DNA was isolated from peripheral leukocytes in 38 HFI patients who were diagnosed by low aldolase B activity in liver biopsy samples, diagnostic response to intravenous (I.V.) fructose challenge, the identification of one HFI allele combined with clinical symptoms, and/or patients of Hispanic descent with typical dietary and clinical histories consistent with HFI. A follow-up analysis by ASO hybridization analysis (see below) was performed on leukocyte DNA from an additional group of suspected patients with one or more symptoms suggestive of HFI.
The specific genotypes of six probands are reported. Patient-34 was diagnosed with HFI by liver biopsy. Patient-50 presented at 1.5 years of age and was diagnosed with HFI by liver biopsy. Patient-249 was a female who presented at six months of age with failure to thrive and fructosemia. Patient-278 presented at three years of age with neonatal giant cell hepatitis of unknown etiology, and was diagnosed with HFI by liver biopsy. Patient-284 was a male who presented at five months of age with lactic acidosis, and was diagnosed with HFI by identification of one HFI allele. Patient-295 was a female who presented at seven months with liver failure following Pedialyte treatment for diarrhea and dehydration, and was diagnosed by identification of one HFI allele and successful exclusion of dietary fructose. This study was approved by the Institutional Review Board and informed consent was obtained from all subjects or their guardians.
Allele-specific oligonucleotide (ASO) Hybridization
Plasmids containing DNA inserts with either wild-type or mutant alleles were constructed from genotyped patient genomic DNA as controls for ASO screening. Each DNA insert was amplified by PCR and cloned into TOPO TA (Invitrogen). All control clones were confirmed by DNA sequencing. Amplified genomic DNA from patient samples and plasmid DNA from control clones (100 ng) were denatured and applied to a Zeta-Probe®
GT nylon membrane (Bio-Rad). Blots were used for ASO hybridization analysis for A149P, A174D, N334K, R59Op, Δ4E4, A337V, and L256P as described previously (Coffee et al 2009
). For new alleles, ASO probes for wild-type (g.–132wt 5′-ATTTTAAGG
ACTGGTTG-3′ and IVS1+1wt 5′-CCCAAACTG
TAAGTAAA-3′) and mutant alleles (g.–132G>A 5′-ATTTTAAGA
ACTGGTTG-3′ and IVS1+1G>C 5′-CCCAAACTC
TAAGTAAA-3′) were used. High stringency washes were performed at the following discriminatory temperatures: g.–132wt at 54 °C, g.–132G>A at 52 °C, IVS1+1wt at 58 °C, and IVS1+1G>C at 56 °C.
DNA Sequence Determination
Genomic DNA was extracted from leukocytes isolated from whole blood as described previously (Orkin et al 1978
). A 982 bp fragment containing the ALDOB
proximal promoter and exon 1 was amplified by PCR (Saiki et al 1985
) using sense and antisense primers (50 μM) 5′-ACTGCGTACAGACACTATACAAC-3′ and 5′-CATAAGGCAGTAGATATGTA-3′, respectively. PCR was carried out in a buffer containing 16 mM (NH4
, 67 mM Tris-HCl, pH 8.8, 200 μM dNTPs, 2 mM MgCl2
, and 5 U/ml of Taq polymerase. Cycling conditions were as follows: 94 °C for 1.5 min; 60 °C for 1 min; 72 °C for 1 min, repeated for five cycles; 94 °C for 1.5 min, 60 °C for 1 min; 72 °C for 3 min, repeated for 25 cycles; 72 °C for 10 min; hold at 4 °C. A 1.2 kb fragment containing the ALDOB
intronic enhancer was amplified similarly by PCR using sense and antisense primers 5′-TAGGATGTAACTTGCAATCC-3′ and 5′-CTGCTCATTGTAGTTGCTCA-3′, respectively. ALDOB
exons 2–9 were PCR-amplified as previously described (Coffee et al 2009
Each PCR product was purified using the NucleoSpin® Extract II silica-membrane columns (Macherey-Nagel) as per manufacturer’s instructions. DNA (0.5 μg) was submitted for sequence determination (≥3 independent times) (GENEWIZ, Inc., South Plainfield, NJ) using several internal primers to ensure overlapping reads along both strands. Sequence results were analyzed using Sequencher® 4.9 software (Gene Codes Corporation).
The pGL3-basic luciferase reporter plasmid (Promega) was used for generation of reporter plasmids for in vivo studies of expression, and the pcDNA3.1(−) mammalian expression plasmid (Invitrogen) was used for studies of splicing. Genomic DNA from non-HFI subjects was used as a template for PCR amplification of a 2,798 bp region from position −264 to +2534. This included the promoter (264 bp), the first exon (72 bp), and part of the first intron (2,462 bp). Primers were designed with 5′ extensions containing Xma I restriction sites for cloning into pGL3-basic at an Xma I site upstream of the luciferase open reading frame. Additionally, a 96-bp fragment from position +4795 to +4890 containing 87 bp upstream of exon 2 and the first nine bases of exon 2 was PCR-amplified from wild type genomic DNA. These primers were designed with 5′ extensions containing Bgl II restriction sites for cloning into pGL3-basic at a Bgl II site between the Xma I site and the luciferase open reading frame. This luciferase reporter plasmid, pProm2, contained a total of 2,893 bp of wild type ALDOB sequence.
Wild type ALDOB
sequence was subcloned into the Eco
R I, Eco
R V, and Bam
H I sites of pcDNA3.1(−) mammalian expression vector (Invitrogen) for analysis of the splicing mutation. A 1.4 kb Eco
R I fragment was isolated from pEE313 (Tolan and Penhoet 1986
). A 3.5 kb Eco
H I fragment was isolated from pBE313 (Tolan and Penhoet 1986
). Both fragments were cloned adjacently into pcDNA3.1(−) resulting in the wild type plasmid, pSplice_wt. The point mutations at g. 132 and IVS1+1 were introduced into pProm2 and pSplice_wt, respectively, using the QuikChange®
Site-Directed Mutagenesis Kit (Stratagene) with primers containing 30–34 complementary bases producing p–132G>A and pSplice_mut, respectively.
The luciferase gene was PCR-amplified from pGL3-basic using a forward primer with a 5′ extension containing a BamH I restriction site enabling cloning in frame with the ALDOB translation start codon and a reverse primer complementary to pGL3-basic sequence downstream of a BamH I restriction site in the vector. The 1.8 kb BamH I fragment was cloned into pSplice_mut creating the plasmid pMut_Luc. Sub-cloning a fragment containing the wild-type splice sequence of exon 1 from pSplice_wt into pMut_Luc created the plasmid pWt_Luc. The correct construction of all plasmids was confirmed by DNA sequencing.
Human kidney-derived A293 cells were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 U (60 μg) of penicillin, and 100 μg of streptomycin (Invitrogen) at 37 °C with 5% CO2. Cells were seeded in 6-well tissue culture plates and transfected in triplicate when they reached approximately 30% confluence. The media was replaced and DNA was mixed with polyethylenimine (PEI) (1 μg/μl, pH 7.2) (Polysciences Inc, Warrington, PA) at a ratio of 1μg DNA:3μl PEI and added to an aliquot of serum-free media. This DNA mixture was incubated at room temperature for 15 min and then added to the cells. Cells were harvested after 42–48 h.
Luciferase activity in transfected A293 cells was measured using the Luciferase Assay System (Promega). Briefly, media was removed and cells were rinsed with phosphate-buffered saline. Reporter Lysis Buffer (Promega) was added to each well and cells were gently scraped and collected. Cells were lysed by freeze thaw, vortexed briefly, and centrifuged for 3 min at 20,800 × g at 4 °C. Supernatant fractions were collected and transferred to a new tube. Luciferase activity was determined by adding 100 μl of Luciferase Reagent (Promega) to 20 μl of cell extract and luminescence was measured for 10 s in a 20/20n Luminometer (Turner BioSystems). Transfection efficiency was measured by adding 20 μl of cell extract to a final volume of 135 μl in a reaction containing 122.5 mM sodium phosphate, pH 7.5, 3 mM O-nitrophenyl-β-D-galactopyranoside, 1.1 mM MgCl2, and 53 mM β-mercaptoethanol and incubated at 37 °C until reactions became yellow. The reactions were stopped by the addition of Na2CO3 to 644 mM and absorbance values were measured at 415 nm. Luciferase values were normalized to transfection levels determined by β galactosidase expression. Error calculated by either standard error of the mean (SEM) from two to four trials as noted. Unpaired Student’s t-test was performed to determine p values.
Preparation of Nuclear Extracts
Human liver-derived HepG2 cells were grown to confluency and cells were collected and resuspended in 0.4 ml of Buffer E (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 1 μg/ml pepstatin A). After 10 min at 4 °C, 5% (v/v) NP-40 was added to 0.6%, cells were vortexed briefly, incubated at 4 °C for 15 min, and centrifuged for 30 s at 20,800 × g. The cytoplasm-containing supernatant fraction was removed and the nuclear pellet was resuspended in 0.1 ml of Buffer E and centrifugation was repeated. The pellet was resuspended in 0.1 ml of 20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 1 μg/ml pepstatin A. Nuclei were vortexed and incubated for 45 min with gentle rocking at 4 °C. The sample was centrifuged for 10 min at 20,800 × g at 4 °C. The supernatant fraction was transferred to a new tube, supplemented with glycerol to a final concentration of 20% (v/v), aliquoted, and stored at −80 °C.
An expression plasmid that contained human C/EBPα cloned into pcDNA3 (Invitrogen) was used for in vitro translation (kind gift from Dr. Geoffrey Cooper). The TNT® Coupled Wheat Germ Extract System (Promega) was used with T7 RNA polymerase to transcribe and translate C/EBP in vitro.
Electrophoretic Mobility Shift Assay (EMSA)
DNA fragments of 24 bp corresponding to the ALDOB
promoter from position −144 to −121 relative to the start of transcription were synthesized (Eurofins MWG Operon, Huntsville, AL) with either wild-type sequence or the point mutant at position −132. Complementary oligonucleotides (1 nmol) were annealed by incubating at 95 °C for 10 min and allowed to cool overnight in 10 mM Tris, pH 7.6, 10 mM NaCl, 2 mM MgCl2
, and 0.1 mM EDTA. Annealed probes were radioactively labeled at the 5′-end by incubation with γ-[32
P ]-ATP and T4 polynucleotide kinase (New England Biolabs) at 37 °C and purified from unincorporated ATP by a Bio-Gel®
P-6 DG (Bio-Rad) column. DNA-binding reactions contained 5 μg of nuclear extract, 1 μg poly dI:dC (Sigma), 1 μg BSA (NEB), and 2 × 106
cpm of radiolabelled ALDOB
promoter probes in 50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM DTT, 5 mM EDTA, and 20% (w/v) glycerol. Samples were incubated at room temperature for 40 min. DNA-protein complexes were resolved on 5% polyacrylamide gels in 25 mM Tris, 0.19 M glycine, 1 mM EDTA, and 2.5% (v/v) glycerol run at 25 mA at room temperature. Gels were dried for 60 min at 80 °C prior to autoradiography. Relative binding was quantified using ImageJ software (Abramoff et al 2004
RNA Extraction and cDNA Synthesis
Total RNA was extracted using TRIzol® Reagent (Invitrogen). RNA (5 μg) was incubated with 2 nM poly(T) primer at 70 °C for 5 min and immediately cooled on ice. M-MLV reverse transcriptase (200 U) (Promega), dNTPs (10 mM), and RNasin® (25 U) (Promega) were incubated in 5x M-MLV reaction buffer (Promega) for 2 h at 42 °C. PCR was performed with 5 μl of this cDNA reaction using the T7 primer (5′-TAATACGACTCACTATAGGG-3′), a reverse primer (5′-TCAGCGGTTTAAACTTAAGC-3′), and 2 mM MgCl2 under the following cycling conditions: 94 °C for 4 min; (94 °C for 30 sec; 60 °C for 30 sec; 72 °C for 2 min) repeat for thirty cycles; hold at 4 °C. PCR products were visualized on agarose gels stained with ethidium bromide.
Southern Blot Hybridization
DNA was separated on 1.5% TAE agarose gel and transferred to a nylon membrane under conditions previously described (Tolan and Penhoet 1986
). The membrane was incubated in a buffer containing 5x SSC, 20 mM NaH2
, pH 7.2, 1 mM EDTA, 7% (w/v) SDS, and 10X Denhardt’s (Ficoll:polyvinylpyrrolidone:BSA (2g each/L)) for 1 h at 37 °C in a rotating hybridization oven. A radiolabeled probe (splice junction 5′-TCCCAAACTATGGCCCAC-3′, exon 1 5′-ACTCTTCTCTCCCAAACT -3′, or exon 2 5′-GGCAATTTCTGAAGAGCT-3′) was added and hybridization proceeded overnight at 37 °C. Blots probed with oligonucleotides for the splice junction, exon 1, and exon 2 were washed with 5x SSC and 0.1% (w/v) SDS at 37 °C, 52°C, and 45 °C, respectively, and autoradiography was performed.