Alpha-Thalassemia in Southern Italy: Characterization of Five New Deletions Removing the Alpha-Globin Gene Cluster

Abstract

α-thalassemia is characterized in about 80% of cases by deletions generated by the presence of duplications and interspersed repeated sequences in the α-globin gene cluster. In a project on the molecular basis of α-thalassemia in Southern Italy, we identified six families, showing an absence of the most common deletions, and normal α-globin gene sequences. Multiplex Ligation-dependent Probe Amplification (MLPA), qRT-PCR, and the sequencing of long-range PCR amplicon have been used for the identification and characterization of new deletions. MLPA analysis for the identification of α- and β-globin rearrangement revealed the presence of five new α-thalassemia deletions. The set-up of qRT-PCR allowed us to delimit the extent of the deletions ranging from about 10 kb to more than 250 kb, two of them being of the telomeric type. The long-range PCR generated a specific anomalous fragment in three deletions, and only several unspecific bands in the other two deletions. The sequencing of the anomalous amplicons revealed the breakpoints of two deletions: the --PA, 34 kb long, identified in two families, and the telomeric --AG, 274 kb long. The anomalous fragment containing the breakpoint of the deletion --FG was partially sequenced, and it was not possible to identify the breakpoints due to the presence of several repetitive Alu sequences. The analysis of the breakpoint regions of the --Sciacca and --Puglia, respectively, are about 10 and 165 kb long, and revealed the presence of repeats that most likely impaired the amplification of a specific fragment for the identification of the breakpoint. MLPA, in association with qRT-PCR and long-range PCR, is a good approach for the identification and molecular characterization of rare or new deletions. Breakpoint analysis confirms that Alu sequences play an important role in favoring unequal crossing-over. Southern Italy shows considerable genetic heterogeneity, as expected with its central position in the Mediterranean basin, favoring migratory flows.

Keywords: --AG; --FG; --PA; --Puglia; --Sciacca; MLPA; alpha-thalassemia; deletion breakpoint characterization; qRT-PCR.

Figure 1

Scheme of the telomeric region on chromosome 16 containing the α-globin gene cluster. In the upper part are reported the genes and the MCS-R, then the location of the MLPA probes (indicated with numbers followed by the letter M) and of the quantitative real-time PCR fragments (indicated with numbers). The five new deletions (--Sciacca, --FG, --PA, --Puglia, and --AG) are indicated in red, the deletions already described are indicated in grey, and the undefined region are in black. Four known deletions --GEO, --SPAN, --NOR, and --CANT are compared with the new deletions --Sciacca and --FG. --PA is compared with two known deletions --MED II and --CAL. The --AG is compared with the --285 and the --JT deletions.

Figure 2

Molecular characterization of the five new alpha-thalassemia deletions. (A–E) MLPA analysis of five new deletions. Comparison between the value obtained from the proband examined and normal control (y-axis). The position of the different probes on the α -globin gene cluster is indicated on the x-axis. In red, the probes with half-dose; in gray, the probes with normal dosage. (F–J) Quantitative real-time PCR analysis of 33 probes (primers are reported in Table S2) posed in regions not tested by MLPA: red diamonds show fragments in half-dose; blue diamonds show normal dosage. The localization of the MLPA probes is indicated by the arrows. (K,L) Sequencing of the long-range gap-PCR fragments. In the middle has been reported the breakpoint of the deletion, above and below, respectively, the regions at 5′ and 3′. The new deletions most likely originated from events of non-homologous recombination mediated by short sequences (highlighted with a box), common to both the extremities and most likely causing the formation of loops. The HGVS code of the --PA is NC_000016.10:g.(146281_146304)_(180074_180097)del33793 and the HGVS code of the --AG is NC_000016.10:g.10001_(284537_284542)del274537.

Figure 3

Molecular characterization of the --FG deletion. (A,B) Product of the long-range GAP-PCR separated on new-Sieve agarose gel. Line 1: carrier of the --FG deletion; line 2: ladder. (C) Scheme of the sequencing analysis of the amplicons. In the upper part have been reported the oligos and the length of the amplicons. Green boxes indicate the sequenced regions, white boxes the un-sequenced regions. The number close to the vertical arrow refer to the last sequenced positions on the chromosome. (D) Probable breakpoint. The boxes with arrows indicate the repeated sequences and the orientation located, respectively, at 5′ and 3′ of the breakpoint. The red dotted lines indicate the possible recombinant event.