Transfusion-Dependent Alpha-Spectrin Deficient Hemolytic Anemia Due to Maternal Uniparental Disomy

Abstract 1030

We studied a girl with hereditary spherocytosis who required in utero transfusions and remained transfusion-dependent after birth. Initial clinical, laboratory, and biochemical characteristics of this patient have been described (Blood 78:3043, 1991). Severe alpha-spectrin deficiency was observed in membranes prepared from her marrow-derived cultured erythroid cells. Since this report, despite splenectomy at age 2 years, the proband remains transfusion-dependent. Our goal was to determine the molecular basis of the patient's transfusion-dependent anemia. Typically, alpha-spectrin deficiency is recessively inherited due to homozygous or compound heterozygous inactivating mutations of the alpha-spectrin gene. Genomic DNA from the proband was amplified using primers flanking the 52 coding exons and promoter region of the alpha-spectrin gene. Capillary electrophoresis-based nucleotide sequencing identified a homozygous nonsense mutation in exon 19 of the alpha-spectrin gene. The mother was heterozygous for this mutation; the father did not carry the mutation. To determine if a deletion involving the alpha-spectrin gene locus leading to homozygosity of the nonsense mutation in the proband was present, array comparative genomic hybridization (aCGH) was performed using the 44K CGH microarray (G4426A; Agilent Technologies, Santa Clara, CA). This array includes 44,000 60-mer oligonucleotides covering the entire human genome at a density of ∼14–15 oligonucleotides/MB. No deletions or rearrangements were identified by aCGH. To interrogate the alpha-spectrin locus at higher resolution, SNP typing of proband and parental genomic DNA was performed using the Illumina HumanHap 550 BeadChip. This array contains >550,000 SNPs and includes twenty intragenic SNPs in the alpha-spectrin gene locus. These studies identified a large region of homozygosity at 1q21, approximately 10 megabases in length, extending from rs6657293 (154,995,473, hg18) to rs6670426 (165,730,530) including the PRCC gene (155,003,897) to the CD247 gene (165,888,000) on chromosome 1q in genomic DNA from the proband. This region of homozygosity includes the alpha-spectrin gene locus and 158 other genes. Analyses of the SNP data also confirmed paternity and maternity, with a P-P-C heritability of 0.9997 and no inheritance violations in the alpha-spectrin gene region. Finally, to exclude the possibility of an intragenic microdeletion in the alpha-spectrin gene locus, copy number profiling using quantitative, real-time PCR was performed. Amplicons included exon 19, the location of the nonsense mutation, as well as exons 2, 17, 40, 52 and the 3'UTR. The proband and both parents had 2 copies of the corresponding alpha-spectrin region at all sites examined. No deletions were identified, excluding the possibility that a small deletion of the SPTA1 gene is the cause of homozygosity of exon 19 in the proband. Together, these data indicate there is partial maternal uniparental disomy of chromosome 1 in the proband and that reduction to homozygosity of the 1q region containing the maternal SPTA nonsense mutation is responsible for the alpha-spectrin deficient hemolytic anemia phenotype. This is the first case of uniparental disomy leading to an erythrocyte membrane-associated hemolytic anemia. The homozygosity associated with uniparental disomy consists of duplicated copies of alleles from a single chromosome, leading to increased risk of homozygosity for deleterious recessive mutations. Uniparental disomy should be considered when unexpected results are obtained during carrier testing in a recessive disorder.