A rare complex rearrangement in the β‐globin gene cluster causing a novel homozygous <sup>G</sup>γ(<sup>A</sup>γδβ)<sup>0</sup>‐thalassemia
Authors: Xueguang Zhang, Yihong Yang, Haixia Zhang, Ze Du, Hongqian Liu, Ying Shen
Journal: American Journal of Hematology (2021)

Thalassemia is one of the most common autosomal recessive genetic diseases and is responsible for significant morbidity and mortality worldwide.1 Large deletions of the α-globin gene cluster cause most of the types of α-thalassemia, and point mutations or short deletions in the β-globin gene cluster are often associated with β-thalassemia. Rarely, β-thalassemia results from gross gene deletion,2 and only three cases with homozygous deletions in the β-globin gene cluster have been encountered thus far, presumably because it is not compatible with fetal survival.3-5 Gγ(Aγδβ)0-thalassemia is a rare type of β-thalassemia characterized by large deletions of the Aγ-, δ-, and β-globin genes with mild microcytosis, hypochromia, and a compensatory increase in fetal hemoglobin (Hb F).6 Most functionally characterized Gγ(Aγδβ)0-thalassemias described to date are heterozygotes that represent simple chromosomal rearrangements in the β-globin gene cluster.7 We herein report a novel homozygous deletion of Gγ(Aγδβ)0-thalassemia associated with a complex rearrangement in the β-globin gene cluster identified in a thalassemia major patient from a Chinese consanguineous family. A 14-year-old Chinese boy from a consanguineous family (Figure 1(A)) was referred to our institution for phenotypes of severe anemia showing jaundice, brown pigmentation of the skin, facial bone alteration, and serious hepatosplenomegaly; in addition, features, such as poor musculature, growth retardation, and failure of sexual development were also prominent; however, there was no history of blood transfusion (Figure 1(B) and (C)). Hematology analysis of this index indicated marked microcytosis [mean cell volume (MCV) 73.8 fL] and hypochromia [mean cell hemoglobin (MCH) 20.0 pg], coupled with low Hb levels (9.2 g/dL), indicating the possibility of thalassemia (Table S1). Iron deficiency (serum ferritin 560 μg/L) and G-6-PD deficiency (20.7 U/gHb) were excluded from this index. Hemoglobin analysis revealed decreased Hb A2 (0.9%) and extremely elevated Hb F (91.5%), and Hb A was absent (Table S1). Blood examinations were also performed on his parents (who were first cousins) and sister who were clinically asymptomatic. Compared to the patient, the three family members exhibited mild microcytosis and hypochromia, and moderately elevated levels of Hb F (Table S1). Noticeably, primary screening of genetic testing for α-thalassemia and β-thalassemia showed absent deletion and mutation in the α-globin gene. Subsequent multiplex ligation-dependent probe amplification (MLPA) analysis revealed a homozygous deletion involving the HBB, HBD, and HGB1 genes in the patient, and his parents showed the heterozygous state of this deletion (Figure S1(A)). Nanopore sequencing with the advantage of long reads was next used to resolve the deletion range and breakpoints more precisely due to the long distance between MLPA probes. Remarkably, a 78.9 kb deleted region in the β-globin gene cluster (chr11:5191125–chr11:5270051) (refer to GRCh37/hg19) was identified with the removal of HBB, OR51V1, HBBP1, HBD, OR52Z1, BGLT3, and partial HBG1 (Figure 1(D), methods in Appendix S1), which represents a novel homozygous Gγ(Aγδβ)0-thalassemia deletion. Quantitative PCR (qPCR) analysis of the copy numbers of HBB, OR51V1, HBBP1, HBD, OR52Z1, BGLT3, and HBG1 further confirmed this deletion (Figure S1(B), primers in Table S2). Gap-PCR results showed that the proband only exhibited the deleted amplicon, while both the deletion and internal control fragment were detected in the carriers, compared with the normal control who could only amplify the internal control (Figure 1(E), methods in Appendix S1). To date, only one homozygote of Gγ(Aγδβ)0-thalassemia has been reported, which is characterized as Chinese Gγ(Aγδβ)0-thal.3 Surprisingly, our patient presented retention of 36 bp (chr11:5237504-chr11:5237539) in the deleted region by sequencing of the PCR fragment of the deleted amplicon (Figure 1(F)). Therefore, the deletion identified in the current study is distinct from the previously reported simple deletion with the removal of a single region of chr11:5191148–chr11:5270051, and the copy number variation (CNV) of our index indicates a complex chromosomal rearrangement event in the β-globin gene cluster representing two deleted fragments (chr11:5191125–chr11:5237503 and chr11:5237540–chr11:5270051) with a link of a 36 bp sequence between them. Next, we tried to explain the potential factors that may contribute to this novel CNV. After analyzing the identity of the sequence around the four breakpoints (chr11:5191125–chr11:5237503 and chr11:5237540–chr11:5270051), there was no significant homology between the 5′ and 3′ junction sequences (Figure 1(F)), indicating that these two deletions were not caused by the non-allelic homologous recombination (NAHR). For the left deletion (chr11:5191125–chr11:5237503), it was associated with the nonhomologous end-joining (NHEJ) event (Figure 1(G)), because few special structures were found around the junction. Intriguingly, microhomology of nucleotide T was observed in both breakpoints (chr11:5191124 and chr11:5237503) (Figure 1(G)), which could promote the NHEJ process.8 Strikingly, microhomology of four nucleotides (TCCA) in breakpoints (chr11:5237541–44, and chr11:5270052–55) of the right deletion (chr11:5237540–chr11:5270051) induced microhomology-mediated break-induced replication (MMBIR) and further caused loss of the fragment between chr11:5237540 and chr11:5270051 (Figure 1(G)). Collectively, this complex chromosomal rearrangement showing discontinuous deletions mixed with 36 bp retention between the two deletions detected in the patient might be induced by MMBIR, which is thought to be the main origin of complex CNVs.9 Here, the extremely high expression of Hb F is causal for patient survival. In adults, high production of Hb F might be associated with hematologic stress, and it is also dependent on various genetic factors.2 Prominently, several single-nucleotide polymorphisms (SNPs) associated with high Hb F levels were identified in the index, including rs7719521 (chr5:151049404), rs4910742 (chr11:5306509), rs1391619 (chr11:5455929), rs2855123 (chr11:5277078), and rs2855126 (chr11:5273147), by whole genome sequencing (WGS) analysis.10-12 In addition, significantly changed mRNA levels of the four transcription factors for Hb F expression were identified in the patient compared to his sister, by RNA-sequencing, including increased expression levels of TAL1, ATF2, and ATF4, which act as positive regulators of Hb F levels,13-15 and decreased LYAR, which is essential for silencing the γ-globin gene.16 Likewise, qPCR assay further validated the altered expression of these four genes in the patient (Figure S1(C), methods in Appendix S1). However, clinical presentation of this patient showed marked hepatosplenomegaly with growth retardation, which reflect that the clinical severity is moderately severe [rather than non-transfusion-dependent type (NTDT)], and the patient should be currently treated as a transfusion-dependent type (TDT) (regular transfusion) according to the current practice guidelines, although he is alive based on the extremely elevated Hb F levels. In conclusion, we report the first observation of a complex CNV as a new cause underlying Gγ(Aγδβ)0-thalassemia, and we noticeably unveil a rare case of a homozygous large deletion in the β-globin gene cluster. Here, this complex CNV resulted from the MMBIR process leading to two discontinuous deletions separated by a short sequence, which presents the evidence for existence of a complex genomic rearrangement in deletional β-thalassemia and also the potential explanations for its occurrence. The factors responsible for the extremely elevated Hb F concentration fundamentally resulting in clinical improvement of this patient are complex, and they might be associated with the nucleotide variations in the candidate site loci highly correlated with the Hb F levels, or the significantly changed expression of the transcription factors of Hb F might also play a role in the maintenance of high Hb F level to a certain extent. Collectively, discovery of the new type of β-thalassemia would provide valuable insights into the limited findings of the molecular mechanism responsible for homozygous deletional β-thalassemia, which are important for the diagnosis and treatment of this disease. This work was supported by grants from the Sichuan Provincial Health Commission (No. 20PJ073). The authors declare no conflict of interest. H.L. and Y.S. designed the study and wrote the manuscript. X.Z., Y.Y., H.Z., and Z.D. performed experiments and analyzed of the data. All authors contributed to the review and approval of the manuscript. This study was conducted following the tenets of the Declaration of Helsinki and ethical approval was obtained from the Ethical Review Board of West China Second University Hospital, Sichuan University. All subjects signed an informed consent. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1 Supporting Information Figure S1 A large deletion in the β-globin gene cluster detected in the family. (A) MLPA analysis of this family. The result showed that the homozygous deletion extends from OR51V1 to HBG1 in the patient (i), and half dosages of these probes located in the same region were observed in his parents (ii and iii) compared to the normal control (iv). (B) qPCR validated the deletion in this family. Results are representative of three independent experiments. Student's t test; *p < 0.05; error bars, s.e.m. (C) qPCR confirmed the significantly changed expression of the transcription factors for Hb F production in the patient compared to his sister. Results are representative of three independent experiments. Student's t test; *p < 0.05; error bars, s.e.m. Table S1 Hematology results for this family. Table S2 The qPCR primers used in the present study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.