Identification of a novel 91.5 kb-deletion (αα)FJ in the α-globin gene cluster using single-molecule real-time (SMRT) sequencing

Abstract Objectives To present a novel 91.5-kb deletion of the α-globin gene cluster (αα)FJ identified by genetic assay and prenatal diagnosis in a Chinese family. Subjects and Methods The proband was a 34-year-old G3P1 (Gravida 3, Para 1) female at the gestational age of 21+ weeks with a history of an edematous fetus. A routine genetic assay (reverse dot blot hybridization, RDB) was performed to detect common thalassemia mutations. Multiplex ligation-dependent probe amplification (MLPA) and single-molecule real-time technology (SMRT) were used to detect rare thalassemia mutations. Results The hematological phenotypes of the proband, her mother, elder sister, husband, daughter, and nephew were consistent with the phenotype of α-thalassemia trait. No mutations were found in these family members by RDB, except for the proband’s husband who carried an α-globin gene deletion --SEA/αα. MLPA results showed that the proband and other α-thalassemia-suspected relatives had heterozygous deletions around the POLR3K-3-463nt, HS40-178nt, and HBA-HS40-382nt probes. The 5′-breakpoint was out of probe scope and could not be determined. SMRT was performed and a 91.5-kb deletion (NC_000016.10: g.39268_130758del) in the α-globin gene cluster (αα)FJ was identified in the proband and other suspected relatives, which could explain their phenotypes. At the proband’s gestational age of 22+ weeks, an amniotic fluid sample was collected and analyzed. As only the 91.5-kb deletion (αα)FJ was identified in the fetus with RDB, MLPA, and SMRT. The proband was suggested to continue the pregnancy. Conclusion We first reported a 91.5-kb deletion (NC_000016.10: g.hg38-chr16:39268-_130758del) of the HS-40 region in the α-globin gene cluster (αα)FJ identified in a Chinese family. Since the HS-40 loss of heterozygosity in combination with the heterozygous deletion --SEA might result in Hb Bart’s hydrops fetalis, routine genetic assay, and SMRT were recommended to individuals at risk for prenatal diagnosis.


Introduction
Thalassemia is a common monogenic recessive hereditary disease that occurs mainly in tropic, subtropical, and Mediterranean areas.It is classified as a-and b-thalassemia based on the globin gene involved [1].Of these two classes, a-thalassemia is an autosomal recessive hematopathy, characterized by microcytic hypochromic anemia.It occurs when deletions or point mutations exist in a-globin genes or a-major regulatory elements (aMREs).Down-regulated or silenced synthesis of a-globin peptides disrupts the balance between the a-and b-globin peptides, causing a-thalassemia in patients [2].Patients with a-thalassemia could present with a broad spectrum of phenotypes, from asymptomatic features to a severe fatal form of anemia.The severity of the disease depends on the number of affected a-globin genes involved.a-Thalassemia is a common disease, with a global prevalence of approximately 5% [3].A meta-analysis showed the overall prevalence of a-thalassemia being 7.88% in mainland China, with a distribution peaking in the south of China which then decreased from south to north, indicating that southern China is a high-risk region for a-thalassemia [4].We also reported a-thalassemia being common in Fujian, a province of southeastern China, with a prevalence of 4.84% [5].The prevention and control of a-thalassemia would be particularly important in these areas.Carrier screening and prenatal diagnosis based on the genetic assay for preventing births of children with severe disease appears to be the most cost-effective means.
With the development of new technologies, increased numbers of a-thalassemia mutations have been reported.There are more than 300 HBA1 mutations and 400 HBA2 mutations recorded in the databank (http://globin.cse.psu.edu/hbvar/menu.html), up to December 2, 2022.However, a-thalassemia is geographically specific.Routine diagnosis of a-thalassemia in Chinese populations depends on the detection of the six most common a-globin mutations (accounting for more than 95% a-thalassemia cases), including three deletions (-SEA , -a 3.7 [right deletion], and -a 4.2 [left deletion]) and three-point mutations (Hb Constant Spring [CS, HBA2: c.427T > C], Hb Westmead [HBA2: c.369C > G], and Hb Quong Sze (QS, HBA2: c.377T > C)].If the patient could not be diagnosed with tests mentioned above, multiplex ligationdependent probe amplification (MLPA) and Sanger sequencing would be used to detect rare or novel deletions and point mutations, respectively.However, the probe scope of MLPA is limited.When the breakpoint locations of the deletion exceed the detection range, the range of the deletion could not be identified with MLPA.Next-generation sequencing (NGS) can detect common, rare, and novel mutations.However, the read length is too short (less than 300 bp) to identify large deletions or homologous recombinations [6].With the characteristics of long read length, high accuracy, no GC preference, and single-molecule resolution, single-molecule real-time technology (SMRT) enables accurate detection of various genetic mutations [7].To date, SMRT has been used in the genetic analysis of monogenic complex genetic diseases including a-thalassemia [8-10].Compared with the traditional PCR-based method, SMRT has a similar ability in the detection of common a-thalassemia mutations and shows a greater advantage in the detection of rare and novel a-thalassemia mutations [6,11,12].In this study, we first reported a case with a 91.5-kb deletion of the a-globin gene cluster (aa) FJ , which was identified by SMRT and verified in family members.

Subjects
The proband was a 34-year-old G3P1 (Gravida 3, Para 1) female at the gestational age of 21 þ weeks with a history of edematous fetus.In April 2022, she came to our center of Fujian Maternity and Child Health Hospital (Fuzhou, China) for prenatal diagnosis.The proband and her husband were suspected to be a-thalassemia carriers.At the gestational age of 22 þ weeks, 10 mL amnionic fluid sample was obtained from the proband by amniocentesis for prenatal diagnosis.Meanwhile, approximately 2 mL EDTA-K2 anticoagulated peripheral blood was collected from the proband and her family members.Blood cell parameters and the hemoglobin (Hb) concentration and components were analyzed on the XN-3000 blood cell analyzer (Sysmex, Shanghai, China), and the Hb electrophoresis apparatus (Capillary S 2, software version 6.2; Sebia, Paris, France), respectively.This study was reviewed and approved by the Ethics Review Committee of Fujian Maternity and Child Health Hospital.Informed consent was signed by all participants or their parents after a detailed description of the purpose of the study.Retrospective data from patients' medical records are not reported in this study.All experiments were conducted in accordance with relevant guidelines and regulations.

Molecular assays
Genomic DNA was extracted from the peripheral blood samples using a genomic DNA isolation kit (Qiagen; Hilden, Germany) following the manufacturer's instructions.Routine genetic assay of thalassemia (including three common a-thalassemia deletions and three common a-thalassemia point mutations and 17 b-thalassemia point mutations) was performed using reverse dot blot hybridization-PCR (RDB-PCR) (Guangdong Kaipu Biotech Co., Ltd., China).Rare thalassemia mutations were detected using MLPA (P1402-B2 HBA; MRC Holland, Amsterdam, Netherlands) and SMRT as previously described [12].Briefly, genomic DNA was amplified by multiple pairs of primers covering almost the entire a-globin gene cluster and its upstream regulatory regions including the HBA1, HBA2, HBB, HBG, and HS40 genes.The PCR products were ligated with barcoded adaptors and used to construct a SMRT bell library using the Sequel Binding and Internal Ctrl Kit 3.0 (Pacific Biosciences of California, Inc., USA).The library was sequenced on the Sequel II platform (Pacific Biosciences of California, Inc., USA) and then processed by CCS software (Pacific Biosciences of California, Inc., USA) and the Pbbioconda package (Pacific Biosciences of California, Inc., USA).Processed reads were aligned to genome hg38.Structural variations were identified according to the HbVar, Ithanet, and LOVD databases.SNVs and indels were identified by FreeBayes1.3.4 (https://www.geneious.com/plugins/freebayes;Biomatters, Inc., San Diego, CA).In this study, breakpoints of the deletion that identified by SMRT were confirmed by Gap-PCR and Sanger sequencing, using primers as follows: forward primer: 5 0 -CACATTCCCATCAGCTTCTAC-3 0 , reverse primer: 5 0 -TTTTGTCTCTGGCTTATTCAC-3 0 .All primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.(Shanghai, China).The PCR product was 428 bp.The Gap-PCR reaction system consisted of 12.5 lL 2 � PCR Mix, 1 lL DNA template, 10 lmol primers 1 lL each, plus ultra-pure water to 25 lL.The amplification conditions were as follows: pre-denaturation at 95 � C for 3 min; 35 cycles of denaturation at 94 � C for 30 s, annealing at 58 � C for 30 s, extension at 72 � C for 30 s; and a final extension at 72 � C for 8 min.The PCR products were analyzed by electrophoresis agarose gels and then sequenced.Breakpoints of the deletion were determined by comparing the PCR product with the standard sequence from GenBank.

Hematological parameters of the family members
Hematological parameters of the family members are shown in Table 1.The proband and her husband presented with mild hypochromic anemia with low levels of mean corpuscular volume (MCV) (64.0 fL and 69.8 fL) and mean corpuscular Hb (MCH) (20.8 pg and 21.4 pg).The Hb A 2 levels were borderline or slightly lower than normal (2.5% and 2.3%), suggesting that the proband and her husband may be diagnosed as a-thalassemia trait.In addition, we found the proband's daughter, mother, elder sister, and elder sister's son all had mild hypochromic anemia, with reduced levels of MCV (60.0 fL, 63.7 fL, 60.4 fL, and 61.6 fL), MCH (19.6 pg, 19.7 pg, 19.1 pg, and 19.5 pg) and borderline or reduced Hb A 2 levels (2.5%, 2.5%, 2.2%, and 2.6%), which were similar to the phenotype of the proband, indicating that this kind of hypochromic anemia might be inherited.The proband's father showed normal hematological parameters (Hb 156 g/L, MCV 95.3 fL, MCH 30.7 pg).And the result of Hb electrophoresis showed a slight decrease in Hb A 2 (2.4%).

Routine genetic assay for thalassemia in the family
A routine genetic assay for thalassemia in family members was shown in Figure S1.No mutations and one deletion -SEA were found in the proband's father (Figure S1A) and husband (Figure S1F), respectively, which were consistent with their phenotypes.However, no common mutations were identified in the proband's mother (Figure S1B), elder sister (Figure S1C), sister's son (Figure S1D), the proband (Figure S1E), and her daughter (Figure S1G), and the cause of their phenotypes remained unknown.

Identification of a novel 91.5-kb deletion (aa) FJ in the family members
MLPA was then applied to the proband and her relatives with mild hypochromic anemia.Results were the same for all, with a signal of reduced copy number around three probes (POLR3K-3-463nt, HBA-HS40-178nt, and HBA-HS40-382nt) (Figure 1), which suggested the existence of LOH in these three regulatory zones.The 5 0 -breakpoint of the deletion could not be determined because it was out of probe scope, and the 3 0 -breakpoint of the deletion was located in the 30-kb zone between HBA-HS40-382nt and HBZ regionup-364nt.SMRT was performed, and identified a 91.5kb deletion in the regulatory region of the a-globin gene ((NC_000016.10:g.39268_130758del) (Figure 2), which might be the cause of microcytic hypochromic anemia in this family.Gap-PCR using specifically designed primers and Sanger sequencing was performed to confirm this deletion.The 428-bp PCR product (Figure 3A) exhibited a new fusion segment with the deletion of a region 91490 bp long from 39268 to 130758, which was termed as (aa) FJ , a novel type of a 0 deletion (Figure 3B).

Prenatal diagnosis for the proband
As shown in the family pedigree (Figure 4), the (aa) FJ deletion of the proband (II2) and her elderly sister (II3) was inherited from their mother.The genotype of the proband's husband (II1) was -SEA /aa.Therefore, we deduced that the edematous fetus (III2) most likely was compound heterozygous for the deletions (aa) FJ and -SEA.The proband had a 25% chance of bearing an edematous fetus again.Therefore, we drew 10 mL of amnionic fluid from the proband at her gestational age of 22 þ weeks for prenatal examination.Fortunately, we only identified the deletion (aa) FJ in the fetus through routine genetic assay of thalassemia  (Figure S2), SMRT, MLPA, and Sanger sequencing (data not shown).

Discussion
Thalassemia is a monogenic disease that is difficult to cure but clinically easier to prevent.a-thalassemia is one of the most important forms and is caused by deletions or mutations of the a-globin gene cluster or its aMRE.The a-globin gene cluster is located at 16p13.3 in the order of 5 0 -f-Wf-Wa2-Wa1-a2-a1-h-3 0 .Four highly conserved elements (R1-R4), which serve as long-range regulatory elements of a-like globin genes, are located at 30-70 kb upstream of a-globin gene cluster and correspond to the erythrocytespecific DNase I hypersensitive sites (HS-48, HS-40, HS-33, and HS-10) (Figure 5).HS-40 is located 40 kb upstream of the human f-globin mRNA CAP site [13].
It is the most important enhancer element, regulating about 90% of a-globin gene expression [14,15].Differences in the number, arrangement, type, and even spatial distance of the DNA binding sites within the enhancer determine the spatial and temporal differences in gene expression [16].Therefore, even though all a-globin genes are normal, the expression of a-globin peptide chain would be down-regulated in patients with HS-40 deletion.Clinically, patients with HS-40 LOH may present with mild microcytic hypochromic anemia, whereas patients with homozygous HS-40 deletion often present with Hb H disease [17][18][19][20][21][22][23][24][25] (Figure 5).It has also been reported that if one spouse is HS-40 LOH and the other carries only two functionally normal a-globin genes located on one allele (such as -SEA /aa), they will have a 25% chance of bearing an  edematous fetus [17].Simultaneous analysis of a-globin genes (HBA1/2) and regulatory regions was deemed necessary for carrier screening and prenatal diagnosis.
In this study, we tried to use MLPA to detect rare or novel thalassemia-related mutations in a proband with a history of bearing edematous fetus and mild microcytic hypochromic anemia, whose genotype showed no aberration in routine genetic assay of thalassemia.The signal of three probes (POLR3K-3-463nt, HBA-HS40-178nt, and HBA-HS40-382nt) of the proband indicated an unclear regional deletion in MLPA, suggesting an LOH in the upstream of the a-globin gene cluster (Figure 1).A heterozygous deletion of HS-40 was considered, which could explain mild microcytic hypochromic anemia in the proband.It was not the first time that rare or novel deletions containing the HS-40 elements were detected by MLPA.Using MLPA, Coelho et al. have detected four different HS-40 deletions in a group of patients with a-thal phenotype, which included two HS-40 large deletions with lengths of 97 kb and at least 116 kb deletion, respectively [18].Nezhat et al. and Luo et al. also reported two rare heterozygous HS-40 deletions, where MLPA probes showed a ratio 0.5 in related probes [17,26].However, breakpoints of these deletions remained unknown because they were out of probe scope.NGS can detect common, rare, and novel mutations, and has been used for genetic screening of thalassemia [27].However, the read length is too short (less than 300 bp) to identify large deletions or homologous recombinations.In contrast, the read length of SMRT can be as long as dozens of kb and it is more accurate in repeat regions and highly homologous regions.Therefore, SMRT can undertake comprehensive and accurate gene detection, thus avoiding misdiagnosis due to the limitations of the conventional assays [28,29].Xu et al. first used SMRT to measure the full length of HBB and HBA1/2 genes and detect common and rare thalassemia genotypes [8].Subsequently, SMRT has been gradually improved to cover a more extensive detecting range.Now, SMRT could analyze the a-globin genes (HBA1/2) and regulatory regions simultaneously [12].Therefore, we performed SMRT on the proband and we finally found a 91.5-kb deletion on chromosome 16 (NC_ 000016.10:g.39268_130758del) (Figure 2), which was confirmed by Gap-PCR and Sanger sequencing and termed it as (aa) FJ , a novel type of a 0 deletion, according to the reports from Capasso, Xu, and Velasco-Rodr� ıguez D [30][31][32].As the association of the (aa) FJ with the -SEA deletion might lead to Hb Bart's hydrops [17], we suggested a prenatal diagnosis for the proband.Fortunately, the fetus only carried the (aa) FJ deletion.We deduced that the phenotype of the fetus may be mild microcytic hypochromic anemia and the proband was suggested to continue the pregnancy.
In summary, we first reported a 91.5-kb deletion (aa) FJ (NC_000016.10:g.39268_130758del) in a Chinese family.As subjects with HS-40 LOH and -SEA might present with Hb Bart's hydrops fetalis syndrome, prenatal diagnosis using the routine genetic assay of thalassemia and SMRT for rare thalassemia mutations were recommended to individuals at risk.

Figure 1 .
Figure 1.The MLPA analysis of a-globin gene in the proband.

Figure 2 .
Figure 2. A novel 91.5-kb deletion (aa) FJ identified in this family by SMRT.

Figure 3 .
Figure 3. Confirmation of the deletion (aa) FJ identified in the family.(A) Amplification of deletion (aa) FJ by Gap-PCR.(B) Sanger sequencing of the Gap-PCR product, indicating the breakpoints of the deletion.

Figure 4 .
Figure 4. Genotypes of family members shown in the family pedigree.

Table 1 .
Analysis of the hematological parameters of the family members.