Genomic and mutational analysis of the mitochondrial trifunctional protein [beta]-subunit (HADHB) gene in patients with trifunctional protein deficiency
Genomic and mutational analysis of the mitochondrial trifunctional protein [beta]-subunit (HADHB) gene in patients with trifunctional protein deficiencyKenji E. Orii1,2,*, Toshifumi Aoyama2, Keiko Wakui3, Yoshimitsu Fukushima3, Hiroaki Miyajima4, Seiji Yamaguchi5, Tadao Orii6, Naomi Kondo1 and Takashi Hashimoto2
1Department of Pediatrics, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500, Japan, 2Department of Biochemistry and 3Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, Matsumoto 390, Japan, 4First Department of Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-31, Japan, 5Department of Pediatrics, Shimane Medical University, Izumo 693, Japan and 6Department of Human Welfare, Chubu Gakuin University, Seki, Gifu 501-32, Japan
Received February 25, 1997;Revised and Accepted May 23, 1997DDBJ/EMBL/GenBank accession nos D86841-D86850
Mitochondrial trifunctional protein (TP), an enzyme of [beta]-oxidation, is a multienzyme complex composed of four molecules of the [alpha]-subunit (HADHA) containing the enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase domains and four molecules of the [beta]-subunit (HADHB) containing the 3-ketoacyl-CoA thiolase domain. An inborn error of this enzyme complex can cause sudden infant death syndrome, acute hepatic encephalopathy or liver failure, skeletal myopathy, or hypertrophic cardiomyopathy. TP deficiency is classified into two different biochemical phenotypes: one represents the existence of both subunits and the lack of only the 3-hydroxyacyl-CoA dehydrogenase activity and the other represents the absence of both subunits and the lack of all three TP activities, although their clinical features are similar. We have identified two Japanese patients with this disorder. Three enzyme activities of TP were undetectable in fibroblasts from these two patients. We detected two mutations in the HADHB gene from two Japanese patients, an exonic single T insertion which created a new cryptic 5' splice site and a G1331A transition (R411K). Patient 1 was a compound heterozygote, while patient 2 was a homozygote of a G1331A transition.
Mitochondrial [beta]-oxidation of fatty acids provides the primary source of energy for the heart and other muscles. The [beta]-oxidation cycle degrades long-chain-fatty acyl-CoA substrates via four enzymatic reactions to produce acetyl-CoA. However, because of a wide range of carbon chain lengths of fatty acyl moieties, multiple enzymes with different but overlapping chain length specificities are involved in this metabolic cycle. Long-chain fatty acyl-CoA is first processed by two enzymes, very-long-chain acyl-CoA dehydrogenase (VLCAD) (1 -3 ) and a trifunctional protein (TP) catalyzing the following three steps (4 ,5 ). Both VLCAD and TP are bound to the inner membrane of mitochondria, and catalyze the CoA-esters produced by carnitine palmitoyltransferase II. Subsequently, chain-shortened acyl-CoAs are further processed by classical enzymes, located in the mitochondrial matrix.
TP is a multienzyme complex composed of four molecules of the [alpha]-subunit (HADHA) with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase domains and four molecules of the [beta]-subunit (HADHB) with the 3-ketoacyl-CoA thiolase domain (6 ). TP deficiencies were first described by Wanders et al. in 1989 (7 ). Clinical manifestations include a Reye-like syndrome of hypoglycemia, coma, skeletal myopathy, cardiomyopathy or sudden infant death (8 ,9 ).
TP deficiency is classified into two different biochemical phenotypes, although their clinical features are similar (8 ,10 -12 ). One is an isolated deficiency of the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity with normal or slightly lesser amounts of both the HADHA and HADHB proteins. There are numerous case reports on the first phenotype, LCHAD deficiency, from Europe and North America (7 -9 ). Mutation analysis of the first phenotype of TP deficiency at the cDNA and DNA level revealed four point mutations on HADHA (8 ,13 -16 ), and a G1528 -> C transversion was considered to be a common missense mutation. In the second group, all three enzyme activities of TP are undetectable with loss of both HADHA and HADHB proteins. However, there are only five case reports of the second phenotype, all from Europe and North America (10 ,12 ,17 -19 ). Mutation analysis of the second phenotype of TP deficiency revealed two splice donor site mutations on the HADHA gene and three point mutations on the HADHB cDNA, from three patients (17 ,18 ). However, no identical mutations on the HADHB gene were reported. Further detailed studies relating to the HADHB gene were not documented because the genomic DNA of HADHB had not yet been described, though the genomic DNA structure of HADHA has been reported (20 ) and the HADHA gene was mapped to 2p23 (21 ). In the present work, we cloned the mitochondrial HADHB gene and localized it to 2p23, the same location as the HADHA gene. When we analyzed findings in two Japanese patients with TP deficiency belonging to the second biological phenotype, we identified two novel mutations on the HADHB gene, one being an exonic single T insertion in exon 9, which created a new cryptic 5' splice site and the other a G1331 -> A transition (R411K) in exon 15. Patient 1 was a compound heterozygote of these two mutations and patient 2 was a homozygote of a G1331 -> A transition.
In attempts to obtain the HADHB gene by genomic PCR, we used oligonucleotide primers (Table 1 ) and found five overlapping PCR-amplified fragments (6F-8R, 8Fb-10Rc, 10F-13R, 13F-14R and 14F-16R) covering part of the HADHB gene. Since we did not obtain the residual portion using PCR, we prepared one DNA (8Fb-10Rc) and two cDNA probes (1F-4R and 13F-16R) by PCR to acquire the residual coding and 5'- and 3'-non-coding regions. We screened ~1 * 106 [lambda]EMBL3 recombinant phages from a Sau3AI human leukocyte genomic library, using the three probes, and obtained five clones. Five PCR fragments (6F-8R, 8F-10Rc, 10F-13R, 13F-14R and 14F-16R) and five clones were sequenced to determine the genomic organization. The coding region of the human HADHB gene consists of 16 exons and 15 introns, spanning ~45 kb. All exon/intron junctions followed the GT-AG rule and were compatible with established consensus sequences (Table 2 ; 22 ). The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with accession nos. D86841-D86850. Sizes of the introns varied; intron 11 is 79 bp long, whereas intron 1 consists of 9.8 kb. Most exons were in the range of 45-188 bp long (Table 2 ). The exon sequences were identical to those of HADHB cDNA (23 ). Fluorescence in situ hybridization (FISH) of the biotin-labeled part of the HADHB gene probe to normal human metaphase chromosomes was used to localize the HADHB locus. FISH signals were detected at position 2p23, the same as in the report of Yang et al. (24 ).
The entire cDNA for the HADHA and HADHB were amplified from mRNAs prepared from a control, the patients and their parents, respectively, as described under Materials and Methods. The coding region of HADHA (1-2289) was amplified by PCR using primers [alpha]1F and [alpha]20R (Table 1 ). The coding region of the HADHB (1-1425) was amplified using primers 1F and 16R (Table 1 ). Six subclones of HADHA cDNA from the patients had normal sequences with the three normal polymorphisms, as reported (18 ,23 ). Six subclones of HADHB cDNA from the patients were sequenced, respectively. There was a G1331 -> A transition (R411K) in four of the six from patient 1 and all six from patient 2. In the other two subclones of patient 1, we found a T825 -> C transition which did not change amino acid residues, and a 36 bp deletion at position 776-811. Thus, patient 1 was thought to be a compound heterozygote and patient 2 probably a homozygote.
1F, 8Fa, 10Rb, and 16R have an EcoRI site at the 5' end, respectively. a1F and a20R were prepared for amplification of HADHA cDNA. Oligonucleotide primers were numbered according to the exon number and nucleotide sequence of HADHB cDNA.
Table 2 Exon/intron organization of the human HADHB gene
Exon
Size (bp)
cDNA position
5' splice donor
Intron
Size (bp)
3' splice acceptor
1
>38
(-46)-(-9)
GCCAAG
gtgaga
1
9800b
ttttag
ATTCCA
2
72
(-8)-64
GATTTT
gtaagt
2
111a
ttccag
CCATAA
3
45
65-109
CCCCAG
gtacag
3
7900b
ttaaag
CTGTCC
4
100
110-209
CACTTC
gtaagt
4
7200b
ccccag
ATATAA
5
45
210-254
GCTTAC
gttaag
5
3100b
ctaaag
GCTGCC
7
88
355-442
CCACAG
gtatgt
7
1600b
ctgtag
GTGTTG
8
188
443-630
CCTGAG
gtaagg
8
333a
tcccag
CTCCCT
9
181
631-811
TACCAG
gtgaaa
9
680a
taatag
GAAAAG
10
122
812-933
TTCTTG
gtaact
10
3000b
tttcag
ACTGAT
11
79
934-1013
TTTGAG
gtaaag
11
79a
acctag
GGATTT
12
48
1014-1061
ACTTGG
gtaggt
12
1100b
ttgcag
ACCAAC
13
88
1062-1149
TTCTCG
gtatgt
13
681a
ttccag
GGTCAG
14
75
1150-1224
ACCAAG
gtgagt
14
450a
agccag
GTTGGA
15
65
1225-1389
GGGCAG
gtacgt
15
3800b
ttacag
GGCCAT
16
>547
1390-1936
The sequence is numbered starting from adenine at translation start codon ATG. Exon sizes were determined from the nucleotide sequence. Approximate intron sizes were determined from the nucleotide sequencea, from genomic mapping and from PCR with exon specific primersb.
Deletion of this region was confirmed by analyzing the amplified 9F-10Rc fragment (Fig. 1 A). Two different 9F-10Rc fragments were obtained from patient 1 and from his mother (Fig. 1 A, lanes P1 and M1), one was the same size as that from the fibroblasts of a normal control, and the other was smaller, with a 36 bp deletion.
We then searched for the cause of the 36 bp deletion at nucleotide position 776-811. A region containing this part of the genomic DNA was amplified as the 9F-10Rc PCR fragment. The 9F-10Rc fragments of genomic DNA from the normal control and patient 1 were both ~883 bp. The nucleotide sequences of the fragments encompassing bases 701-903 were then determined. A single T insertion at nucleotide position 777 (Fig. 1 C), which was 36 bp upstream from the 5' splice site of intron 9, was detected in five of the eight subclones. The other three subclones had the same sequence as that of the normal control. Since an AfaI site was obtained by a single T insertion at nucleotide position 777, we amplified short PCR fragments (9F-9R) of genomic DNA from the normal control, patient 1, and his parents and digested them with AfaI, followed by electrophoresis (Fig. 1 B). As a result, patient 1 and his mother were found to have a single heterozygous T insertion at nucleotide position 777.
We then did in vivo splicing to determine if the single T insertion at nucleotide position 777 on the genomic DNA would cause a 36 bp deletion (Fig. 2 ). We amplified the 8Fa-10Rb PCR fragment (1336 bp) of genomic DNA from the normal control and patient 1, including the 5' splice site of intron 8 and the 3' splice site of intron 9 (Fig. 2 A). These PCR fragments were inserted into pCXN2 expression vectors (25 ) and transfected to CHO cells. We obtained mRNA from these stably transformed CHO cells and performed reverse transcriptase (RT) PCR using 9F and 10Ra primers. We obtained two kinds of RT-PCR fragments from these CHO cell lines. One was the same size as that from the normal control fibroblasts (Fig. 2 B, lane FC) of CHO cells expressing a normal fragment (lane CN) and the other (lane CM) from CHO cells expressing a mutant fragment was smaller than the normal (lane CN). That smaller fragment was the same size as the lower band for patient 1, with the 36 bp deletion (lane FP). The upper bands of 168 bp were considered to be non-specific as they were seen in all five lanes. In lane CC, the 168 bp band was detected. Hamster [beta]-subunit cDNA sequences are thought to be similar to those in humans (23 ). Thus, the single T insertion at nucleotide position 777 on the genomic DNA apparently caused the 36 bp deletion, by abnormal splicing.
Normal HADHB cDNA (pCXN2-[beta]N) and mutant cDNA with a G1331 -> A transition (R411K) (pCXN2-[beta]M) were stably expressed, separately, in fibroblasts from patient 1, using the pCXN2 expression vector (25 ). The amounts of cDNA integrated into the host genome and the expressed mRNA in the independent two cell lines were similar (Fig. 4 A and B). HADHA and HADHB proteins were not detected in cells from patient 1 (Fig. 4 C, lane P), but both HADHA and HADHB proteins were detected (lane PN) when the normal HADHB protein was expressed in fibroblasts from patient 1. In contrast, HADHA and HADHB proteins were not detected in fibroblasts expressing mutant HADHB cDNA (lane PM). When analyzing TP-dependent three enzyme activities, there could be a problem of significant contribution to activities by other enzymes such as mitochondrial crotonase, short-chain 3-hydroxyacyl-CoA dehydrogenase, short-chain 3-ketoacyl-CoA thiolase and unknown enzymes (10 ,11 ,27 ). As shown in Table 3 , patient 1's fibroblasts represent significant residual activities by other enzymes, and the activity pattern is similar to that in other patients' fibroblasts with TP deficiency (11 ,18 ). All three enzyme activities of TP in patient 1's cells expressing normal HADHB cDNA increased to the control level (Table 3 ). In contrast, those in patient 1's fibroblasts expressing the mutant HADHB cDNA were similar to those of the untransfected patient 1 cells (Table 3 ). Therefore, the G1331 -> A transition in patients 1 and 2 also caused disease.
We have found only two reports on molecular analysis of samples from patients belonging to the second phenotype. One was a deletion of exons 2 and 3 (71 and 113 bp) of HADHA due to two HADHA donor splice site mutations in one patient (17 ), and the other was three point mutations of HADHB cDNA from two Caucasian patients (G182 -> A, G740 -> A, A788 -> G), as described by Ushikubo et al. in 1996 (18 ). Therefore, we cloned the human HADHB gene and carried out mutation analysis at the genomic DNA level.
We found two novel mutations on the HADHB gene from two Japanese patients with the second phenotype of TP deficiency, one was an exonic single T insertion in exon 9 which created a new cryptic 5' splice site and the other was a G1331 -> A transition (R411K) in exon 15. Patient 1 was a compound heterozygote who inherited one allele with the G1331 -> A transition (R411K) from his father and the other allele with a single T insertion at nucleotide position 777, which caused a 36 bp deletion with a loss of 12 amino acids from G227 to P238, from his mother. Patient 2 was homozygous for the G1331 -> A transition. This evidence is consistent with the consanguinity of patient 2's family.
We did in vivo splicing experiments to verify that a single T insertion on the genomic DNA was the cause of the 36 bp deletion in exon 9. Normal mRNA was generated in CHO cells transfected with pCXN2-T(-) which has the normal sequence (Fig. 2 B, lane CN). However, a mRNA fragment with a 36 bp deletion was generated in CHO cells transfected with the pCXN2-T(+) carrying the single T insertion at nucleotide position 777 (lane CM). This means that a new cryptic 5' splice site in exon 9 is created by insertion of a single T and results in a 36 bp deletion. Various facets of exon structure are considered to play a role in splice site selection and alternative splicing. In patient 1, a new exonic cryptic 5' splice site (AAG/GTACTC) was suboptimal compared with the authentic 5' splice site (CAG/GTGAAA) of intron 9. However, there was a 7 nt (AAAGATA) direct repeat within a purine-rich region in the 5' end of exon 10, which we considered to be an exonic splicing enhancer (ESE) sequence. Since the efficiency of exon recognition is weaker than that of constitutive exons due to suboptimal splice sites and length, it is strengthened by the ESE that binds to putative splicing regulators (30 ,31 ). We therefore considered that the ESE sequence in exon 10 activated the new cryptic 5' splice site in exon 9 of patient 1.
We expressed HADHB cDNA in patient 1's fibroblasts using lipofection, the objective being to verify that the R411K mutation causes TP deficiency. As expression of TP protein requires formation of a stable complex (18 ), the expression system we used is more suitable for analysis of TP deficiency. When analyzing the TP-dependent three enzyme activities, there is a problem of significant contribution to the activities by other enzymes such as mitochondrial crotonase, short-chain 3-hydroxyacyl-CoA dehydrogenase, short-chain 3-ketoacyl-CoA thiolase and unknown enzymes (10 ,11 ,27 ). As shown in Table 3 , patient 1's fibroblasts represent significant residual activities, 41% of hexadecenoyl-CoA hydratase activity and 24% of 3-ketohexadecanoyl-CoA dehydrogenase activity in control fibroblasts. In other patients with TP deficiency, two patients' fibroblasts, both having disease-causing mutations on only the HADHB protein, represent considerable residual activities, 59-74% of hexadecenoyl-CoA hydratase activity and 41-50% of 3-ketohexadecanoyl-CoA dehydrogenase activity in control fibroblasts (18 ). Additionally, another patient's fibroblasts, having disease-causing mutations on only the HADHA protein, also exhibit significant residual activities, 59% of hexadecenoyl-CoA hydratase activity and 35% of 3-ketohexadecanoyl-CoA dehydrogenase activity in control fibroblasts (18 ). These data clearly indicate that the location of mutations, whether on HADHA or HADHB, does not affect residual activities. Since the activity pattern in patient 1's fibroblasts in this study was similar to that in other patients' fibroblasts (11 ,18 ), we considered that there were no TP-dependent three enzyme activities in the fibroblasts, a notion also supported by the results of immunoblot analysis (Fig.4 C; 32 ). When mutant HADHB cDNA (pCXN2-[beta]M) was expressed in patient 1's fibroblasts, all three enzyme activities remained unchanged (Table 3 ). When normal HADHB cDNA (pCXN2-[beta]N) was expressed in patient 1's fibroblasts, all three enzyme activities of TP increased to the control level (Table 3 ). As shown by immunoblotting (Fig. 4 C), both HADHA and HADHB proteins were detected at a level similar to that seen in control fibroblasts when normal HADHB cDNA (pCXN2-[beta]N) was expressed in patient 1's fibroblasts (lane PN). This increase in the three enzyme activities was apparently due to an increase of normal HADHB protein in patient 1's fibroblasts. However, neither the HADHA nor the HADHB protein was detected when mutant HADHB cDNA (pCXN2-[beta]M) was expressed (lane PM). Thus, the R411K mutation apparently accounts for the TP deficiency.
In the present study, we confirmed two novel mutations of HADHB at the genomic DNA level in two patients belonging to the second biological phenotype, as described above. Common mutations on the HADHB gene have heretofore never been detected. However, it was known that the enzyme complex of TP could not be formed due to the five mutations on the HADHB gene (18 ,32 ). Additionally, three of five mutations on the HADHB gene were located within 50 bp in exon 9. These findings suggest that these mutations in exons 4, 9 and 15, especially exon 9, are on the important regions to consider with regard to structures and intersubunit contacts for heterooctamer formation of an enzyme complex.
Hexadecenoyl-CoA prepared by the mixed anhydride method (33 ) was converted to 3-ketohexadecanoyl-CoA, using the fatty acid oxidation multienzyme complex from Pseudomonas fragi (34 ). Purified human mitochondrial TP and its antibodies were prepared as described (11 ). Moloney murine leukemia virus (M-MLV) reverse transcriptase and culture medium were purchased from GIBCO/BRL. AmpliTaqTM DNA polymerase and DNA Sequencing Kit was purchased from Perkin-Elmer. LA Taq was purchased from Takara. Restriction and modification enzymes were purchased from Takara and Toyobo. All other reagents were of the purest analytical grade available.
For the genomic PCR, we used oligonucleotide primers (6F, 8R, 8Fb, 10F, 10Rc, 13F, 13R, 14F, 14R and 16R; Table 1 ), based on sequence of the HADHB cDNA (23 ). Genomic DNA (1 [mu]g) from human peripheral blood leukocytes was used as a template. Genomic fragments were amplified in a mixture (50 [mu]l) of 50 mM Tris-Cl (pH 8.3 at 25oC), containing 2.0 mM MgCl2, 0.25 mg/ml BSA, 2.5 mM each of dATP, dCTP, dGTP and dTTP, 20mer oligonucleotide primers as indicated (10 pmol each) and 2.5 U Takara LA Taq. Thirty cycles of PCR reactions were run using a DNA Thermal Cycler (Perkin Elmer). The first denaturation was for 2 min at 96oC. Each cycle consisted of 1 min denaturation at 98oC, 2 min annealing at 55oC, and 10 min extension at 72oC. The final extension continued for 20 min at 72oC. The PCR product was separated by electrophoresis on a 1% (w/v) agarose gel and extracted using a Geneclean II kit (BIO 101). Isolated PCR products (50 ng) were ligated into the pT7blue T-Vector (Novagen) for subcloning. The PCR product or the subcloned plasmid DNA was sequenced by dideoxy chain termination (35 ), using a DNA sequencing kit and an analysis software package (Perkin Elmer). A Sau3AI human leukocyte genomic library containing 1 * 106 [lambda]EMBL3 recombinant phages, obtained from Dr S. Tomatsu at Gifu University was plated on Escherichia coli K803 and transferred to nylon membranes (HybondTM-N+, Amersham). A genomic PCR-amplified fragment (8Fb-10Rc), and two PCR-amplified cDNA fragments (1F-4R and 13F-16R) were labeled with [[alpha]-32P]dCTP by the Megaprime DNA labeling system (Amersham) and used as probes. The nylon membranes were washed three times in 2* SSC, 0.1% SDS at 65oC for 20 min. The dried membranes were exposed to X-ray films, and positive clones were picked up. Secondary and tertiary rounds of screening were similarly conducted. Thereafter, the phage clones were subcloned into the pT7blue vector (Novagen) or pBluescript® II phagemid SK+ (Stratagene) at appropriate restriction sites and sequenced.
Fluorescence in situ hybridization (FISH) was done as described (36 ), using a biotin-labeled HADHB gene probe. A cosmid probe containing a part of HADHB genome clone (6 kb) was used as a HADHB gene probe. Background noise due to repetitive sequences of Alu and L1 was eliminated using human placental DNA (10 mg/ml) (37 ).
Fibroblasts from the normal controls, patients 1 and 2, and parents of patient 1 were collected after trypsinization and sonicated in 50 mM potassium phosphate (pH 7.5), 0.2 M NaCl, 0.1% (v/v) hexamethylphosphoric triamide, 2 mM 2-mercaptoethanol, 0.5 mM EDTA, 0.5% Tween 20. After standing on ice for 30 min, the cell lysate was centrifuged at 18 000 g for 10 min. Enzymes in the supernatant were assayed. The enoyl-CoA hydratase activity was assayed by following a decrease in absorbance of hexadecenoyl-CoA at 280 nm (38 ). The 3-hydroxyacyl-CoA dehydrogenase activity was measured by 3-ketohexadecanoyl-CoA-dependent NADH oxidation (39 ,40 ). The 3-ketodecanoyl-CoA thiolase activity in the thiolytic reaction was assayed by following a decrease in absorbance at 303 nm (11 ). One unit of the enzyme was defined as the amount that converted 1 [mu]mol of substrate per min at 30oC.
Protein concentration was determined by a modification (41 ) of the procedure of Lowry et al. (42 ). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (43 ) on a 10% gel. Samples were immunoblotted as described by Towbin et al. (44 ). Protein was detected using an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Jackson Immuno Research Laboratories) and an ImmunoPure NBT/BCIP Substrate KitTM (Pierce).
Details on patients 1 and 2 have been reported by Orii et al. (32 ) and Miyajima et al. (29 ), respectively. Patient 1's parents were unrelated, whereas patient 2's parents were second-degree cousins. Skin fibroblasts were obtained from patients 1 and 2, patient 1's parents and from healthy Japanese men. Skin fibroblasts and CHO cells were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) FCS, 0.1 mM non-essential amino acids (GIBCO/BRL), 1* antibiotic-antimycotic solution (GIBCO/BRL) and 4.5 mg/ml glucose.
RNA was extracted from fibroblasts and CHO cells using acid guanidinium thiocyanate-phenol-chloroform (45 ). Total RNA (8 [mu]g) isolated from the CHO cells was electrophoresed on a 1% (w/v) agarose gel containing 6% formaldehyde. RNA on the gel was transferred to a nylon membrane (Hybond N+) and hybridized with the DIGTM-RNA probe. Detection of RNA was performed using a DIGTM-nucleic acid-detection kit (Boehringer-Mannheim). Total RNA (5 [mu]g) was dissolved in 50 [mu]l of 50 mM Tris-HCl pH 7.5, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTPs, 10 [mu]g/ml oligo(dT)16 and 200 U of M-MLV reverse transcriptase, and the preparation was incubated at 37oC for 1 h. The reaction was stopped by incubation at 95oC for 10 min, then 10 [mu]l of 15 mM EDTA was added. The mixture was stored at -20oC.
Genomic DNA was isolated by the sodium iodide method, using a DNA extractor WB kit (Wako). PCR proceeded using various pairs of oligonucleotides, as shown in Table 1 . Genomic DNA (20 [mu]g) isolated from fibroblasts was digested with EcoRI and electrophoresed on a 1% (w/v) agarose gel. DNA on the gel was transferred to a nylon membrane (HybondTM-N+; Amersham) and hybridized with PCR fragments labeled for 16 h with [[alpha]-32P]dCTP using the Megaprime DNA labeling system (Amersham). The nylon membranes were washed three times with 1* SSC, 0.1% SDS at 65oC for 20 min, then membranes were exposed to X-ray films for 1-10 days.
The expression plasmid pCXN2 (25 ) was provided by Dr J. Miyazaki at Tohoku University. The 1F-16R PCR-amplified fragments from patient 1 and control cDNA were digested with EcoRI and inserted into the EcoRI site of pCXN2. The 8Fa-10Rb PCR-amplified fragments from patient 1 and control genomic DNA were digested with EcoRI and inserted into the EcoRI site of pCXN2.
CHO cells were transfected by calcium phosphate coprecipitation (46 ). Two days later, CHO cells were selected in medium containing 0.5 mg/ml G418 (Sigma), as described by Niwa et al. (25 ). The G418-selected transfectant was selected and further cultured in the medium for 3 weeks. Skin fibroblasts from the patient were transfected with 1 [mu]g of plasmid DNA, 5 [mu]l of Lipofectamine (GIBCO/BRL) and 200 [mu]l Opti-Mem I (GIBCO/BRL). Three days later, fibroblasts were selected in medium containing 0.5 mg/ml G418 and further cultured in the medium for 6 weeks.
Genomic DNA from the normal control and patient 1 was amplified using primers 8Fa and 10Rb with an EcoRI linker at the 5' end (Table 1 ), digested with EcoRI, then inserted into the EcoRI site of pCXN2 expression vectors (25 ). An insert of partial DNA from patient 1 was sequenced to confirm the presence of a single T insertion. We prepared expression vectors, pCXN2-T(-), which included the normal fragment from the normal control and pCXN2-T(+), which included the mutant fragment from patient 1. These vectors were stably expressed in CHO cells. The cDNA isolated from these cells was separately transfected with wild pCXN2, pCXN2-T(+), and pCXN2-T(-), and amplified using primers 9F and 10Ra (Table 1 ).
Two reverse primers, 15Rw and 15Rm corresponding to a position 1350-1331 on HADHB cDNA (Fig. 3 A), were prepared according to Newton et al. (26 ). The 15Rw reverse primer was a wild-type oligonucleotide with one base mismatch (G1336 -> A) and the 15Rm reverse primer was a mutant-type oligonucleotide with two base mismatches (C1331 -> T and G1336 -> A). Genomic DNA from the normal control, the patient and his parents was amplified using two sets of the primers: a set of 14F and 15Rw to detect the normal allele, and 14F and 15Rm to detect the mutant allele.
We thank M.Ohara for helpful comments, and A.Uchiyama, M.Souri, K.Kamijo and K.O.Orii for technical advice. This study was supported by a Grant-in-Aid for Scientific Research (B) and (C) from the Ministry of Education, Science, Sports and Culture of Japan and a Research Grant for Intractable Diseases from the Ministry of Health and Welfare of Japan.
1 Izai, K., Uchida, Y., Orii, T., Yamamoto, S., and Hashimoto, T. (1992) Novel fatty acid [beta]-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase. J. Biol. Chem., 267, 1027-1033.MEDLINE Abstract
2 Aoyama, T., Souri, M., Ushikubo, S., Kamijo, T., Yamaguchi, S., Kelley, R.I., Rhead, W.J., Uetake, K., Tanaka, K., and Hashimoto, T. (1995) Purification of human very-long-chain acyl-coenzyme A dehydrogenase and characterization of its deficiency in seven patients. J. Clin. Invest., 95,2465-2473.MEDLINE Abstract
3 Aoyama, T., Ueno, I., Kamijo, T., and Hashimoto, T. (1994) Rat very-long-chain acyl-CoA dehydrogenase, a novel mitochondrial acyl-CoA dehydrogenase gene product, is a rate-limiting enzyme in long-chain fatty acid [beta]-oxidation system. J. Biol. Chem., 269,19088-19094. MEDLINE Abstract
4 Carpenter, K., Pollitt, R.J., and Middleton, B. (1992) Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria. Biochem. Biophys. Res. Commun., 183,443-448.MEDLINE Abstract
5 Uchida, Y., Izai, K., Orii, T., and Hashimoto, T. (1992) Novel fatty acid [beta]-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein. J. Biol. Chem., 267, 1034-1041.MEDLINE Abstract
6 Kamijo, T., Aoyama, T., Miyazaki, J., and Hashimoto, T. (1993) Molecular cloning of the cDNA for the subunits of rat mitochondrial fatty acid [beta]-oxidation multienzyme complex: structural and functional relationships to other mitochondrial and peroxisomal [beta]-oxidation enzymes. J. Biol. Chem., 268, 26452-26460.MEDLINE Abstract
7 Wanders, R.J.A., Duran, M., IJlst, L., de Jager, J.P., van Gennip, A.H., Jakobs, C., Dorland, L., and Van Sprang, F.J. (1989) Sudden infant death and long-chain 3-hydroxyacyl-CoA dehydrogenase. Lancet,i, 52-53.
8 IJlst, L., Ushikubo, S., Kamijo, T., Hashimoto, T., Ruiter, J.P.N., de Klerk, J.B.C., and Wanders, R.J.A. (1995) Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: high frequency of the G1528C mutation with no apparent correlation with the clinical phenotype. J. Inher. Metab. Dis., 18, 241-244.MEDLINE Abstract
9 Sewell, A.C., Bender, S.W., Wirth, S., Münterfering, H., IJlst, L., and Wanders, R.J. A. (1994) Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: a severe fatty acid oxidation disorder. Eur. J. Pediatr., 153, 745-750.MEDLINE Abstract
10 Jackson, S., Kler, R.S., Bartlett, K., Briggs, H., Bindoff, L.A., Pourfarzam, M., Gardner-Medwin, D., and Turnbull. D. M. (1992) Combined enzyme defect of mitochondrial fatty acid oxidation. J. Clin. Invest., 90, 1219-1225.MEDLINE Abstract
11 Kamijo, T., Wanders, R.J.A., Saudubray, J.M., Aoyama, T., Komiyama, A., and Hashimoto, T. (1994) Mitochondrial trifunctional protein deficiency: catalytic heterogeneity of the mutant enzyme in two patients. J. Clin. Invest., 93, 1740-1747.MEDLINE Abstract
12 Wanders, R.J.A., IJlst, L., Poggi, F., Bonnefont, J.P., Munnich, A., Brivet, M., Rabier, D., and Saudubray, J.M. (1992) Human trifunctional protein deficiency: a new disorder of mitochondrial fatty acid [beta]-oxidation. Biochem. Biophys. Res. Commun., 188, 1139-1145.
13 IJlst, L., Wanders, R.J.A., Ushikubo, S., Kamijo, T., and Hashimoto, T. (1994) Molecular basis of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: identification of the major disease-causing mutation in the [alpha]-subunit of the mitochondrial trifunctional protein. Biochim. Biophys. Acta, 1215,347-350.MEDLINE Abstract
14 IJlst, L., Ruiter, J.P.N., Vreijling, J., and Wanders, R.J.A. (1996) Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: a new method to identify the G1528C mutation in genomic DNA showing its high frequency (~90%) and identification of a new mutation (T2198C). J. Inher. Metab. Dis., 19,165-168.MEDLINE Abstract
15 Isaacs, J.D., Sims, H.F., Powell, C.K., Bennett, M.J., Hale, D.E., Treem, W.R., and Strauss, A.W. (1996) Maternal acute fatty liver of pregnancy associated with fetal trifunctional protein deficiency: Molecular characterization of a novel maternal mutant allele. Pediatr. Res., 40, 393-398.MEDLINE Abstract
16 Sims, H.F., Brackett, J.C., Powell, C.K., Treem, W.R., Hale, D.E., Bennett, M.J., Gibson, B., Shapiro, S., and Strauss, A.W. (1995) The molecular basis of pediatric long chain 3-hydroxyacyl-CoA dehydrogenase deficiency associated with maternal acute fatty liver of pregnancy. Proc. Natl. Acad. Sci. USA, 92, 841-845.MEDLINE Abstract
17 Brackett, J.C., Sims, H.F., Rinaldo, P., Shapiro, S., Powell, C.K., Bennett, M.J., and Strauss, A.W. (1995) Two [alpha] subunit donor splice site mutations cause human trifunctional protein deficiency. J. Clin. Invest., 95,2076-2082.MEDLINE Abstract
18 Ushikubo, S., Aoyama, T., Kamijo, T., Wanders, R.J.A., Rinaldo, P., Vockley, J., and Hashimoto, T. (1996) Molecular characterization of mitochondrial trifunctional protein deficiency: formation of the enzyme complex is important for stabilization of both [alpha]- and [beta]-subunits. Am. J. Hum. Genet., 58, 979-988.MEDLINE Abstract
19 Dionisi-Vici, C., Garavaglia, B., Burlina, A.B., Bertini, E., Saponara, I., Sabetta, G., and Taroni, F. (1996) Hypoparathyroidism in mitochondrial trifunctional protein deficiency. J. Pediatr., 129, 159-162.MEDLINE Abstract
20 Zhang, Q.X., and Baldwin, G.S. (1994) Structures of the human cDNA and gene encoding the 78 kDa gastrin-binding protein and of a related pseudogene. Biochim. Biophys. Acta, 1219, 567-575.MEDLINE Abstract
21 IJlst, L., Ruiter, J.P.N., Hoovers, J.M.N., Jakobs, M.E., and Wanders, R.J.A. (1996) Common missense mutation G1528C in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Characterization and expression of the mutant protein, mutation analysis on genomic DNA and chromosomal localization of the mitochondrial trifunctional protein [alpha] subunit gene. J. Clin. Invest., 98, 1028-1033.MEDLINE Abstract
22 Shapiro, M.B., and Senapathy, P. (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res., 15, 7155-7174.MEDLINE Abstract
23 Kamijo, T., Aoyama, T., Komiyama, A., and Hashimoto, T. (1994) Structural analysis of cDNA for subunits of human mitochondrial fatty acid [beta]-oxidation trifunctional protein. Biochem. Biophys. Res. Commun., 199, 818-825.MEDLINE Abstract
24 Yang, B-Z., Heng, H.H.Q., Ding, J-H., and Roe C.R. (1996) The genes for the [alpha] and [beta] subunits of the mitochondrial trifunctional protein are both located in the same region of the human chromosome 2p23. Genomics, 37, 141-143.
25 Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108, 193-200.MEDLINE Abstract
26 Newton, C.R., Graham, A., Heptinstall, L.E., Powell, S.J., Summers, C., Kalsheker, N., Smith, J.C., and Markham, A.F. (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res., 17, 2503-2516.MEDLINE Abstract
27 Jackson, S., Schaefer, J., Middleton, B., and Turnbull, D.M. (1995) Characterization of a novel enzyme of human fatty acid [beta]-oxidation: a matrix-associated, mitochondrial 2-enoyl-CoA hydratase. Biochem. Biophys. Res. Commun., 214, 247-253.MEDLINE Abstract
28 Shaefer, J., Jackson, S., Dick, D.J., and Turnbull, D.M. (1996) Trifunctional enzyme deficiency: adult presentation of a usually fatal [beta]-oxidation defect. Ann.. Neurol., 40, 597-602.
29 Miyajima, H., Orii, K.E., Shindo, Y., Hashimoto, T., Shinka, T., Kuhara, T., Matsumoto, T., Shimizu, H., and Kaneko, E. (1997) Mitochondrial trifunctional protein deficiency associated with recurrent myoglobinuria in adolescence. Neurology, in press.
30 Humphrey, M.B., Bryan, J., Cooper, T.A., and Berget, S.M. (1995) A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon. Mol. Cell. Biol., 15,3979-3988.MEDLINE Abstract
31 Tanaka, K., Watakabe, A., and Shimura, Y. (1994) Polypurine sequences within a downstream exon function as a splicing enhancer. Mol. Cell. Biol., 14, 1347-1354.MEDLINE Abstract
32 Orii, K.E., Aoyama, T., Souri, M., Jiang, L.L., Orii, K.O., Hayashi, S., Yamaguchi, S., Kondo, N., Orii, T., and Hashimoto, T. (1996) Formation of the enzyme complex in mitochondria is required for function of trifunctional [beta]-oxidation protein. Biochem. Biophys. Res. Commun., 219, 773-777.MEDLINE Abstract
33 Wieland, T., and Rueff, L. (1953) Synthese von S-[beta]-oxybutyryl- und S-acetacetyl-coenzyme A. Angew. Chem., 65, 186-187.
34 Imamura, S., Ueda, S., Mizugaki, M., and Kawaguchi, A. (1990) Purification of the multienzyme complex for fatty acid oxidation from Pseudomonas fragi and reconstitution of the fatty acid oxidation system. J. Biochem., 107, 184-189.
35 Sanger, F., Nicklen, S., and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74, 5463-5467.MEDLINE Abstract
36 Aoyama, T., Wakui, K., Fukushima, Y., Orii, K.O., and Hashimoto, T. (1996) Assignment of the human mitochondrial very-long-chain acyl-CoA dehydrogenase gene (LCACD) to 17p13 by in situ hybridization. Genomics, 37,144-145.
37 Hatada, I., Inazawa, J., Abe, T., Nakayama, M., Kaneko, Y., Jinno, Y., Niikawa, N., Ohashi, H., Fukushima, Y., Iida, K., Yutani, C., Takahashi, S., Chiba, Y., Ohishi, S., and Mukai, T. (1996) Genomic imprinting of human p57KIP2 and its reduced expression in Wilms' tumors. Hum. Mol. Genet., 5,783-788.MEDLINE Abstract
38 Furuta, S., Miyazawa, S., Osumi, T., Hashimoto, T., and Ui, N.(1980) Properties of mitochondrial and peroxisomal enoyl-CoA hydratases from rat liver. J. Biochem., 88,1059-1070.MEDLINE Abstract
39 Osumi, T., and Hashimoto, T. (1979) Occurrence of two 3-hydroxyacyl-CoA dehydrogenases in rat liver. Biochim. Biophys. Acta, 574, 258-267.MEDLINE Abstract
40 Osumi, T., and Hashimoto, T. (1980) Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver. Arch. Biochem. Biophys., 203, 372-383.MEDLINE Abstract
41 Markwell, M.A., Haas, S.M., Bieber, L.L., and Tolbert, N.E. (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem., 87, 206-210.MEDLINE Abstract
42 Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275.
43 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227,680-685. MEDLINE Abstract
44 Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76, 4350-4354.MEDLINE Abstract
45 Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162,156-159.MEDLINE Abstract
46 Graham, F.L., and van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus IV DNA. Virology, 52, 456-467.MEDLINE Abstract
*To whom correspondence should be addressed at: Department of Biochemistry, Shinshu University School of Medicine, 3-3-1 Asahi, Matsumoto, Nagano 390, Japan. Tel: +81 263 37 2603; Fax: +81 263 37 2604; Email: kenjior-gif@umin.u-tokyo.ac.jp
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