4 and - -THAI in a single family. The total frequency for these alleles was 0.293% in southern Guangxi, suggesting that the thalassemia clinical center in this region should utilize a screening kit that allows detection of these types of deletions for a more comprehensive evaluation of thalassemia risk.

Rapid prenatal diagnosis of aneuploidy for chromosomes 21, 18, 13, X, and Y using segmental duplication quantitative fluorescent PCR (SD-QF-PCR).

BACKGROUND: In our previous studies, the rapid diagnosis of aneuploidy has been achieved using the segmental duplication molecular markers-based SD-QF-PCR technique. However, it is also insufficient due to the drawbacks including less detection loci and incompetence in single-tube detection. METHODS: In this paper, we developed 13 new segmental duplications as molecular markers, as well as designed 13 pairs of primers and 1 fluorescence-labeled universal primer, which could detect chromosome aneuploidies in one PCR tube. RESULTS: Two hundred and thirty samples were detected using SD-QF-PCR, the samples were collected from individuals with trisomy 21 (n=16); trisomy 18 (n=4); trisomy 13 (n=3); 45,X (n=3); 47,XXY (n=2); 47,XYY (n=2); suspected mosaic 46,XX/46,XY (n=2); and unaffected controls (n=198). CONCLUSIONS: The detection results of SD-QF-PCR were consistent with those of conventional karyotype analysis. SD-QF-PCR based on the newly developed segmental duplications enables the single-tube and multi-locus simultaneous detection on the number of chromosomes 13, 18, 21, X and Y. Therefore, this technique offers a new alternative for the diagnosis of chromosome aneuploidies.

Identification of the -α(2.4) Deletion in One Family and in One Hb H Disease Patient in Guangxi, People's Republic of China.

The 2.4 kb (or -α(2.4)) deletion in the α-globin gene cluster (NG_000006.1) is an α(+)-thalassemia (α(+)-thal) allele. The molecular basis of -α(2.4) is a deletion from 36860 to 39251 of the α-globin gene cluster. It was reported by three research groups in 2005, 2012 and 2014, respectively. In routine thalassemia screening studies by this research group, we found an individual with the -α(2.4)/αα genotype and an Hb H (ß4) disease patient whose genotype was - -(SEA)/-α(2.4). Samples from the parents of the carrier of the -α(2.4)/αα genotype were collected to perform pedigree analysis, and the proband's mother's genotype was diagnosed to be - -(SEA)/-α(2.4). The research revealed that the -α(2.4) allele exists in the population of southern Guangxi, People's Republic of China.

Identification of a variation in the IVSII of α2 gene and its frequency in the population of Guangxi.

OBJECTIVE: During thalassemia screening, a previously unidentified α2 gene variation in α-globin gene cluster was isolated. This variation was distinct from other variations known to confer thalassemia as assessed by conventional thalassemia genotype analysis. Because the sample in the thalassemia screening was positive (MCV=83.6fL, MCH=26.1pg/cell, Hb=11.3g/dL), further analysis was required. MATERIAL AND METHODS: MLPA (multiplex ligation-dependent probe amplification) and sequencing were used for analysis, and a qPCR system was designed for the frequency study. RESULTS: The MLPA result showed that there was a mutation or small fragment deletions between 34247 (160bp probe) and 34618 (196bp probe) in α-globin gene cluster (NG_000006.1). Through sequencing, this variation was identified as HBA2: c.301-24delGinsCTCGGCCC. The gene polymorphisms similar to HBA2:c.301-24delGinsCTCGGCCC are α121 and α212. Since α212 is unrelated to microcytosis, and the structure of HBA2: c.301-24delGinsCTCGGCCC is similar to α212, this change is more appropriately considered as a polymorphism. The allele frequency of HBA2: c.301-24delGinsCTCGGCCC is 0.184% in this region. CONCLUSIONS: There is a certain ratio for HBA2:c.301-24delGinsCTCGGCCC carriers among the Chinese population. The HBA2:c.301-24delGinsCTCGGCCC variant results in an abnormal result from MLPA analysis. Investigators performing thalassemia screening in Guangxi region should be aware of the HBA2:c.301-24delGinsCTCGGCCC variant to avoid misinterpretation of the MLPA results.

Diagnosis of a Family with the Novel -α(21.9) Thalassemia Deletion.

The Qinzhou α-thalassemia (α-thal) or -α(21.9) deletion was first described at the Qinzhou Maternal and Child Health Care Hospital, Qinzhou, Guangxi, People's Republic of China (PRC) in 2013. The molecular biological mechanism by which this allele leads to α-thal involves the deletion of a 21.9 kb DNA fragment of the α-globin gene cluster (NG_000006.1), designated as -α(21.9). During routine screening, a new family with -α(21.9) was found by the research group. This is the first time that an adult patient with the -α(21.9)/αα genotype and a 6-month-old baby with the -α(21.9)/- -(SEA) (Southeast Asian) genotype were detected in one family. The discovery of this family demonstrates that there is a certain risk for the Qinzhou α-thal deletion in the southern regions of Guangxi Province, PRC. The detection of the adult patient with the -α(21.9)/αα genotype and the analysis of hematological data are important supplements for -α(21.9) research. Additionally, Hb Bart's (γ4) and Hb H (ß4) were detected in the 6-month-old, confirming that the baby with the -α(21.9)/- -(SEA) genotype also carries Hb H disease. The analysis of this family verifies that the -α(21.9) deletion is an α(+)-thal allele.

Rapid detection of Down's syndrome using quantitative real-time PCR (qPCR) targeting segmental duplications on chromosomes 21 and 11.

OBJECTIVE: Development of a qPCR test for the detection of trisomy 21 using segmental duplications. METHODS: Segmental duplications in the TTC3 gene on chromosome 21 and the KDM2A gene on chromosome 11 were selected as molecular markers for the diagnostic qPCR assay. A set of consensus primers selected from the conserved regions of these segmental duplications were used to amplify internal diverse sequences that were detected and quantified with different probes labeled with distinct fluorescence. The copy numbers of these two fragments were determined based on the ΔCq values of qPCR. The results of qPCR for prenatal and neonatal screening of Down's syndrome were compared with the conventional karyotype analysis by testing 82 normal individuals and 50 subjects with Down's syndrome. RESULTS: The ΔCq values of segmental duplications on chr21 and 11 ranged between 0.33 and 0.75 in normal individuals, and between 0.91 and 1.18 in subjects with Down's syndrome. The ΔCq values of these two segmental duplications clearly discriminated Down's syndrome from normal individuals (P<0.001). Furthermore, the qPCR results were consistent with karyotype analysis. CONCLUSION: Our qPCR can be used for rapid prenatal and neonatal screening of Down's syndrome.

Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36); trisomy 18 (n = 6); trisomy 13 (n = 4); 45, X (n = 5); 47, XXX (n = 3); 48, XXYY (n = 2); and unaffected controls (n = 40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.

Rapid diagnosis of aneuploidy in chromosomes 13, 18, 21, X and Y by quantitative fluorescence-PCR combined with short tandem repeat and fluorescence-labeled homologous gene quantitative­PCR using 4-color fluorescently labeled universal primers.

The present study aimed to develop a rapid diagnostic test of aneuploidy in chromosomes 13, 18, 21, X and Y through a program combining short tandem repeat (STR) typing with fluorescence-labeled homologous gene quantitative­polymerase chain reaction (fHGQ-PCR), which avoids misjudgment risks by using one method alone. Furthermore, fluorescently labeled universal primers not only ensure the accuracy of the results but also reduces the cost of fluorescent labels. The verification of DNA extracted from samples confirmed by karyotype analysis with quantitative fluorescence (QF)-PCR shows that the results obtained using the QF-PCR program are consistent with the results of karyotype analysis in rapidly diagnosing the aneuploidy of chromosomes 13, 18, 21, X and Y.

Detection of three common α-thalassemia in non-deletion types and six common thalassemia in deletion types by QF-PCR.

OBJECTIVE: Thalassemia is one of the most common monogenic hereditary diseases in tropical and subtropical regions. An effective way to avoid the birth of severe thalassemia patients is to strengthen the thalassemia screening of couples before wives are pregnant. Thalassemia gene carriers can be diagnosed by molecular biology in order to conduct effective guidance for fertility. DESIGNS AND METHODS: For --(SEA) and --(THAI) of α-thalassemia and HPFH-SEA and DBT of ß-thalassemia, we design the fGap-PCR primer; for α(CS)α, α(QS)α and α(WS)α, we design the fAS-PCR primer; for -α(3.7)and -α(4.2), we design the QF-PCR primer; and lastly, we use universal primers and multiple-tailed primers to make a single-tube QF-PCR system. RESULTS: When the QF-PCR system is used to diagnose 123 screening samples of thalassemia genotyping, the typing result is consistent with conventional diagnosis of Gap-PCR and PCR-RDB. CONCLUSIONS: Compared with conventional Gap-PCR and PCR-RDB, this QF-PCR system is easy to operate, has high precision, and can diagnose genotypes in a large scale. Its automatic operation is more suitable for the large-scale screening of the thalassemia gene.