Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique.
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PROVIDER: S-EPMC6761473 | BioStudies |
REPOSITORIES: biostudies
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Project description:We propose antenatal blood tests using high-resolution DNA melting (HRM) analysis for beta thalassemia mutation detection after hemoglobin A2 estimation as a modified strategy for the identification of beta thalassemia at-risk couples. Antenatal blood samples of 1,115 couples were transferred from the antenatal care clinic. Hemoglobin A2 was quantified, and proportions ?3.5% were further assessed for beta thalassemia mutation using HRM analysis. Twelve types of beta thalassemia mutations, including hemoglobin E, were identified. There were 23 couples who were detected as at-risk. All at-risk couples were identified within 7 working days after sample receipt. Prenatal diagnosis revealed 6 affected fetuses. One fetus was homozygous CD17 (AT), and five fetuses exhibited beta0 - thalassemia/hemoglobin E disease. These results were consistent with the outcomes calculated using the Hardy-Weinberg equation. Antenatal blood tests for mutation detection using high-resolution DNA melting analysis after hemoglobin A2 estimation is a feasible laboratory method for the recruitment of couples with a fetus that is at risk for beta thalassemia. This modified strategy is cost-effective and may be beneficial for use in a beta thalassemia prevention program.
| S-EPMC6036980 | BioStudies
Project description:<h4>Background</h4>Thalassemia is a group of inherited autosomal recessive hemolytic anemia disease caused by reduced or absent synthesis of globin chain/chains of hemoglobin. Only few studies showed the molecular characterization of α- and β-thalassemia in Meizhou city of China.<h4>Methods</h4>A total of 22,401 individuals were collected; hematological and hemoglobin electrophoresis analysis and thalassemia genetic testing were performed.<h4>Results</h4>Eleven thousand and thirty (49.24%) cases with microcytosis (mean corpuscular volume (MCV) < 82 fl), 11,074 (49.44%) cases with hypochromia (mean corpuscular Hb (MCH) < 27 pg) in 22,401 subjects, 11,085 cases with abnormal hemoglobin results were identified in subjects aged ≥6 months. 7,322 (32.69%) subjects harbored thalassemia mutations, including 4,841 (21.61%) subjects with α-thalassemia, 2,237 (9.99%) with β-thalassemia, and 244 (1.09%) with α-thalassemia combined β-thalassemia. 18 genotypes of α-thalassemia mutations and 27 genotypes of β-thalassemia mutations were characterized. The most frequent α gene mutation was --<sup>SEA</sup> (64.69%), followed by -α<sup>3.7</sup> (19.93%), -α<sup>4.2</sup> (7.73%), α<sup>CS</sup> α (3.97%), and α<sup>WS</sup> α (2.83%). The six most common β-thalassemia mutations were IVS-II-654 (C>T) (39.79%), CD41-42 (-TCTT) (33.02%), -28 (A>G) (10.38%), CD17 (A>T) (9.08%), CD27-28 (+C) (2.14%), and CD26 (G>A) (2.02%). In addition, MCV and MCH were sensitive markers for α- and β-thalassemia except for -α<sup>3.7</sup> /αα, -α<sup>4.2</sup> /αα, α<sup>CS</sup> α/αα, α<sup>WS</sup> α/αα, and β<sup>Cap+40-43</sup> /β<sup>N</sup> .<h4>Conclusions</h4>The --<sup>SEA</sup> , -α<sup>3.7</sup> , and -α<sup>4.2</sup> deletions were the main mutations of α-thalassemia, while IVS-II-654 (C>T), CD41-42 (-TCTT), -28 (A>G), and CD17 (A>T) mutations of β-thalassemia in Meizhou. There were some differences in thalassemia mutation frequencies in Meizhou city from other populations in China.
| S-EPMC8649333 | BioStudies
Project description:<h4>Background</h4>Thalassemia is one of the most common genetic diseases in southern China. Accurate population frequency data regarding the occurrence and distribution of thalassemia are important for designing appropriate prevention strategies for thalassemia. This study aims to reveal the molecular spectrum, ethnic and geographical distribution of thalassemia in the southern area of Hainan Province, China.<h4>Methods</h4>A total of 9813 suspected carriers of thalassemia were screened for genetic analysis by using the PCR-reverse dot blot hybridization method targeting three known deletions of α-thalassemias (--<sup>SEA</sup>, -α<sup>3.7</sup>, and -α<sup>4.2</sup>), three nondeletional mutations of α-thalassaemias (α<sup>CS</sup>, α<sup>QS</sup>, and α<sup>WS</sup>) and the 17 most common mutations of β-thalassaemias in the Chinese population.<h4>Results</h4>Approximately 6,924 subjects were genetically diagnosed as thalassemia carriers or patients, including 5812 cases of α-thalassemia (83.9%), 369 cases of β-thalassemia (5.3%), and 743 cases of α-composite β-thalassemia (10.7%). A total of 21 distinct genotypes were identified among the 5,812 α-thalassemia carriers, -α<sup>4.2</sup>/αα, -α<sup>3.7</sup>/αα, and -α<sup>3.7</sup>/-α<sup>4.2</sup> were the most common α-thalassemia genotypes. The most frequent β-thalassemia genotype was β<sup>CD41-42</sup>/β<sup>N</sup>, with a notable proportion of 69.6%, followed by the β<sup>-28<i>M</i></sup> /β<sup>N</sup>, β<sup>IVS-II-654</sup>/β<sup>N</sup>, β<sup>CD71-72</sup>/β<sup>N</sup>, β<sup>E</sup>/β<sup>N</sup>, and β<sup>CD17</sup>/β<sup>N</sup> genotypes. In addition, 37 genotypes were detected among the 743 cases of both α- and β-thalassemia mutations. The α-thalassemia genotypes were most commonly found in the Li people, who accounted for 73.5% of α-thalassemia carriers. The β-thalassemia genotypes were most commonly identified in the Han people, who accounted for 59.4% of β-thalassemia carriers. Among the subjects carrying both α- and β-thalassemia variations, only three ethnic minorities were identified, including the Li, Han, and Miao people, accounting for 82.0, 17.4, and 0.7%, respectively.<h4>Conclusions</h4>Our study indicates that there is high genetic heterogeneity, geographical and ethnic differences in thalassemia in populations in the southern area of Hainan Province. These findings will be helpful in guiding genetic counseling and prenatal diagnosis of thalassemia in Hainan Province.
| S-EPMC9245522 | BioStudies
Project description:PURPOSE: Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis (PND) giving couples at risk a chance to start a pregnancy with a disease-free baby. This study aimed to develop a new PGD protocol for alpha-thalassemia(-SEA) mutation, the commonest Mendelian disorder. PATIENTS AND METHODS: Multiplex fluorescent PCR was employed for mutation, contamination and linkage analysis. A couple experienced termination of pregnancy following positive PND decided to join the project. RESULTS: Novel primers for alpha-thalassemia(-SEA) mutation amplifying 5 DNA fragments were developed. Two PGD cycles were performed, resulting in an un-affected baby. PND confirmed the heterozygous result. From 24 embryos, 87.5% of affected genotype were of best quality compared to 0% and 18.2% of those with normal and heterozygous, respectively. CONCLUSIONS: A novel PCR protocol for the common alpha-thalassemia(-SEA) mutation is reported. This test should be widely applicable. Interestingly, a potential effect of alpha-thalassemia(-SEA) mutation on preimplantation embryonic development was noticed.
| S-EPMC3252417 | BioStudies
Project description:OBJECTIVE:The aim of the study was to explore genotype distribution thalassemia and G6PD deficiency in Meizhou city, China. METHODS:A total of 16 158 individuals were involved in thalassemia genetic testing. A total of 605 subjects were screened for common Chinese G6PD mutations by gene chip analysis. Genotypes and allele frequencies were analyzed. RESULTS:A total of 5463 cases carried thalassemia mutations were identified, including 3585 cases, 1701 cases, and 177 cases with ?-, ?-, and ? + ?-thalassemia mutations, respectively. --SEA (65.12%), -?3.7 (19.05%), and -?4.2 (8.05%) deletion were the main mutations of ?-thalassemia, while IVS-II-654(C ? T) (40.39%), CD41-42(-TCTT) (32.72%), -28(A ? G) (10.11%), and CD17(A ? T) (9.32%) mutations were the principal mutations of ?-thalassemia in Meizhou. There were significant differences in allele frequencies in some counties. Genetic testing for G6PD deficiency, six mutation sites, and one polymorphism were detected in our study. A total of 198 alleles with the mutation were detected among 805 alleles (24.6%). G6PD Canton (c.1376 G ? T) (45.96%), G6PD Kaiping (c.1388 G ? A) (39.39%), and G6PD Gaohe (c.95 A ? G) (9.09%) account for 94.44% mutations, followed by G6PD Chinese-5 (c.1024 C ? T) (4.04%), G6PD Viangchan (c.871G ? A) (1.01%), and G6PD Maewo (c.1360 C ? T) (0.51%). There were some differences of the distribution of G6PD mutations among eight counties in Meizhou. CONCLUSIONS:The --SEA , -?3.7 , and -?4.2 deletion were the main mutations of ?-thalassemia, while IVS-II-654(C ? T), CD41-42(-TCTT), -28(A ? G), and CD17(A ? T) mutations were the principal mutations of ?-thalassemia in Meizhou. G6PD c.1376 G ? T, c.1388 G ? A, and c.95 A ? G were the main mutations of G6PD deficiency. There were some differences of the distribution of thalassemia and G6PD mutations among eight counties in Meizhou.
| S-EPMC7171329 | BioStudies
Project description:Thalassemia is a dangerous hematolytic genetic disease. In south China, ?24% Chinese carry alpha-thalassemia or beta-thalassemia gene mutations. Given the fact that the invasive sampling procedures can only be performed by professionals in experienced centers, it may increase the risk of miscarriage or infection. Thus, most people are worried about the invasive operation. As such, a noninvasive and accurate prenatal diagnosis is needed for appropriate genetic counseling for families with high risks. Here we sought to develop capture probes and their companion analysis methods for the noninvasive prenatal detection of deletional and nondeletional thalassemia.Two families diagnosed as carriers of either beta-thalassemia gene or Southeast Asian deletional alpha-thalassemia gene mutation were recruited. The maternal plasma and amniotic fluid were collected for prenatal diagnosis. Probes targeting exons of the genes of interest and the highly heterozygous SNPs within the 1Mb flanking region were designed. The target capture sequencing was performed with plasma DNA from the pregnant woman and genomic DNA from the couples and their children. Then the parental haplotype was constructed by the trios-based strategy. The fetal haplotype was deduced from the parental haplotype with a hidden Markov model-based algorithm.The fetal genotypes were successfully deduced in both families noninvasively. The noninvasively constructed haplotypes of both fetuses were identical to the invasive prenatal diagnosis results with an accuracy rate of 100% in the target region.Our study demonstrates that the effective noninvasive prenatal diagnosis of alpha-thalassemia and beta-thalassemia can be achieved with the targeted capture sequencing and the haplotype-assisted analysis method.
| S-EPMC6016721 | BioStudies
Project description:Expanded carrier screening (ECS) has been demonstrated to increase the detection rate of carriers compared with traditional tests. The aim of this study was to assess the potential value of ECS for clinical application in Southern China, a region with high prevalence of thalassemia and with diverse ethnic groups, and to provide a reference for future implementations in areas with similar population characteristics. A total of 10,476 prenatal/preconception couples from 34 self-reported ethnic groups were simultaneously tested and analyzed anonymously for 11 Mendelian disorders using targeted next-generation sequencing. Overall, 27.49% of individuals without self-reported family history of disorders were found to be carriers of at least 1 of the 11 conditions, and the carrier frequency varied greatly between ethnic groups, ranging from 4.15% to 81.35%. Furthermore, 255 couples (2.43%) were identified as carrier couples at an elevated risk having an affected baby, sixty-five of which would not have been identified through the existing screening strategy, which only detects thalassemia. The modeled risk of fetuses being affected by any of the selected disorders was 531 per 100,000 (95% CI, 497-567 per 100,000). Our data demonstrate the feasibility of ECS, and provide evidence that ECS is a promising alternative to traditional one-condition screening strategies. The lessons learned from this experience should be applicable for other countries or regions with diverse ethnic groups.
| S-EPMC6336873 | BioStudies
Project description:?-Thalassemia is the most common autosomal recessive single-gene disorder in Sardinia, where approximately 10.3% of the population is a carrier. Prenatal diagnosis is carried out at 12 weeks of gestation via villocentesis and is commonly aimed at ascertaining the presence or absence of the HBB variant c.118C>T, which is the most common in Sardinia. In this study, we describe for the first time the application of semiconductor sequencing to the non-invasive prenatal diagnosis of ?-thalassemia in 37 couples at risk for this variant. In particular, by using a haplotyping-based approach with a hidden Markov model (HMM) and a dedicated pipeline, the two parental haplotypes most likely inherited by the foetus could be established in 30 out of 37 cffDNA samples. Specifically, the paternally inherited haplotype was correctly determined in all 30 of the samples, while the maternal haplotype was incorrectly predicted in six of the 30 genotyped samples. The lack of informative SNPs hampered the inference of both parental haplotypes in the remaining seven samples. As shown in previous studies, we have confirmed that the haplotyping-based approach represents a valuable resource, as it improves the detection of both parental haplotypes inherited by the foetus. In general, our results are encouraging, as we have proven that NIPD is also feasible in couples who are at risk for a monogenic disorder and share the same variant.
| S-EPMC5437905 | BioStudies
Project description:Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude ?-thalassemia major that uses SNPs linked to the normal paternal ?-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (--(SEA)) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (--(SEA)) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (--(SEA)) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4-78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in ?-thalassemia major, and could further be employed to test for other diseases due to gene deletion.
| S-EPMC3182989 | BioStudies
Project description:We used a modified 5i/L/FA system to generate transgene-free naïve iPSCs directly from the fibroblasts of a patient suffering from β-thalassemia and further demonstrated efficient gene correction with a CRISPR/Cas9 system, which provides an improved strategy for personalized treatment of β-thalassemia. Overall design: A modified 5i/L/FA system was used to generate transgene-free naïve iPSCs directly from the fibroblasts of a patient suffering from β-thalassemia.
2016-12-01 | GSE69319 | GEO