The present study reports the identification and a comparative analysis of the hematological and molecular properties of Hb variants resulting from the same mutation but residing on a different α-globin gene. Our effort was facilitated from the extensive and diverse sample and comprehensive data recording of large diagnostic centers and reference laboratories participating in this study, most of which are members of the ITHANET network (http://www.ithanet.eu).
Structural variants have provided important insights into α-globin gene transcriptional regulation and translation. In particular, using in vitro translation experiments, it has been shown that the HBA2 gene encodes two- to threefold more protein that the HBA1 gene [13]. However, a more detailed study of heterozygous cases for an α-globin gene structural variant showed that the average abnormal Hb percentage in heterozygotes with HBA2 and HBA1 mutations correspond to a α2/α1 ratio of 1.19/1, respectively [14]. This ratio, however, was obtained from the cumulative analysis of all α-globin chain variants, and the relative position of the variant amino acid in the α-globin chain or the relative stability of the variant Hb was not taken into account. The latter may impact on the final amount of α-globin chain, particularly for unstable variants, the translational efficiency or the altered efficiency in hemoglobin tetramer formation. Although the Hb variant carriers reported herein are phenotypically silent, this study provides a much safer approach, since the comparison is more direct and performed between identical mutations in the α-globin genes, yielding identical globin chains. To our knowledge, this is the first attempt to comparatively analyze the mutations in gene paralogues leading to an identical protein product based on data collected from such a large multiethnic sample, not only for the α-globin but also for other gene families.
Our data show that the α2/α1 ratio of the average percentage of the abnormal Hb in heterozygotes with HBA2 and HBA1 mutations, respectively, varies from 0.98 (Hb J-Paris-I) to 1.67 (Hb Gerland; Tables 2 and 3). Moreover, comparison of the α2/α1 ratio of the average percentage of the abnormal Hbs showed that stable and mildly unstable abnormal Hbs nicely cluster in two distinct groups with average α2/α1 ratios of 1.15 ± 0.10 and 1.60 ± 0.09, respectively (Fig. 2). Notably, in all cases summarized in Tables 2 and 3, the percentage of variant Hb has been calculated using cation-exchange HPLC. Therefore, although these values come from different centers and published reports, the use of similar analytical method, apparatus, and analysis software makes these values virtually comparable. Also, the large number of Hb Winnipeg cases studied in this context statistically strengthens the above claims (Table 2). These data are comparable with previous results from heterozygous cases for 24 different α-globin chain variants [14] but sharply contradicts with a previous study [13], which indicated that a mutated HBA2 gene yielding a variant α-globin chain is expressed at a two- to threefold higher level than a mutated HBA1 gene. The main supporting data for this claim were the Hb levels of Hb J-Oxford (40%) and Hb Hasharon (35%), which are comparable to levels expected for Hb variant/α-thalassemia compound heterozygotes (see also Table 3). However, these data were obtained from in vitro translation experiments, and hence, this discrepancy is most likely due to the experimental approach rather that directly measuring Hb variant levels in vivo.
How do these variants occur? Although recurrent mutational events can be a likely cause for some of them, interallelic gene conversion event is the most plausible cause that might have resulted in the same mutation being “transferred” into different genomic contexts. A handful of examples also exist in the human β-like globin genes (reviewed in [15]), as well as in other human multigene families [16]. In favor for this assumption is the fact that 13 out of 14 Hb variants described herein are within exons 1 and 2. These exons, and not exon 3, have been shown to be involved in gene conversion events, as the 3′ end of the human α-globin gene conversion tract is located in intron II (Fig. 3).
Importantly, however, the ethnic background of the family or the individual where the mutation is found cannot be always indicative for the likely mechanism that resulted in the mutation in question. For example, the plethora of Hb Winnipeg observed in the French population may indicate recurrent gene conversion events. However, in the cases of Hb Gerland and Hb Stanleyville-II, although the variants have been reported in two families from France and Southeast Asia and in Caucasian and Black families, respectively, recurrent mutations and (recurrent) gene conversion events are equally likely mechanisms to have generated the mutations in the HBA2 and HBA1 genes, and thorough haplotype analysis is required to favor one mechanism over the other.
Apart from gene conversion events, recurrent unequal crossovers cannot be completely ruled out as a likely cause of these Hb variants. Unequal crossovers have been previously shown to be frequent in the human α-globin gene family [17, 18]. In particular, two successive unequal crossover events, i.e., from a normal (αα) to a rearranged [e.g., deletional (α-) or triplicated (ααα)] chromosome and back, will be undistinguishable from a gene conversion event and have the same consequence, e.g., transfer of a DNA segment from one α-globin gene to another. The rearranged (intermediate) chromosome bearing the variant nucleotide can either persist in the population and, therefore, be found, as in the case of Hb Frankfurt [5] or can subsequently be lost.
In general, gene conversion events can be identified from neighboring to the mutation paralogous sequence variants (PSVs) that are also transferred along with the mutation. Therefore, PSVs are valuable, as they provide insights regarding the length of the gene conversion tract. The Cretan type of non-deletional hereditary persistence of fetal hemoglobin stands as a representative example [19, 20]. HBA2 and HBA1 genes have several PSVs outside the 1,436-bp α-globin gene converted region, spanning from positions −868 to +568 relative to the genes’ transcriptional initiation site [4], but very few PSVs within this DNA segment (depicted as “^” in Fig. 3; see also Supplementary data). Therefore, it is very difficult to unambiguously distinguish these recombinational events from recurrent mutations. At present, there is only one well-documented mutagenic gene conversion event in the human α-globin gene locus (Hb I; p.K16E) identified in both HBA1 and HBA2 genes in cis [6]. This resembles Hb F-Charlotte [HBG1:p.I75T and p.A136E; 21], contrary to Hb F-Waynesboro and Hb F-Lesvos [22], which resulted from the same mutation but in the HBG2 gene (HBG2:p.I75T). Also, two variant α-globin gene alleles consisting of a small DNA segment from the other α-globin gene paralogue (α212 and α121) are most likely the result of a single crossover between a normal and a recombinant allele, although a non-reciprocal gene conversion event cannot be completely ruled out [23]. In all cases, extended haplotype analysis around these variants is an absolute requirement, although this is beyond the scope of the present study.
From a formal genetic point of view, despite the fact that these mutations lead to an identical globin chain, they must be considered as different mutational events, since the nucleotide change occurs in another gene. These situations often cause ambiguities when depositing relevant data in locus-specific databases, like HbVar [1]. The same situation is also reported for two mutations leading to α-thalassemia, namely the c.95+5G>A mutation, found on both α-globin genes and the c.1A>G (initiation codon mutation), found on both HBA2 and HBA1 genes and in a hybrid α-globin gene in the context of α-3.7 thalassemia chromosome. In addition, apart from the Hb variants reported herein, there are also Hb variants for which the amino acid change is identical, but the nucleotide change is different. Such Hb variants are clearly the products of independent mutational events, namely Hb G-Philadelphia (p.N68K; HBA2:c.207C>A or HBA1:c.207C>G), Hb J-Broussais (p.K90N; HBA2:c.273G>T or HBA1:c.273G>C), and Hb Manitoba (p.S102R; HBA2:c.307A>C or HBA2:c.309C>A or HBA1:c.309C>A; also documented as Hb Manitoba I, III, and II, respectively). Therefore, we propose the use of an appropriate suffix in the corresponding variant’s name as a solution to this issue. We propose the use of the [A1] suffix for Hb variants due to HBA1 gene and [A2] suffix for HBA2 gene mutations, e.g., Hb Winnipeg [A1] and Hb Winnipeg [A2], referring to the HBA1:c.226G>C and HBA2:c.226G>C mutations, respectively. This proposed nomenclature is different than the one used for δ-globin chain variants, e.g., Hb A2-Yialousa, to avoid confusion.