Figure 1.
Figure 1. Summary of the molecular analysis of a novel interstitial upstream deletion. (A) The (αα)ZW deletion and its relative position within chromosome 16p13.3. Coordinates (in kb) according to GenBank no. NT_037 887.4 are depicted above the line. Erythroid DHS are shown as black arrows. Numbered gray boxes represent known genes9 located in this subtelomeric region in addition to the α globin genes (labeled with Greek symbols). The small gray bar below the line represents (αα)ZW deletion and the black bar represents the shortest region of overlap (20.4 kb) derived from previously characterized upstream deletions.5 The (αα)ZW deletion provides a new 3′ limit for minimal upstream sequences required for full α globin expression (14.7 kb, dashed line). The small white and gray boxes below the line represent probes used for Southern blotting (white) and MLPA (gray):10 (I) 757 × 758, (87 755-88 337); (II) 4a, (90 548-90 603); (III) 755 × 756, (96 671-97 389); (IV) 5a (103 695-103 774); (V) 767 × 768, (107 503-108 177); (VI) 769 × 770 (108 517-109 159); and (VII) 6a (120 541-120 590). Four small black boxes show the multispecies conserved sequences-regulatory elements (MCS-R1 to -R4). (B) Characterization of (αα)ZW breakpoint. The 3′ breakpoint of the (αα)ZW chromosome was characterized after BglII, XbaI, SacI, and BclI digestion, hybridized with combined probes V and VI. Breakpoint fragments were identified with SacI and BclI demonstrating that the 3′ breakpoint lies between coordinates 105 036-107 043. No breakpoint fragment was detected using probe III, showing that this region was deleted from the (αα)ZW chromosome. The 5′ breakpoint (coordinate 90 512-91 701) was identified using NcoI, PstI, HpaI, and HindII using probe I (not shown). C indicates control; P, patient. (C) Gap-polymerase chain reaction (PCR) amplified a 572-bp fragment specific to the (αα)ZW chromosome. Using forward (ZW-2F; 5′-GCTTAGGGGAAACTGCAGGTG-3′) and reverse (ZW-2R; 5′-AGGCAGACTGCACTTCATTGTTTA-3′) primers, the PCR product was amplified in peripheral blood DNA (PB-DNA) in triplicate. The PCR did not amplify a normal chromosome. M indicates 100-bp marker (New England Biolabs, Hitchin, United Kingdom). (D) Direct sequencing of the breakpoint fragment demonstrates the sequence adjoining between coordinates 90 777 and 106 774 (leftward and rightward to the arrow respectively). (E) Marked decrease in mRNA levels from the (αα)ZW chromosome. An RNase protection assay was used to analyze total RNA extracted from chromosome 16 x mouse erythroleukemia, interspecific hybrids. This showed a severely reduced level of human α globin expression (133-bp protected band) in both uninduced (U) and hemin-induced (I) cells containing the mutant (M) but not the normal (N) chromosome 16. The 93-bp protected fragment represents the mouse α globin mRNA as an internal control.

Summary of the molecular analysis of a novel interstitial upstream deletion. (A) The (αα)ZW deletion and its relative position within chromosome 16p13.3. Coordinates (in kb) according to GenBank no. NT_037 887.4 are depicted above the line. Erythroid DHS are shown as black arrows. Numbered gray boxes represent known genes9  located in this subtelomeric region in addition to the α globin genes (labeled with Greek symbols). The small gray bar below the line represents (αα)ZW deletion and the black bar represents the shortest region of overlap (20.4 kb) derived from previously characterized upstream deletions.5  The (αα)ZW deletion provides a new 3′ limit for minimal upstream sequences required for full α globin expression (14.7 kb, dashed line). The small white and gray boxes below the line represent probes used for Southern blotting (white) and MLPA (gray):10  (I) 757 × 758, (87 755-88 337); (II) 4a, (90 548-90 603); (III) 755 × 756, (96 671-97 389); (IV) 5a (103 695-103 774); (V) 767 × 768, (107 503-108 177); (VI) 769 × 770 (108 517-109 159); and (VII) 6a (120 541-120 590). Four small black boxes show the multispecies conserved sequences-regulatory elements (MCS-R1 to -R4). (B) Characterization of (αα)ZW breakpoint. The 3′ breakpoint of the (αα)ZW chromosome was characterized after BglII, XbaI, SacI, and BclI digestion, hybridized with combined probes V and VI. Breakpoint fragments were identified with SacI and BclI demonstrating that the 3′ breakpoint lies between coordinates 105 036-107 043. No breakpoint fragment was detected using probe III, showing that this region was deleted from the (αα)ZW chromosome. The 5′ breakpoint (coordinate 90 512-91 701) was identified using NcoI, PstI, HpaI, and HindII using probe I (not shown). C indicates control; P, patient. (C) Gap-polymerase chain reaction (PCR) amplified a 572-bp fragment specific to the (αα)ZW chromosome. Using forward (ZW-2F; 5′-GCTTAGGGGAAACTGCAGGTG-3′) and reverse (ZW-2R; 5′-AGGCAGACTGCACTTCATTGTTTA-3′) primers, the PCR product was amplified in peripheral blood DNA (PB-DNA) in triplicate. The PCR did not amplify a normal chromosome. M indicates 100-bp marker (New England Biolabs, Hitchin, United Kingdom). (D) Direct sequencing of the breakpoint fragment demonstrates the sequence adjoining between coordinates 90 777 and 106 774 (leftward and rightward to the arrow respectively). (E) Marked decrease in mRNA levels from the (αα)ZW chromosome. An RNase protection assay was used to analyze total RNA extracted from chromosome 16 x mouse erythroleukemia, interspecific hybrids. This showed a severely reduced level of human α globin expression (133-bp protected band) in both uninduced (U) and hemin-induced (I) cells containing the mutant (M) but not the normal (N) chromosome 16. The 93-bp protected fragment represents the mouse α globin mRNA as an internal control.

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