Third-Generation Sequencing as a New Comprehensive Technology for Identifying Rare α- and ß-Globin Gene Variants in Thalassemia Alleles in the Chinese Population.

CONTEXT.­: Identification of rare thalassemia variants requires a combination of multiple diagnostic technologies. OBJECTIVE.­: To investigate a new approach of comprehensive analysis of thalassemia alleles based on third-generation sequencing (TGS) for identification of α- and ß-globin gene variants. DESIGN.­: Enrolled in this study were 70 suspected carriers of rare thalassemia variants. Routine gap-polymerase chain reaction and DNA sequencing were used to detect rare thalassemia variants, and TGS technology was performed to identify α- and ß-globin gene variants. RESULTS.­: Twenty-three cases that carried rare variants in α- and ß-globin genes were identified by the routine detection methods. TGS technology yielded a 7.14% (5 of 70) increment of rare α- and ß-globin gene variants as compared with the routine methods. Among them, the rare deletional genotype of -THAI was the most common variant. In addition, rare variants of CD15 (G>A) (HBA2:c.46G>A), CD117/118(+TCA) (HBA1:c.354_355insTCA), and ß-thalassemia 3.5-kilobase gene deletion were first identified in Fujian Province, China; to the best of our knowledge, this is the second report in the Chinese population. Moreover, HBA1:c.-24C>G, IVS-II-55 (G>T) (HBA1:c.300+55G>T) and hemoglobin (Hb) Maranon (HBA2:c.94A>G) were first identified in the Chinese population. We also identified rare Hb variants of HbC, HbG-Honolulu, Hb Miyashiro, and HbG-Coushatta in this study. CONCLUSIONS.­: TGS technology can effectively and accurately detect deletional and nondeletional thalassemia variants simultaneously in one experiment. Our study also demonstrated the application value of TGS-based comprehensive analysis of thalassemia alleles in the detection of rare thalassemia gene variants.

[Genetic analysis of a case with a supernumerary marker derived from chromosome 9].

OBJECTIVE: To delineate a small supernumerary marker chromosome (sSMC) derived from chromosome 9 with combined cytogenetic and molecular methods. METHODS: For a pregnant woman with fetal ultrasound revealing left ventricular punctate hyperechoic echo, and a high risk for monosomy or partial deletion of chromosome 8, chromosome 9 trisomy, monosomy or partial deletion of chromosome 11 by non-invasive prenatal testing, and an abnormal MOM value revealed by mid-term serum screening, amniocentesis was performed for G banded chromosomal analysis and single nucleotide polymorphism array (SNP-array) assay. Peripheral blood samples of the woman and her spouse were also collected for the above tests. In addition, the woman was further subjected to C banding karyotyping analysis and fluorescence in situ hybridization (FISH) assay. RESULTS: The G-banded karyotype of the pregnant women was 47,XX,+mar[20]/46,XX[80], whilst C-banding analysis showed a deep stain in the middle of the sSMC (suggestive of centromeric region) and light stain at both ends (suggestive of euchromatism). FISH combined with DAPI banding analysis using 9pter/9qter probes revealed a karyotype of 47,XX,+mar.ish i(9)(9p10)(9p++)[2]/46,XX[18], whilst SNP-array has revealed a 68.1 Mb duplication in the 9p24.3q13 region. A database search has suggested the duplication to be likely pathogenic. No abnormality was found in her fetus and spouse by karyotyping and SNP-array analysis. CONCLUSION: Through combined cytogenetic and molecular genetic analysis, a sSMC derived from chromosome 9 was delineated, which has enabled genetic counseling for the couple.

[The value of combined detection of HbA2 and HbF for the screening of thalassemia among individuals of childbearing ages].

OBJECTIVE: To assess the application value of combined detection of HbA2 and HbF for the screening of thalassemia among a population of childbearing age in Quanzhou, Fujian, and determine the optimal cut-off values for the region. METHODS: Capillary hemoglobin electrophoresis and genetic testing for α and ß globin gene mutations were simultaneously carried out on 11 428 patients with suspected thalassemia. Statistical methods were used to analyze the distribution of various types of thalassemia and compare the performance of HbA2 and HbF measurement for the screening of various types of thalassemia. The optimal cut-off values for HbA2 and HbF were determined with the ROC curves. RESULTS: 4591 patients with α, ß, and αß compound thalassemia were identified by genetic testing. The most common genotypes for α and ß thalassemia included --SEA/αα and ß654/ßN, ß41-42/ßN, and ß17/ßN. The ROC curves were drawn to compare the performance of HbA2 screening for α-, ß-, αß-compound, static α-, mild α-, and intermediate α-thalassemia, and the maximum area under the curves was 0.674, 0.984, 0.936, 0.499, 0.731, 0.956, and the optimal cut-off values for HbA2 were 2.45%, 3.25%, 3.65%, 2.95%, 2.55%, 1.75%, respectively. CONCLUSION: HbA2 is an efficient indicator for identifying intermediate types of α-, ß-, and αß compound thalassemia. The combination of HbA2 and HbF measurement can effectively detect carriers for ß-thalassemia mutations.

[Genetic Testing for Alpha and Beta Thalassemia in Children in Quanzhou Region of Fujian Province in China].

OBJECTIVE: To analyze the genotypes and distribution of thalassemia in children in Quanzhou Region so as to provide reference for the prevention and control of thalassemia. METHODS: A total of 1 302 children with suspected thalassemia were collected from January 2014 to April 2020 in Quanzhou Region. The deletional α-thalassemia was detected by Gap-PCR, and DNA reverse dot blot (RDB) hybridization was used to detect α- and ß-thalassemia mutations. RESULTS: In the 1 302 cases, 667 cases were identified as thalassemia carriers, and the positive detection rate was about 51.23%. Among them, 380 cases of α-thalassemia gene were detected, and --SEA/αα was the most common genotype with the composition rate about 69.21%. Forty-two cases were identified as HbH disease, and -α3.7/--SEA was the most common genotype. While, 274 cases were identified as ß-thalassemia, and ßIVS-Ⅱ-654/ßN (35.40%) and ßCD41-42/ßN (33.94%) were the most common genotypes. Seventeen cases of ß-thalassemia major/intermedia were identified, and the most common genotypes were ßIVS-Ⅱ-654/ßIVS-Ⅱ-654 and ßIVS-Ⅱ-654/ßCD17. Meanwhile, 13 cases of α- complex ß- thalassemia were detected. Among them, 1 case of ß-thalassemia gene rare mutation Term CD+32 was firstly detected in Fujian Province, and 1 case of CD14-15 mutation was firstly detected in Quanzhou Region. In addition, 3 cases of abnormal hemoglobin disease were identified, in which 2 cases were Hb Q-Thailand and 1 case was Hb G-Honolulu. CONCLUSION: There are various genotypes of thalassemia in children in Quanzhou Region, and many children with thalassemia major or intermedia. Therefore, further prevention and control of thalassemia need to be strengthened for reducing the birth of thalassemia major or intermedia.

Identification of a new ß-thalassaemia variant Term CD+32(HBB: c.32A>C) in two Chinese families.

AIMS: ß-Thalassaemia is an inherited blood disorder caused by mutations in the ß-globin gene cluster. Molecular characterisation of ß-thalassaemia is essential for its diagnosis and management. More and more rare and novel mutations have been reported. METHODS: Two Chinese families with ß-thalassaemia from Fujian Province were recruited in this study. The phenotypes of the probands were confirmed through haematological analysis. Routine molecular analysis of thalassaemia was employed to identify the common mutations of thalassaemia. The rare and novel mutations were detected by direct DNA sequencing. RESULTS: In family 1, the proband, a Chinese woman aged 31 years, showed elevated level of haemoglobin A2 (HbA2). No common mutations associated with ß-thalassaemia were detected, whereas a rare mutation Term CD+32(HBB: c.32A>C) was identified through DNA sequencing. Subsequent investigation of the ß-thalassaemia mutation in her family showed that her mother, her brother as well as her nephew also carried this mutation. In addition, both the proband's husband and her son carrying the rare --THAI mutation exhibited decreased levels of MCH, MCH and HbA2. In family 2, the proband, a child aged 1 year, showed elevated level of HbA2, but had no common mutations of ß-thalassaemia. The proband was identified carrying the mutation Term CD+32(HBB: c.32A>C), which was inherited from his mother. CONCLUSIONS: In this study, we first report a rare ß-thalassaemia mutation in Fujian Province, Southeast China. Moreover, our study also identified this rare mutation in humans. This finding has helped broaden the spectrum of ß-thalassaemia mutations in our region and suggested that this rare mutation may be more prevalent in the Chinese population.

Molecular analysis of α-thalassemia and ß-thalassemia in Quanzhou region Southeast China.

AIMS: Thalassemia is one of the most prevalent inherited disorders in south China. However, there still has no comprehensive research on molecular characterisation of α-thalassemia and ß-thalassemia in the Quanzhou region of Fujian province, a city with high incidence of thalassemia in Southeast China. METHODS: A total of 11 668 cases were collected in Quanzhou region from January 2013 to June 2019. The deletions of α-thalassemia were detected by Gap-PCR, α-thalassemia and ß-thalassemia mutations were detected by DNA reverse dot blot hybridisation. Rare thalassemia gene testing and DNA sequencing were performed to detect rare and novel thalassemia mutation for suspected rare thalassemia carriers. RESULTS: Among 11 668 subjects, 4796 (41.10%) subjects were diagnosed with thalassemia. 3298 (28.27%) subjects were α-thalassemia carriers, 26 types of α-thalassemia mutations were identified, with the common α-thalassemia genotypes being --SEA/αα (71.47%), -α3.7/αα (17.13%) and -α4.2/αα (3.49%). 1407 (12.06%) subjects were ß-thalassemia carriers, 18 types of ß-thalassemia mutations were identified. The common five genotypes of ß-thalassemia were ßIVS-II-654/ßN (36.53%), ßCD41-42/ßN (30.28%), ßCD17/ßN (17.13%), ßCD26/ßN (5.12%) and ß-28/ßN (4.62%). Additionally, 91 (0.78%) subjects with composite α-thalassemia and ß-thalassemia were identified. Furthermore, 9 α-thalassemia and ß-thalassemia gene mutations (CAP +40-43 (-AAAC), IVS-I-1 (G>T), IVS-I-5 (G>C), SEA-HPFH, CD53 (-T), CD37 (A>G), -90 (C>T), CD3 (T>C), -α6.9) were identified for the first time in the region. Among them, CD53 (-T), CD37 (A>G) and -90 (C>T) mutations were identified for the first time in Fujian province. Moreover, CD3 (T>C), -α6.9 mutations were first identified in Chinese individual. CONCLUSIONS: Quanzhou region of South China has high incidence of thalassemia mutations. In this study, several cases of rare thalassemia mutations have been identified, providing reference for clinical consultation. The completion of this study is of great significance to strengthen the prevention and control of thalassaemia in the Quanzhou region.

Molecular analysis of a large novel deletion causing α+-thalassemia.

BACKGROUND: α-thalassaemia is an inherited blood disorder caused by mutations in the α-globin gene cluster. Recognizing the pathogenic α-globin gene mutations associated with α-Thalassemia is of significant importance to thalassaemia's diagnosis and management. METHODS: A family with α-thalassaemia from Fujian, China was recruited for this study. The phenotype was confirmed through haematological analysis. Commercially available Gap-PCR genotypic methods were employed to identify the known deletions causing α-thalassemia. MLPA analysis was used to study the novel mutations; this was then confirmed through DNA sequencing and bioinformatics analysis. RESULTS: The proband of the family belonged to Southeast Asian type (--SEA) thalassaemia. None of the known mutations associated with α-thalassaemia were detected in this family's genetics, whereas a novel 6.9 kb deletion (16p13.3 g.29,785-36,746) covering the α2 gene on the globin gene cluster was identified with MLPA and confirmed through Sanger Sequencing. This data led us to propose a novel pathogenic deletion associated with α-thalassemia: -α6.9 /--SEA. CONCLUSIONS: A novel α-thalassaemia deletion was identified in members of a Chinese family and subsequently analyzed. This finding has helped broaden the spectrum of pathogenic mutations leading to the development of α-thalassaemia, paving the way for improved disease diagnosis and management.

[Analysis a family with partial Xq deletion].

OBJECTIVE: To analyze partial deletion of the long arm of X chromosome in a family and explore the mechanism underlying its phenotypes. METHODS: G-banding technique was employed to analyze the karyotypes of the subjects, and fluorescence in situ hybridization (FISH) was used to analyze their X chromosomes with Xpter, Xqter and WCPX probes. RESULTS: The karyotypes of the proband, her mother and her fetus were all 46,X,del(X)(q24). Combined FISH and karyotyping analysis suggested that the proband and her fetus both carried a Xq24q27.3 deletion. CONCLUSION: The Xq24q27.3 deletion carried by the family is closely related with premature ovarian failure but not with short stature, gonadal dysgenesis and primary amenorrhea.