Fig. 4.
Fig. 4. 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.253 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.

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.

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