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.

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.

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.

[Construction and identification of the chromosome specific DNA library.].

The objective of the present study was to construct a human chromosome 21 specific DNA library for further use in research of genetic disease. Human chromosome 21 microdissected from the peripheral blood cells were subjected to repeatedly incubation in gradient temperature bath to release DNA. The library of chromosome 21 was constructed using the DNA fragment of 100-500 bp and 500-2 000 bp recovered from the products of DOP-PCR. Florescence in situ hy-bridization (FISH) and dot blotting analyses were carried out to assess the chromosome 21 specificity of the DNA library. The results indicated that DNA of chromosome 21 was released easily after repeatedly incubation in gradient temperature bath. Recovery of DNA fragments from DOP-PCR in different size ranges improved the efficiency of cloning of large fragments. Both FISH and dot blotting analyses revealed that the DNA library constructed in this study was chromosome 21-specific. This DNA library facilitates identification and investigation of the chromosome 21 related abnormality.