β-thalassemia major evolution from β-thalassemia minor is associated with paternal uniparental isodisomy of chromosome 11p15

Abstract

β-thalassemia major can be caused by homozygosity or compound heterozygosity for β-globin gene mutations (HBB gene). Most cases are inherited from parents who both have diseased alleles of the HBB gene. We report a patient with late-onset β-thalassemia major that evolved from β-thalassemia minor in which only one of her parents had the diseased HBB gene. To study the cause of β-thalassemia major in this patient, we performed the 100K single nucleotide polymorphism genotyping assay, fluorescence in situ hybridization, and DNA methylation analysis of the imprinting genes near the HBB gene. The results showed a loss of heterozygosity in the region of chromosome 11p14.3 to 11p15.5, which perfectly matched one allele of her father. Our study demonstrates that paternal uniparental isodisomy of chromosomal 11p15.5 is associated with the β-thalassemia major in this patient. Key words: β-thalassemia major, uniparental isodisomy, mosaicism.

Introduction

Thalassemia is the most common recessive single gene disease in humans which is caused by inheritance of an affected allele from both parents.1 The people of Taiwan have a high prevalence of β-thalassemias.2 Various types of molecular defects, most of which are point mutations, affect the expression of the β-globin gene (HBB gene) (GenBank accession n. NC_000011.8) causing β-thalassemia (MIM# 141900). β-thalassemia major is caused by inheritance of two diseased alleles of the HBB gene, one from each parent, and is characterized by severe anemia at an early age, transfusion dependency, and life-limiting complications of iron overload. More than 20 different β-thalassemia mutations are reported in the Taiwanese population, and four of the mutations account for more than 90% of mutant alleles.2,3 We report a β-thalassemia major patient diagnosed at 28 years of age, in which β-thalassemia major developed from β-thalassemia minor.

Design and Methods

Patient

The patient, a 28-year-old woman did not report a history of anemia but first received medical attention for progressive dyspnea and easy fatigue in October, 2005. Her hemogram showed the following results: hemoglobin (Hb) of 2.1 g/dL, RBCs of 1.03 × 10/L, hematocrit of 8.4%, and mean corpuscular volume of 68.3 fl. The percentages of the Hb fractions on Hb electrophoresis were Hb A1, 86.8%, Hb A₂, 5.0%, and Hb F 8.2% (after packed RBC transfusion). The patient’s childhood blood counts were not available, but at 20 years of age (October 1998) her hemoglobin level (8.6 g/dL) and mean corpuscular volume (62.4 fL) were low. Family history revealed that her father also had microcytic hypochromic anemia, with an elevated Hb A₂ level. Upon the diagnosis of β-thalassemia major, further studies were approved by the Institutional Review Board of Kaohsiung Medical University, and written informed consent was obtained from the patient and her family.

DNA preparation and genotyping

DNA was isolated from peripheral blood mononuclear cells, bone marrow, hair follicles, and oral mucosal epithelium by standard methods. The polymerase chain reaction (PCR)-restriction fragment length polymorphism and direct sequencing for the HBB gene mutation analyses were performed using the protocols described by Chang et al.2

RNA preparation and globin messenger RNA quantification

RNA was extracted from peripheral reticulocytes and amplification of α/β globin mRNA by reverse transcription PCR was followed by silver staining of the PCR products according to the protocol described by Lin et al.4

High-density single nucleotide polymorphism genotyping arrays

The single nucleotide polymorphism (SNP) genotyping assay was performed according to the Affymetrix Gene-Chip Mapping 100K Assay Manual. The Affymetrix Mapping 100 K Set contained 116,204 SNPs (consisting of the 50 K Xba and the 50 K Hind arrays that each contained approximately 58,000 SNPs).5 (Data submitted to the GEO repository with Accession Number GSE7847; user name d730093, password m12045).

Fluorescence in situ hybridization

Metaphase spreads of peripheral blood mononuclear cells were prepared for fluorescence in situ hybridization (FISH) using standard methods.6 Slides were then hybridized with three biotinylated fluorescence isothiocyanate-labeled, plasmid DNA probes (pHS1234 miniLAR, puLCR/γ and pβ) from the human β-globin gene region on chromosome 11 (11p15.5). The insert of miniLAR, with a size of 8.3 kb, detected the β-locus control region (β-LCR). The puLCR/γ , with an insert size 10.1 kb, detected the β-LCR and the γ region of the β-gene cluster, whereas the 4.9 kb pβ insert detected the β-globin region of the β-gene cluster.

Loss of imprinting of the H19 differentially methylated region

Bisulphite treatment of genomic DNA was performed as previously described.7 Bisulphite-modified DNA was amplified by PCR. CpG methylase (Sss I)-treated genomic DNA was used as the positive control. To ensure the specificity of the H19 differentially methylated region (DMR) primer sets for bisulphite DNA treatment, amplification using unmodified genomic DNA samples from the proband was also carried out. The primers for the H19 DMR were: forward 5’-TGTTGAAGGTTGGGGAG ATGGGA-3’ and reverse 5’-CCCAAACCATAACACTAAAACCC-3’ (GenBank accession n. NC_000011.8). The PCR products were sequenced using the ABI 310 DNA Sequencer.

Results and Discussion

We found that the patient had homozygous HBB [c.52A>T] + [c.52A>T] (homozygous codon 17 A → T) mutations. Her father had a heterozygous HBB [c.52A>T] + [wt] mutation. However, her mother had a normal HBB genotype. Surprisingly, DNA from her hair follicles and oral mucosal epithelium was heterozygous for the HBB [c.52A>T] mutation. The results of PCR-restriction fragment length polymorphism genotyping are shown in Figure 1A. The peripheral reticulocyte α/β globin mRNA ratio was 34.8 and compatible with the diagnosis of β-thalassemia major.

The homozygosity of the HBB gene mutation [c.52A>T] + [c.52A>T] in the patient and a heterozygous HBB mutation [c.52A>T] + [wt] in her father were discrepant with Mendelian law. Because the acquired chromosome 11p15.5 deletion is frequently observed in a variety of malignancies,8 and acquired deletion of the HBB gene is also described by Badens and by Galanello et al.,9,10 we suspected that the patient may have a hemizygosity caused by acquired deletion of chromosome 11p15.5 harboring the HBB gene. However, FISH analysis showed that both chromosome 11s had positive hybridization signals with all three probes studied (β-LCR, HBG and HBB gene probes), indicating that the HBB gene cluster was probably intact (Figure 1B). To further clarify the cause of the patient’s homozygous HBB mutation, we genotyped the family using a SNP array. The results showed a loss of heterozygosity in the region of chromosome 11p14.3 to 11p15.5 (166 informative markers mapped to the physical distance of 2.73 Mb to 22.39 Mb) which perfectly matched one allele of the father (Figure 2A). Nevertheless, the patient’s hair follicle and oral mucosal cell DNA showed heterozygosity in this region. The results suggest that paternal uniparental isodisomy and mosaicism11,12 of chromosomal region 11p14.3-11p15.5 could be associated with the β-thalassemia major in this patient.

Since the SNP genotyping assay covered only a small part of chromosomal region 11p15.5, we extended the study to the more distal segment of 11p15.5. We investigated the methylation status of the differentially methylated region upstream of the H19 gene (H19 DMR) using the bisulphite direct sequencing method. H19 DMR controls the imprinting of the maternally expressed H19 and the paternally expressed insulin-like growth factor 2 (IGF2) genes (physical distance of 1.97 Mb to 2.11 Mb).13,14 Loss of imprinting (LOI) of H19 DMR (hypermethylated) is associated with the silencing of paternally derived H19 and the activation of paternally derived IGF2. The methylation status analysis of H19 DMR showed LOI in our patient’s peripheral blood and bone marrow DNA whereas no LOI was found in the patient’s oral mucosal epithelium DNA or her parent’s DNA samples (Figure 2B). The evidence for this study shows that β-thalassemia major in our patient was associated with mosaic paternal iUPD of chromosome 11 p15. Firstly, the HBB [c.52A>T] + [c.52A>T] mutation was only found in the patient’s hemopoietic tissue, not in her other tissues, and only one of her parents had the HBB [c.52A>T] mutation. Secondly, the SNP assay showed shared SNP homozygosity in the chromosome region from 11p14.3 to11p15.5, which perfectly matched one allele of the patient’s father, and this phenomenon was not observed in other tissues. Thirdly, two copies of the HBB gene were found during FISH analyses, and the results confirmed that the homozygous informative SNPs were derived from paternal uniparental isodisomy. These results suggest mosaicism of the two different clones of cells from the heterozygous and homozygous HBB [c.52A>T] mutations.15 The paternal uniparental isodisomy of the 11p15 clone contained the HBB [c.52A>T] + [c.52A>T] mutation and the LOI of H19 DMR would result in IGF2 activation and overexpression, and could play a role in cellular proliferation and growth over the clone with the heterozygous HBB [c.52A>T] mutation. Through proliferation, this clone became the only hemopoietic cell in the bone marrow and peripheral blood and resulted in the β-thalassemia major phenotype, a phenomenon found in a variety of malignancies.16,17

Figure 1.(A) PCR-RFLP results for diagnosis of HBB gene [c.52A>T] mutations. Lane 1 is the uncut control fragment of 170 bp. Lanes 2–5 show the results from the patient’s peripheral blood (PB), bone marrow (BM) (homozygote of 147 bp), hair follicles, and oral mucosal epithelium (heterozygote of 170 and 147 bp). Lanes 6–9 are the results of the patient’s father (heterozygote of 170 and 147 bp), mother (normal), elder brother, and younger brother. Lanes 10, 11 are the normal control and positive control for heterozygosity of the HBB gene [c.52A>T] mutation. Lane M represents a 100-bp ladder marker. (B) Fluorescence in situ hybridization analysis of patient’s PB metaphase spreads. The location of the three biotinylated FITC-labeled, plasmid DNA probes (pHS1234 miniLAR, puLCR/γ and pβ) related to HBB gene cluster on chromosome 11 is shown at upper panel. Positive hybridization signal (red fluorescent signal indicated) with miniLAR probe on the terminal short arm of both chromosome 11s (11p15.5) is see at panel a, positive hybridization signals also observed with puLCR/γ probe and p‚ probe on 11p15.5 of both chromosome 11s respectively (b and c). The results showed that each PB cell carried two copies of HBB genes with no deletion found using the above three gene probes.

Figure 2.(A) Haplotype screening of the informative biallelic markers with chromosomal locations in the patient’s PB, hair follicles, oral mucosa cells, and PB from the patient’s father and mother. Twenty-nine representative SNPs of the 166 homozygosity SNPs on chromosome 11p are shown. DNA mutation name, reference SNP identity number, and the respective genotypes of the allelic markers are presented (AA: homozygous for one allele, BB: homozygous for the other allele, AB: heterozygous).The chromosomal locations of the SNPs are shown in the left column. Markers on 11p14.3-15.5 show homozygosity and are not interrupted by heterozygous SNPs in the patient’s blood. (B) Direct sequencing results of the 6259~6287 CpG site of the H19 DMR. Modified DNA of patient’s peripheral blood with all Cs in CpG dinucleotides remaining as C (underlined, upper panel), and patient’s father with heterozygous methylation, one methylated (C) and one unmethylated (T) allele (lower panel).

We conclude that the patient’s β-thalassemia major involved inheritance of paternal uniparental isodisomy of chromosome 11p15 harboring the HBB [c.52A>T] allele, which was mixed with β-thalassemia minor mosaicism caused by normal biparental inheritance. Though homozygosity of autosomal recessive gene mutations was described in uniparental disomy of other chromosomes, homozygosity of HBB gene mutations associated with paternal uniparental isodisomy of 11p15 is reported here for the first time. It is likely that more cases will be recognized in areas with high frequencies of β-thalassemia. Paternal uniparental isodisomy of chromosome 11p15 should be considered in cases of unexplained late-onset of β-thalassemia major, and analysis of HBB gene mutations should not be restricted to the blood only, but should also investigate other tissues.

Acknowledgments

we thank the patient and her family for contributing to this study.We also thank Dr. C.-K. James Shen, Institute of Molecular Biology, Academia Sinica, Taiwan, for providing the pHS1234 miniLAR, puLCR/g and pb DNA used as FISH probes

Footnotes

• Funding: this work was supported by research grants NHRI-EX96-9207SI from the National Health Research Institute, Taiwan and KMUH95-5N12 from Kaohsiung Medical University Hospital, Taiwan.
• Authorship and Disclosures T-CL designed the study, participated in data collection, analysis, and interpretation, wrote the draft of the manuscript, and approved the final version; J-GC participated in designing the study, and in data collection, analysis, and interpretation, and approved the final version; W-CT, I-WC, C-SC and C-CL participated in data collection, analysis and interpretation, and approved the final version of the report. The authors reported no potential conflicts of interest.

• Received September 4, 2007.
• Revision received December 8, 2007.
• Accepted January 7, 2008.