1. α-Thalassemia resulting from a negative chromosomal position effect
by Virginia M. Barbour, Cristina Tufarelli, Jacqueline A. Sharpe, Zoe E. Smith, Helena Ayyub, Cynthia A. Heinlein, Jacqueline Sloane-Stanley, Karel Indrak, William G. Wood, and Douglas R. Higgs
Blood
Volume 96(3):
August 1, 2000
©2000 by American Society of Hematology

2. Structure of the terminal region (approximately 300 kb) of the human chromosome 16p.The oval on the left represents the telomere.
Structure of the terminal region (approximately 300 kb) of the human chromosome 16p.The oval on the left represents the telomere. Previously described genes (4-203) are shown as black boxes above the line (transcribed toward the centromere) or below the line (transcribed toward the telomere). The α-globin regulatory element is shown as a white box (approximate coordinates 103   850). The embryonic (ζ) and fetal/adult (α) genes are indicated. Below the chromosome, the positions of previously characterized DNaseI hypersensitive sites (DHSs) and CpG islands (labeled A-N) are shown. Below this is a graph of the percent ofAlu sequences per 3 kb. Dashed vertical lines represent the 5′ and 3′ extents of the ZF deletion. The scale is in base pairs. Coordinate 1 is the first nucleotide in the chromosomal sequence described in Flint et al.3
Virginia M. Barbour et al. Blood 2000;96:
©2000 by American Society of Hematology

3. Details of the region around the −ZFbreakpoints
Details of the region around the −ZFbreakpoints.The positions of the Alu elements orientated toward (Up) or away from (Down) the telomere are shown.
Details of the region around the −ZFbreakpoints.The positions of the Alu elements orientated toward (Up) or away from (Down) the telomere are shown. Below this, tandem repeats, other repeats, and DNaseI HS and CpG islands (labeled F-L) are shown. The ζ, α, and θ1genes are transcribed toward the centromere, and the gene16PHQG;16 is transcribed toward the telomere. The α−ZF deletion is shown as a black bar below the chromosome. Genomic mapping localized its 5′ breakpoint between an HpaI site (at coordinate 164 012) and a SacI site (at coordinate 164 356), beyond the polyA addition site of the α2-globin gene. Pulsed field gel electrophoresis of a Not1 α-specific fragment, approximately 380 kb, indicated that the deletion extends for approximately 18 kb (data not shown). The 3′ breakpoint was localized between a BglII site (coordinate 180 096) and a BamHI site (coordinate 182 417). Forward 280 and reverse 279 primers were designed from the breakpoint regions, and a 928-bp fragment spanning the breakpoint was amplified (data not shown) in the propositus (Z.F.) and his mother (H.F.). DNA sequence analysis demonstrated that the breakpoints lie between coordinates 164  and 182  and arose via an illegitimate recombination event (sequence available on request). Five previously described deletions that remove overlapping segments of this region are denoted a-e14; none of these silence α gene expression, although deletion a, −α3.7, only removes 1 α gene. Cosmids described in the text are shown at the bottom of the figure. The black box attached to cDH2 represents the α-globin regulatory element (HS –40).
Virginia M. Barbour et al. Blood 2000;96:
©2000 by American Society of Hematology

4. Expression studies of cDH2
Expression studies of cDH2.Nuclease protection assays to analyze human -globin gene expression in (A) the abnormal interspecific hybrid (ZF2) and (B) adult peripheral blood from lines of transgenic mice carrying the cDH2 construct.
Expression studies of cDH2.Nuclease protection assays to analyze human -globin gene expression in (A) the abnormal interspecific hybrid (ZF2) and (B) adult peripheral blood from lines of transgenic mice carrying the cDH2 construct. Two samples from different individuals from line α84 are shown.
Virginia M. Barbour et al. Blood 2000;96:
©2000 by American Society of Hematology

5. Analysis of the pattern of methylation along the −ZF chromosome
Analysis of the pattern of methylation along the −ZF chromosome.(A) CpG islands (A-N, see also Figure 1) were analyzed with a combination of enzymes and probes, which were previously described.2 53 In each panel, the left-hand lane is the chosen digest (eg...
Analysis of the pattern of methylation along the −ZF chromosome.(A) CpG islands (A-N, see also Figure 1) were analyzed with a combination of enzymes and probes, which were previously described.2 53 In each panel, the left-hand lane is the chosen digest (eg, BglII) using DNA from the peripheral blood of an unaffected individual. The middle lane represents a double digest incorporating a methylation-sensitive enzyme (eg,BglII/SacII) using DNA from the peripheral blood of an unaffected individual. The third lane represents the same double digest (eg, BglII/SacII) using DNA from the peripheral blood of Z.F. The presence of 2 variably sized bands using IZHVR reflects the 2 different VNTR alleles detected with this probe. (B) Analysis of the CpG islands (H and I) associated with the α-globin genes using DNA from peripheral blood. In each case the left-hand lane is aPstI digest, and the right-hand lane is aPstI/EagI digest. (Only EagI cuts unmethylated sites.) N indicates an unaffected individual; ZF, the propositus; AF, the unaffected father; and HF, the affected mother. ND is a patient in whom an α gene was inactivated by a nondeletional form of α-thalassemia, demonstrating that such mutations do not alter the pattern of methylation. Note that both CpG islands (H and I) are examined in this assay. The different signal intensities of uncut DNA (methylated) to cut DNA (unmethylated) in Z.F. (αα/α−ZF) and H.F. (ααα/α−ZF) is explained by their different genotypes. Only the α−ZF chromosome is methylated at CpG island H. (C) Analysis of DNA from a sample of semen from Z.F. (D) Analysis of the CpG islands (H and I) in EBV-transformed lymphocyte lines from an unaffected individual (N), Z.F., and a MEL16 hybrid containing only the abnormal copy of chromosome 16 (ZF2). (E) Bisulphite modified sequence analysis of DNA from the peripheral blood of the propositus (ZF) and his unaffected father (AF). During bisulphite treatment of DNA, unmethylated cytosines were converted to uracil and subsequently PCR-amplified as thymidine. Methylated cytosines were resistant to this conversion and hence were amplified as cytosine. Arrows indicate that whereas all cytosines were converted to thymidines in A.F., many remain unconverted (methylated) in Z.F. Note that only the cytosines on one allele in Z.F. are methylated.
Virginia M. Barbour et al. Blood 2000;96:
©2000 by American Society of Hematology

6. Analysis of endonuclease sensitivity in the −ZF chromosome
Analysis of endonuclease sensitivity in the −ZF chromosome.DNA from nuclei of (A) EBV lymphocytes, (B) an interspecific MEL hybrid containing the α−ZF chromosome (ZF2), and (C) the erythroid cell line K562.
Analysis of endonuclease sensitivity in the −ZF chromosome.DNA from nuclei of (A) EBV lymphocytes, (B) an interspecific MEL hybrid containing the α−ZF chromosome (ZF2), and (C) the erythroid cell line K562. The DNA was incubated with increasing amounts of Hinf I (“Materials and methods”) and analyzed with probes around gene no. 6 (RA0.6, approximate coordinate 67 000), which encodes the ubiquitously expressed MPG gene, the α-globin regulatory element HS –40 (approximate coordinate 103 000), and the α-globin gene. Whereas the site associated with HS –40 is clearly present in ZF2, the sites are missing from the silenced α-globin promoter.
Virginia M. Barbour et al. Blood 2000;96:
©2000 by American Society of Hematology

7. Analysis of DNase1 hypersensitive sites along the −ZF chromosome
Analysis of DNase1 hypersensitive sites along the −ZF chromosome.DNA from the nuclei of an interspecific MEL hybrid containing the α−ZF chromosome (ZF8) or a normal chromosome (JY5-4) using probes and enzymes that detect the hypersensitive sites at the CpG...
Analysis of DNase1 hypersensitive sites along the −ZF chromosome.DNA from the nuclei of an interspecific MEL hybrid containing the α−ZF chromosome (ZF8) or a normal chromosome (JY5-4) using probes and enzymes that detect the hypersensitive sites at the CpG islands associated with the α2-globin promoter (H) and the surrounding CpG islands B, G, E, and K (see Figure1). Analysis of HSs at E and H in normal hybrids (JY5-4) and abnormal hybrids (ZF8) shows that although the constitutive site at E is sensitive in both, the HS at the silenced α promoter is insensitive.
Virginia M. Barbour et al. Blood 2000;96:
©2000 by American Society of Hematology

8. The pattern of replication along the −ZFchromosome
The pattern of replication along the −ZFchromosome.(A) Replication of the human 16p region was assayed by FISH as set out in Smith and Higgs4 using a set of 12 cosmids spanning this region.
The pattern of replication along the −ZFchromosome.(A) Replication of the human 16p region was assayed by FISH as set out in Smith and Higgs4 using a set of 12 cosmids spanning this region. The pattern in an interspecific hybrid containing a normal copy of chromosome 16 (closed boxes) is compared with a hybrid containing the α−ZF chromosome (open circles). In the α−ZF chromosome, there is no value for Cos 12 (midpoint at approximate coordinate 177 000) because this region is deleted. Black dashed lines indicate the mean percent doublet scores for the early replicating control cosmids (top line) and late replicating control cosmids (bottom line). Below, the key features of the 16p region are shown. (B) Replication timing of control cosmids. The mean value for all early (p48) and late (cSamD4) mouse controls4 are represented as columns. Values for controls measured in the hybrid containing the α−ZFchromosome are shown as a black diamond with lines representing plus or minus 1 SD.
Virginia M. Barbour et al. Blood 2000;96:
©2000 by American Society of Hematology