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, 33 (6), 715-25

Mutations in the Promoter Region of the Aldolase B Gene That Cause Hereditary Fructose Intolerance


Mutations in the Promoter Region of the Aldolase B Gene That Cause Hereditary Fructose Intolerance

Erin M Coffee et al. J Inherit Metab Dis.


Hereditary fructose intolerance (HFI) is a potentially fatal inherited metabolic disease caused by a deficiency of aldolase B activity in the liver and kidney. Over 40 disease-causing mutations are known in the protein-coding region of ALDOB. Mutations upstream of the protein-coding portion of ALDOB are reported here for the first time. DNA sequence analysis of 61 HFI patients revealed single base mutations in the promoter, intronic enhancer, and the first exon, which is entirely untranslated. One mutation, g.-132G>A, is located within the promoter at an evolutionarily conserved nucleotide within a transcription factor-binding site. A second mutation, IVS1+1G>C, is at the donor splice site of the first exon. In vitro electrophoretic mobility shift assays show a decrease in nuclear extract-protein binding at the g.-132G>A mutant site. The promoter mutation results in decreased transcription using luciferase reporter plasmids. Analysis of cDNA from cells transfected with plasmids harboring the IVS1+1G>C mutation results in aberrant splicing leading to complete retention of the first intron (~5 kb). The IVS1+1G>C splicing mutation results in loss of luciferase activity from a reporter plasmid. These novel mutations in ALDOB represent 2% of alleles in American HFI patients, with IVS1+1G>C representing a significantly higher allele frequency (6%) among HFI patients of Hispanic and African-American ethnicity.


Figure 1
Figure 1. DNA sequence analysis reveals novel mutations in the 5′-UTR of ALDOB
Left, DNA sequence analysis of a 981 bp fragment of the 5′-end of ALDOB performed with the primer 5′-AGCTGTATGACTTAAGGGAACCTCC-3′ revealed a mutation at position −132 in patient 50, indicated by an (N). Right, DNA sequence analysis of a similar fragment from patient 295 performed with the primer 5′-ATCAATCTTGGGCATTTTGCCACCT-3′ revealed a mutation at position IVS1+1, indicated by an (N). The corresponding nucleotide sequence is written above each peak with G (black), A (green), T (red), and C (blue) in corresponding colors.
Figure 2
Figure 2. The DNA sequence of the ALDOB promoter region
The sequence of human ALDOB from position −246 to +85 is shown with exon 1 capitalized. The start of transcription is indicated with a forward arrow at position +1. Nucleotides within the promoter that are conserved across vertebrates are indicated with an asterisk (*). The boxed sequence represents the TATA box. DNA sequences containing known and proposed cis-elements and their corresponding transcription factors are denoted with brackets under the sequence. Known single nucleotide polymorphisms are indicated with an inverted carrot (v) (from 5′ to 3′: SNP rs35386088, rs12337537, rs17551011, and rs71866381 as defined by NCBI). The novel mutations are indicated with upward arrows showing the nucleotide change.
Figure 3
Figure 3. In vitro EMSA analysis
Panel A, Nuclear extract (10 μg) prepared from A293 cells was incubated with radiolabelled DNA probes that contained either wild type sequence or g.–132G>A mutant sequence (2 × 105 cpm) indicated at the top. Indicated at the bottom, lanes 1 and 6 contained no nuclear extract. Lanes 2 and 7 contained 0.1 pmole of the labeled oligo. Lanes 3 & 8 and 4 & 9 contained 100x and 200x the amount of cold oligo, respectively. Lanes 5 and 10 contained 100x cold non-self competition. Panel B, same as panel A except using nuclear extract from HepG2 cells. Panel C, HepG2 nuclear extract (5 μg) was incubated with 0.04 pmoles (~1.5 × 105 cpm) of radiolabeled wild type oligo as indicated at top. Lane 1 contained no nuclear extract. Lane 2 contained nuclear extract. Lanes 3–10 were incubated with 10x, 25x, 50x, and 100x amounts of the indicated cold oligo. Autoradiography was performed at −90 °C for 16–20 h.
Figure 4
Figure 4. IVS1+1G>C splicing mutant causes intron retention
A293 cells were transfected with 5 μg of either wild type plasmid (pSplice_wt) or IVS1+1G>C mutant plasmid (pSplice_mut). After 40–48 h, total RNA was extracted and RT-PCR was performed. Transcript size was analyzed by PCR analysis and visualized on a DNA agarose gel used for Southern Blot. Panel A: PCR-amplified cDNA from cells transfected with wild type plasmid (Lane 1) and IVS1+1G>C mutant plasmid (Lane 2). The DNA was transferred to a nylon membrane and probed with oligonucleotides complementary to the splice junction (Panel B), exon 1 (Panel C), and exon 2 (Panel D). Blots were exposed for autoradiography at −90 °C for 3 h, 8 h, and 16 h, respectively. On the left, the lower arrow indicates the position of migration of a 248 bp product expected for a correctly spliced PCR-amplified cDNA synthesized from the pSplice_wt. The upper arrow indicates the position of migration of a >5.0 kb product expected for a PCR-amplified cDNA synthesized from pSplice_mut without splicing.

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