Ju Long

The diagnosis and molecular analysis of a novel 21.9kb deletion (Qinzhou type deletion) causing α+ thalassemia.

α-Thalassemia is a common single-gene genetic disease that can cause Hb Barts hydrops fetalis and Hb H disease in tropical and subtropical regions. When examining conventional thalassemia genes, an only detected --(SEA) genotype sample needs further analysis. In doing so, we found a novel 21.9kb deletion (Qinzhou type deletion). The deletion position of the novel 21.9kb deletion is from 14373bp to 36299bp of the α-globin gene cluster (NG_000006.1); thus, there exists a 21927bp sequence deletion, into which a 29bp sequence is added. After sequence analysis, a group of Gap-PCR primers were synthesized to diagnose this novel thalassemia genotype. Through pedigree analysis, we deduced that the propositus obtained the novel alleles from her mother. The genotype of this propositus is --(SEA)/-α(21.9) and its phenotype conforms to the characteristics of Hb H disease, establishing that the combination between -α(21.9) genotype and α(0) genotype can lead to Hb H disease. By molecular analysis, we established that this case fits the characteristic of an α(+) thalassemia genotype.

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.

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 β-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.

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

Abstract The Qinzhou α-thalassemia (α-thal) or –α21.9 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 –α21.9. During routine screening, a new family with –α21.9 was found by the research group. This is the first time that an adult patient with the –α21.9/αα genotype and a 6-month-old baby with the –α21.9/– –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 –α21.9/αα genotype and the analysis of hematological data are important supplements for –α21.9 research. Additionally, Hb Bart’s (γ4) and Hb H (β4) were detected in the 6-month-old, confirming that the baby with the –α21.9/– –SEA genotype also carries Hb H disease. The analysis of this family verifies that the –α21.9 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 Downs syndrome were compared with the conventional karyotype analysis by testing 82 normal individuals and 50 subjects with Downs 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 Downs syndrome. The ΔCq values of these two segmental duplications clearly discriminated Downs 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 Downs syndrome.

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 quantitative‑PCR 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 quantitative‑polymerase 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.

Identification of the −α2.4 Deletion in One Family and in One Hb H Disease Patient in Guangxi, People’s Republic of China

Abstract The 2.4 kb (or −α2.4) deletion in the α-globin gene cluster (NG_000006.1) is an α+-thalassemia (α+-thal) allele. The molecular basis of −α2.4 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 −α2.4/αα genotype and an Hb H (β4) disease patient whose genotype was – –SEA/−α2.4. Samples from the parents of the carrier of the −α2.4/αα genotype were collected to perform pedigree analysis, and the proband’s mother’s genotype was diagnosed to be – –SEA/−α2.4. The research revealed that the −α2.4 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.

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

Abstract 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.