|
|
Blood, Vol. 109, Issue 12, 5491-5493, June 15, 2007
A complex splicing defect associated with homozygous ankyrin-deficient hereditary spherocytosis
Blood Edelman et al.
109: 5491
Supplemental materials for: Edelman et al, Vol 109, Issue 12, 5491-5493
Files in this Data Supplement:
- Table S1. Oligonucleotide primers and temperature conditions for denaturing high performance liquid chromatography analysis of the ankyrin-1 gene (PDF, 14 KB)
- Table S2. Oligonucleotide primers (PDF, 8.98 KB)
- Table S3. Ankyrin gene variants detected by denaturing high performance liquid chromatography (PDF, 8.87 KB)
- Table S4. Human ankyrin-1 wild type and ankyrinAnkara minigene mRNA isoforms (PDF, 18.3 KB)
- Figure S1. Minigene analyses of ankyrinAnkara mRNA splicing (PDF, 73.6 KB) -
Construction of ankyrin minigenes. Top. A partial restriction map of the region of the ankyrin gene locus. Human ankyrin minigenes were created using a 96,737bp human ankyrin-1 genomic DNA BAC clone (CTD-2310N4) that contains intron 1 to intron 37 as a HindIII fragment (41652402-41749138). Shotgun cloning of this BAC yielded a KpnI fragment from intron 5 to intron 20. The Kpn I sites near exon 5 and 20, utilized for creation of minigenes, are shown, as are oligonucleotide primers used for reverse transcription and PCR amplification, denoted by the letters (Table S2). Bottom. Cartoons of the wild type and mutant ankyrinAnkara mingenes. A CMV promoter (Pcmv) and bovine growth hormone polyadenylation signals (BGH pA) were added to the wild type and ankyrinAnkara genomic DNA fragments. B. RT-PCR products from transfected K562 cells. Lane 1. RT-PCR products prepared from mock-transfected K562 cells demonstrated the expected size of the normally spliced product derived from endogenous ankyrin gene. Lane 2. RT-PCR derived from RNA of K562 cells transfected with the wild type ankyrin minigene demonstrated the expected size of normally spliced product. Lane 3. RT-PCR products derived from RNA of K562 cells transfected with the mutant ankyrinAnkara minigene. The minor bands in mock and WT-transfected cells, lanes 1 and 2, are nonspecific products that varied in migration and intensity in each experiment. We were unable to subclone any of these nonspecific bands to assign a molecular identity. In mutant minigene-transfected cells, lane 3, bands of altered size were observed that were consistent in migration and intensity from experiment to experiment; subclones were easily and consistently obtained, allowing assignment of molecular identity. C. PhosphorImager quantitation of K562 cell RT-PCR products.
- Figure S2. Detection of the ankyrinAnkara mutation (PDF, 242 KB) -
A. The ankyrinAnkara mutation creates an Alu I recognition site, AGˇCT, allowing discrimination of wild type and mutant alleles by Alu I restriction enzyme digestion. B. Primers flanking the mutation, 5′-GTCAGCTCAGCATTCCTGTAACCAG-3′ and 5′-CACACGTTGTTATCCAGCACACCAG-3′, were used to amplify a 299 bp fragment from genomic DNA from the proband, his parents, and a control. Amplification products were digested with Alu I, fractionated by electrophoresis in a 3% agarose gel, and visualized by ethidium bromide staining. There is an Alu I site located in the sense primer used for PCR amplification. This cleaves a 7bp fragment off the 5′ end of both mutant and control PCR products, and explains the slightly faster migration of the AluI-digested control amplification product (cf undigested product). The 7 bp fragment is not visualized on a 3% agarose gel. C. Amplification products from a normal control were digested by Alu I, yielding a band of 292 bp. Digestion of amplification products from the parents yielded fragments of 292 bp, as well as fragments of 256 bp and 29 bp, indicating a wild type allele and a mutant allele. Like the 7bp fragment, the 29 bp fragment is not visualized on a 3% agarose gel. Alu I digestion of amplified DNA from the proband yielded only the 256 bp fragment, indicating homozygosity for the mutant allele. M indicates marker.
|
|
