| Background and objectivesThalassemia is hereditary microcytic hypochromic anemia characterized by quantitative reduction in gene expression and reduced amount of either the a-globin gene leading to a-thalassemiaor the β-globin genes giving rise to β-thalassemia. In human,2α-and2β-globin chains (HbA) make up the major of red cells in postnatal life and globin is coded by4a and2β genes located on16p13.3and11p15.4respectively. Approximately90%of a-thalassemia results from deletions involving a-globin genes or the control region of the a-globin gene cluster. About95%of β-thalassemia mutations are point mutations in the β-globin gene cluster.In general, the anemia of thalassemia is mild in the carrier (healthy heterozygote), but could be hemolytic and very severe in patients of thalassemia intermedia. The most accepted definition of thalassemia intermedia is involving the transfusion. If a patient starts to become transfusion dependent after the age of three either at large intervals than the usual monthly or just sporadically, it’s defined as thalassemia intermedia.The phenotypes of a-thalassemia are diverse and related to the degree of a-globin expression loss. The loss of only one a gene expression is named a+-allele (-a/) and the reduction of both a genes is α0-allele (--/).Mild anemia occurs with one or two a-genes loss (-α/αα,-/αα,-α/-α). Loss of three a-genes expression causes HbH disease (--/-a) with intermediate to severe anemia. No expression of a-gene leads to a perinatal death, that is Hb Bart’s Hydrops Fetalis (--/--).There is25%chances to have an HbH-disease fetus for a α0-thalassemia carrier and a+-thalassemia carrier. Therefore it is essential to investigate various α0-deletions for prenatal diagnosis.Recently, deletions involving the a-globin gene cluster selected by malaria are common, while a-globin gene duplications are easy to be ignored unless it’s inherited together with β-globin gene defect associated with β-thalassemia. The excessive a-globin gene expression might aggravate the phenotype of one β-globin gene defect carrier to intermediate β-thalassemia. C.L.Harteveld described two intermediate P-thalassemia cases with a-globin gene duplication defect.In this study, we describe two cases of intermediate thalassemia including a-and β-thalassemia in two Chinese families. The intermediate a-thalassemia case in family B is a compound heterozygote for αCSα and a novel large deletion, which removes235kb from the telomere to the HBQ1gene on the short arm of chromosome16. The intermediate β-thalassemia case in family A is a compound heterozygote for βCD41-42and rare282kb duplication from the telomere to the LUC7L gene on the short arm of chromosome16. We identified the breakpoints of the deletion and the duplication by the improved array CGH approach. The functional effect was accessed at the mRNA level by real-time quantitative reverse-transcript PCR (RT-PCR) technology and at the protein level by the globin chain analysis. Materialss and methodsSubjects and hematologic analysisIn family A, the proband was a7-year-old girl who was from Shaoyang city of Hunan Province, southern China. She was first diagnosed with β-thalassemia intermedia at4years old according to the severe anemia (Hb54g/L, MCV68.4fL, MCH16.7pg HbA26.8%and HbF6.3%), jaundice and splenomegaly (data obtained more than one year after the latest transfusion). She received blood transfusion four times sporadically since she was11months old. Iron deficiency was excluded. Common a-globin gene mutations and24known β-globin gene mutations in the Chinese population were performed and we found a CD41-42(-TCTT) normal mutation inherited from his mother which couldn’t explain his phenotype. Therefore, we investigated the proband and his parents.In family B, the proband was an8-year-old boy who was the offspring of unrelated parents that lived in Liuzhou City, Guangxi Province of southern China. He looked pale when he was6months old and received blood transfusion sporadically until last year. In last one year, he received blood transfusion monthly. He showed a proportional short stature, a slight yellow tinge to the skin, serve hepatomegaly and splenomegaly. He displayed severe anemia phenotype with the following hematological parameters:Hb45g/L, MCV73.5fL, MCH19.2pg, HbA22.3%and HbF0.7%(data obtained one month later after the last transfusion). Analysis of his hemoglobin showed an Hb H band. Iron deficiency was excluded. The common a-thalassemia deletion mutations(--SEA/,-α3.7/and-α4.2/) and six non-deletion mutations(αQSα/,αCSα/,αWSα/,αcd30α/,αCD31α/and αCD59α/) were scanned and revealed only one αCSα/mutation inherited from his father, suggesting an unknown defect allele inherited from his mother. So the proband and his parents were referred to our laboratory for further investigation. Probands and their parents’ peripheral blood samples were collected using EDTA as anticoagulant with informed consent. Hematological parameters were analyzed on an automated cell counting (Model Sysmex F-820; Sysmex Co Ltd, Kobe, Japan) and quantification of Hb was performed on the capillary electrophoresis (CE) device (Capillarys, Sebia, Montpellier, France).Molecular diagnosisGenomic DNA was isolated from white blood cells collected from peripheral blood by a standard phenol/chloroform method. The genotype of known mutations of both a-globin and β-globin genes in the Chinese population was analyzed using protocols described previously. Multiplex ligation-dependent probe amplification (MLPA) assay (SALSA P140-B2kit; MRC-Holland, Amsterdam, Netherlands) was performed according to the instruction of the manufacturer to determine the gene dosage of a gene cluster.DNA copy number variations in whole genome from peripheral blood lymphocytes were analyzed using Agilent60-mer oligonucleotide microarrays for array-based comparative genomic hybridization (aCGH) analysis. Labeling, hybridization and washing of test and reference DNA was performed according to the manufacturer’s protocols. Microarray images were analyzed by using Feature Extraction software (version10.10, Agilent Technologies). Classification of gain and loss was based on the software’s segmentation algorithm, as described before. Dual-PCR was performed to determine the breakpoints as described previously.To confirm the deletion breakpoints in family A, known DNA sequences around the breakpoints were used to design F1, R1and F2, R2which were amplified across the breakpoints. In family B, we used the Gap-PCR method with primers around the5’and3’breakpoints. The PCR products were transformed into E.coli DH5a competent cells and selected monoclonal colony PCR products were sequenced directly.RNA and globin chain analysisTotal cellular RNA was extracted using Trizol reagent (Gibco BRL, Gaithersburg, MD, USA), according to the manufacturer’s instruction. Complementary DNA (cDNA) synthesis was performed by using the First-Strand cDNA Synthesis Kit (Toyobo Co., Ltd., Osaka, Japan). The mRNA expression level of a and β-globin alleles was measured by SYBR Green-based relative quantitative RT-PCR method, and the β-actin gene served as a control for assessment of equivalent RNA loading. Five independent tests for each of samples with eight different α-globin genotypes (a normal people, a SEA deletion carrier, Ⅰ1,Ⅰ2, Ⅱ1in family A, Ⅰ1, Ⅰ2, Ⅱ2, Ⅱ3, Ⅲ1, Ⅲ3, Ⅲ4in family B) were conducted in order to calculate the mean mRNA concentration. The primers for amplification of the a-globin mRNA were described before.Fresh blood samples were diluted with5volumes of0.9%NaCl saline and washed three times by3000r/min for5min each. The washed red cells were mixed with an equal volume of deionized water and0.4volume CCl4and then cell debris were removed by centrifugation at6000r/min for10min. Globin chain was determined by RP-HPLC, as described previously.ResultsIdentification of a rare duplication (aaaa282) in family A and a novel deletion (--235) in family BIn family A, the proband Ⅱ1, a7-year-old girl with anemia phenotype Hb61g/L, was identified a common βCD41-42mutation inherited from her mother through convenient analysis of a-thalassemia mutation, suggesting an unknown defect in the other chromosome inherited from her father. According to the MLPA result, a new large duplication including the a-globin gene cluster was showed out of the probe range. Based on the array CGH result, a complete duplication of the a-globin gene cluster was found spanning a region of approximately282kb starting from the telomere of chromosome16short arm. The position of the breakpoints was further identified by Dual-PCR method mentioned before with designing a few pairs of primers throughout the breakpoint regions. The reproducible PCR products with upstream primers F1, R1and downstream primers F2, R2were transformed into DH5a competent cells and selected monoclonal colony PCR products were sequenced directly. The products of upstream and downstream breakpoints are about350bp and800bp respectively, uniquely present in the heterozygote’s DNA. Comparison with the normal sequence showed that the282,199bp duplication segment from the telomere was inserted into the position between94054bp and94055bp. In addition, the downstream breakpoint was found to be joined with a nucleotide A. Moreover, sequences in94038-94053and282184-282199are the same which is GGTGGCTCACACCTGT. And sequencing result showed that the duplication mutation was inherited from her father. This duplication defect increases the number of the a-globin genes from4to6in this patient, explaining the intermediate β-thalassemia status.In family B, the proband Ⅲ1, a8-year-old boy, showed anemia phenotype with Hb45g/L (one month after the latest transfusion). The convenient analysis of a-thalassemia mutation only identified a normal acsa mutation inherited from his father. The MLPA result showed a new large deletion including the a-globin gene cluster out of the probe range. Based on the array CGH result, we found a reduction in gene copy number of chromosome16short arm from the telomere to about235kb. The position of the breakpoints was further identified by gap-PCR with primers F and R. This PCR product, about600bp, uniquely detected in case Ⅰ1, Ⅱ2, Ⅱ3,Ⅲ1and Ⅲ3, was sequenced directly. Compared to the normal sequence, there was a235,259bp deletion in length from telomere. This deletion removes the whole a-globin gene cluster on the allele and explains the HbH phenotype, a severe intermediate a-thalassemia.Phenotypic features of the two novel rearrangementsIn family A, sample Ⅰ1with the αααα282/αα genotype displayed the normal hemoglobin level and the microcytic hypochromic anemia phenotype with reduced MCV (77.4fL) and MCH (26.1pg). Clinically, the cutoff value of MCV is80fl and that of MCH is27pg. This carrier also has normal HbA2concentration (2.6%) and HbF concentration (0.6%). Sample Ⅱ1is compound heterozygous for αααα282and βCD41-42) and exhibited symptoms of the intermediate β-thalassemia trait with decreased Hb (61g/L), MCV(63.7fL), MCH(18.3pg) and increased HbA2(4.5%), HbF(7.7%). Sample Ⅰ2showed the typical phenotype of βCD41-42/p with reduced mean cell volume (MCV), mean cell hemoglobin (MCH), and elevated HbA2level.In family B, the proband Ⅲ1and case Ⅱ3were both compound heterozygote in which only one functional a gene remained. They displayed severe HbH disease phenotype with the--235/αCSα genotype. The proband received transfusion monthly since last year and Ⅱ3had transfusion sporadically. Three members of this family (Ⅰ1,Ⅱ2and Ⅲ3) were single heterozygotes of the--235deletion. They showed microcytic hypochromic anemia phenotype with reduced MCV and MCH and the value of HbA2was close to the cutoff value. The carriers had normal hemoglobin level (120g/L in Ⅱ2) or slightly reduced level (103g/L in Ⅰ1and115g/L in Ⅲ3). Expression level of a-globin mRNA decreased in the heterozygoteTo verify the functional consequences of the duplication in Family A and the deletion in Family B, we measured the α/β mRNA expression level ratio using quantitative RT-PCR method. When the normal control (αα/αα, β/β) was defined as1.0, the mean α/β mRNA ratio was showed in tablel. In family A, the ratio of proband Ⅱ1(αααα282/αα, βCD41-42/β) is4.913while her father (αααα282/αα, β/β)2.672and her mother (αα/αα, βCD41-42/β)2.422, which suggested a significant increasing of the a-globin mRNA due to the addition of two a genes on this abnormal chromosome (αααα282).In family B, the proband Ⅲ1and Ⅱ3(both-235/acsa) showed the ratio as0.161and0.179respectively, while the ratios in Ⅰ1, Ⅱ2and Ⅲ3(all--235/αα) were0.586,0.524and0.663. And the Ⅰ2and Ⅲ4(acsa/aa) showed a ratio of0.899and0.654, while the ratio of an unrelated--SEA/αα sample is0.527. This result indicated that the a-globin mRNA level of the deletion heterozygotes was decreased when compared with normal people, which suggested a significant reduction of the a-globin mRNA due to the absence of two a genes on this abnormal chromosome.Discussion and conclusionIt has been known that the additional a-genes have aggravating effect upon the mild heterozygous β-thalassemia phenotype. It has been reported that the effect of a single or a couple of additional a-genes on the homozygous P-thalassemia is very severe. The type of additional a-genes defect is often disregarded unless the excess of a-globin gene is inherited together with a β-globin gene defect associated with β-thalassemia. Harteveld et al. reported in2008a case of young β-thalassemia heterozygous female with the intermediate P-thalassemia status who had6a genes due to a260kb duplication starting75kb5’of the α2-globin gene to185kb downstream. In the study of family A, we identified a novel large duplication of282 kb from the telomere of the short arm in chromosome16, resulting with6a genes. This duplication case compound heterozygote with β41-42showed a severe intermedia β-thalassemia. The clinical phenotype of the carrier showed no anemia but reduced mean cell volume (MCV) and mean cell hemoglobin (MCH). The RT-RNA analysis showed the α/βmRNA ratio of the heterozygote is elevated compared to the normal people lower than the level of compound heterozygote.There was a high frequency of a-thalassemia in Southeast Asia and South China populations. Approximated50deletions from the a-globin cluster delete both a-globin genes either completely or partially which results in no a-chain synthesis. In most Chinese patients, a-thalassemia is caused by three common deletions (--SEA,-α4.2and-α3.7) and one common acsa point mutation. In a-thalassemia, there are two main types of HbH disease:deletional HbH (--/-α) and nondeletional HbH (--/αTα). Analysis of clinical and molecular features in HbH patients from different populations revealed that the nondeletional type usually has more severe phenotypes than those deletional types. The large deletion--235/αα described in this study of family B, begins from the telomere and removes the whole a-globin gene cluster and all the regulatory elements, causing HbH disease when compound heterozygote with acsa in the proband Ⅲ1and Ⅱ3, which means it a nondeletional HbH disease. They showed severe clinical phenotype with HbH detected. The proband had been transfused monthly in last one year and the Ⅱ3received transfusion sporadically. The relation of genotype and phenotype reflected by the proband and Ⅱ3is in accordance with the common view that patients with nondeletional HbH disease often had more severe clinical features than those with deletional HbH disease. Three members of this family are heterozygote of--235deletion. They had similar hematological parameters with--SEA carriers. RT-PCR analysis showed that the a-globin mRNA of the heterozygotes was decreased when compared to the normal people and similar with the-SEA carrier, while the level of the compound heterozygotes (-235/acsa) decreased markedly compared to the-SEA carrier.Large segment deletions including the gene cluster can be caused by nonhomologous recombination or replication errors. In family A, the duplication downstream sequence near to the breakpoint (94038-94053) is the same16bp with the upstream sequence near to the breakpoint (282184-282199). It can be site-specific recombination but more study should be performed, to confirm it. In family B, no appreciable homo logy between the breakpoints flanking sequences was found, which indicated that this deletion should result from nonhomologous recombination. Based on the clinical features, the telomere deletion and duplication including several highly conserved genes, have showed that the deletion and the duplication have no additional effect on their respective carriers.Being clinically no anemia in the aaaa282carrier, we postulate that this kind of duplications could be more frequently occurring than expected, explaining part of the many unclear cases of severe thalassemia intermedia in (3-thalassemia carriers. When the--235deletion combined with another common a-thalassemia deletion or point-mutation, it would lead to HbH disease or even Hb Bart’s hydrop died in pregnancy. The rare mutation screening in prenatal diagnosis shouldn’t be ignored to decrease reproductive risk. We described a novel deletion and a rare duplication with the breakpoints identified. Our results not only extended the spectrum of thalassemia but also provided new cases for the genomic recombination study. |
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