![Fig. 1. Molecular analysis of ALAS2 and clonal analysis of hematopoiesis. / (A) Pedigree of the family. Circles denote female family members; squares, male family members; and symbols with diagonal lines, deceased members. The proband is denoted by an arrow. All women were heterozygotes for the ALAS2 mutation; the only available male was hemizygote. (B) Clonal analysis of hematopoiesis using HUMARA assay on DNA from peripheral blood leukocytes. The − and + signs indicate sample aliquots undigested (−) or digested (+) with the methylation-sensitive restriction endonuclease HpaII.HUMARA alleles (indicated by arrow) are represented by the lower band in the proband's sample (I-2, homozygous woman), and by the 2 lower bands in the other samples (heterozygous women). Upper bands probably derive from intrastrand secondary structures due to the high G + C content. In HpaII digested samples from all the heterozygous women, the lower allele (paternally derived) is amplified more than the upper allele (maternally derived) compared with undigested samples. This indicates that the maternally derivedHUMARA allele (carrying the mutant ALAS2) was less methylated (more active) and more digested by HpaII than the paternally derived allele. In conclusion, unbalanced X-chromosome inactivation, leading to prevalent inactivation of the paternally derived chromosome (carrying the normal ALAS2allele), occurred in hematopoietic cells from these women. (C) Wild-type and mutant ALAS2 mRNA expression in reticulocytes from 4 family members. RNA was isolated from peripheral blood reticulocytes and reverse transcribed into cDNA; arrows indicate nucleotide 1236 of ALAS2 cDNA. III-1 (hemizygous grandson): As expected, only mutant cDNA (carrying A [adenine] at position 1236) was amplified. I-2 (proband): Only mutant cDNA was amplified, indicating that most of the reticulocyte ALAS2 mRNA derived from the mutant allele. II-2 (elder daughter): Only wild-type cDNA (carrying [guanine] at position 1236) was amplified, indicating that most of the reticulocyte ALAS2 mRNA derived from the wild-type allele. III-2 (granddaughter): Both wild-type and mutant cDNA were amplified. (N indicates the ambiguity resulting from overlapping signals corresponding to both guanine and adenine.)](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/96/13/10.1182_blood.v96.13.4363/5/h82400461001.jpeg?Expires=1735513644&Signature=DH8REW~ZgjKYbHb1JVqPnCEcSiZYHk4FKgKGf0vfYKEiCEVfJZ2ujfH18bbIJ54jUroNPxX79VOgqWDya6awDE1pdTwYPNY48G7mijpL00cKmfreG~sPt54wBHbds9UBrO6TqcRuOWEkbEgvkoJ33~Cvz0V6orQ-zS~qTN4j3ax6clk1x4R-1zDRJkZLbdrO4YoRkYvRfOdcb~EjdfgvJNkLfzA7kTn9dL6tubtFE15estK8EV7AvPm38gbTRHrAt5Xe0nReENsuxYOQ8uRJ9KLt~S6G~ghOLINMqpXSl9t1O3BQJJN31uF90UdhX2JqSPv0DQUq3aT5oguWh6wcBg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Molecular analysis of ALAS2 and clonal analysis of hematopoiesis.
(A) Pedigree of the family. Circles denote female family members; squares, male family members; and symbols with diagonal lines, deceased members. The proband is denoted by an arrow. All women were heterozygotes for the ALAS2 mutation; the only available male was hemizygote. (B) Clonal analysis of hematopoiesis using HUMARA assay on DNA from peripheral blood leukocytes. The − and + signs indicate sample aliquots undigested (−) or digested (+) with the methylation-sensitive restriction endonuclease HpaII.HUMARA alleles (indicated by arrow) are represented by the lower band in the proband's sample (I-2, homozygous woman), and by the 2 lower bands in the other samples (heterozygous women). Upper bands probably derive from intrastrand secondary structures due to the high G + C content. In HpaII digested samples from all the heterozygous women, the lower allele (paternally derived) is amplified more than the upper allele (maternally derived) compared with undigested samples. This indicates that the maternally derivedHUMARA allele (carrying the mutant ALAS2) was less methylated (more active) and more digested by HpaII than the paternally derived allele. In conclusion, unbalanced X-chromosome inactivation, leading to prevalent inactivation of the paternally derived chromosome (carrying the normal ALAS2allele), occurred in hematopoietic cells from these women. (C) Wild-type and mutant ALAS2 mRNA expression in reticulocytes from 4 family members. RNA was isolated from peripheral blood reticulocytes and reverse transcribed into cDNA; arrows indicate nucleotide 1236 of ALAS2 cDNA. III-1 (hemizygous grandson): As expected, only mutant cDNA (carrying A [adenine] at position 1236) was amplified. I-2 (proband): Only mutant cDNA was amplified, indicating that most of the reticulocyte ALAS2 mRNA derived from the mutant allele. II-2 (elder daughter): Only wild-type cDNA (carrying [guanine] at position 1236) was amplified, indicating that most of the reticulocyte ALAS2 mRNA derived from the wild-type allele. III-2 (granddaughter): Both wild-type and mutant cDNA were amplified. (N indicates the ambiguity resulting from overlapping signals corresponding to both guanine and adenine.)
