Main

The mitochondrial β-oxidation spiral is the major pathway for energy production in heart and skeletal muscle and is essential for ketogenesis in the liver. β-Oxidation of fatty acids requires four sequential enzyme activities: a fatty acyl-CoA dehydrogenase, a 2,3-enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. Each cycle ofβ-oxidation produces a molecule of acetyl-CoA and a fatty acyl-CoA shortened by two carbons. This acetyl-CoA can either enter the tricarboxylic acid pathway for energy generation in tissues such as heart and slow-twitch muscle or be converted to ketone bodies by the liver. For long chain substrates, the third step in β-oxidation is catalyzed by LCHAD. LCHAD activity is part of TFP, a multienzyme complex associated with the inner mitochondrial membrane(1). TFP is a hetero-octamer composed of 4 α- and 4 β-subunits. The α-subunit contains long chain enoyl-CoA hydratase activity on the N-terminal region and LCHAD activity on the C-terminal region. The β-subunit is the long chain 3-ketoacyl-CoA thiolase.

Deficiencies of the β-oxidation spiral enzymes in children produce sudden unexpected death, acute hepatic encephalopathy with hypoketotic hypoglycemia (a Reye-like syndrome), or cardiomyopathy(2). The hepatic phenotype bears a striking similarity to maternal AFLP both histologically and clinically(3, 4). We and others(5–7) noted a potential association between children with deficiency of LCHAD activity and pregnancies complicated by AFLP. We subsequently characterized the molecular basis of this disorder in three families with maternal AFLP and pediatric LCHAD deficiency(8). We report here a novel mutation in the TFPα-subunit in a fourth family with maternal AFLP and pediatric LCHAD and TFP deficiency. This provides further evidence of the pathophysiologic link between these two disorders and for heterogeneity of mutations in LCHAD deficiency.

METHODS

Case history. The patient was a 43-y-old Caucasian woman with a history of two previous uncomplicated term pregnancies that resulted in the delivery of healthy boys. She presented at 35 wk of gestation with complaints of nausea, vomiting, extreme fatigue, and malaise. She was normotensive with no evidence of edema. Laboratory evaluation, with the normal range of values for each test in parentheses, revealed AST, 293 U/L (14-43 U/L); bilirubin, 5.0 mg/dL (0-1.3 mg/dL); platelet count, 75,000/mm3(140,000-350,000/mm3); prothrombin time, 15.3 s (11.4-13.3 s); and fibrinogen, 164 mg/dL (300-600 mg/dL). Serologic testing for hepatitis A, B, and C was negative, and no proteinuria was detected. An abdominal ultrasound was consistent with diffuse hepatic steatosis. Maternal blood or urine organic acid analysis was not done at this time. A 35-wk male infant was delivered by emergent cesarean section for fetal distress. The mother had modest postoperative bleeding requiring reexploration, and she developed pulmonary edema. She recovered rapidly and is currently well. The infant required brief respiratory support, but was discharged at 6 d of age. At 4 mo of age, he developed a viral upper respiratory infection with vomiting, irritability, and decreased oral intake. He rapidly became lethargic and developed coma with hypoglycemia (32 mg/dL) and a metabolic acidosis. He was resuscitated with i.v. glucose. Further studies revealed elevated AST (230 U/L), and a urine organic acid profile showed elevated C6-C14 3-hydroxy-fatty acids and C6-C14 3-hydroxy-dicarboxylic acids. The increase in long chain 3-hydroxy-fatty acids is virtually diagnostic of LCHAD or TFP deficiency(2). Computed tomography of the abdomen revealed an enlarged liver with diffuse hepatic steatosis, and a chest x-ray demonstrated cardiomegaly. He was started on a low fat diet and L-carnitine supplementation. He is now 5 y of age with normal growth, development, and exercise capability. However, he has suffered several mild and transient episodes of muscle pain, myoglobinuria, and hypotonia and has developed retinitis pigmentosa. He does not have any clinical neuropathy.

Cell lines, enzyme assays, DNA, and RNA isolation. Blood was collected from all family members, and genomic DNA was isolated from leukocytes by alkaline lysis and proteinase digestion(9). Skin fibroblasts were obtained from all family members and maintained in minimal essential medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics. RNA was isolated using the guanidine isothiocyanate method with an added DNase digestion to reduce DNA contamination(10). LCHAD activity using the reverse reaction and long chain 3-ketoacyl-CoA thiolase were assayed by the method of Venizelos et al..(11) with protein extracts of cultured fibroblasts.

SSCV analysis of α- subunit exons. PCR amplification of genomic DNA was done with the following primer pairs corresponding to intronic sequences flanking the exons (artificially engineered restriction sites are underlined): exon 15, sense 5′-cctagcagagaaggaagcttctcaggttcctc-3′ and antisense 5′-agtcttattagaagcttttcaaaaactctgc-3′; exon 16, sense 5′-gggccagtgagctcatgttctcttcttgg-3′ and antisense 5′-tgcaaacactctggagagctcaatacca-3′.

SSCV analysis of the PCR-amplified, genomic DNA was accomplished using a nondenaturing gel electrophoresis according to published protocols(12). After restriction enzyme digestion, the PCR products were placed in plasmid vectors, and nucleotide sequence analysis on each exon was performed on 4-8 subclones using the dideoxy chain termination method.

RT-PCR amplification of mRNA and ASO hybridization. First strand cDNA was synthesized from fibroblast RNA with oligo(dT) as a primer for RT. This product was used as the template for two subsequent PCR amplifications with the following internal (nested) coding region primers with artificially engineered restriction sites underlined: first round, sense 5′-acagaattcacagcgtatgccatgactattcc-3′ (bp 800-827), antisense 5′-residual oligo(dT); and second round, sense 5′-cctgtgcaagaagaataaatttggagctccac-3′ (bp 1040-73), antisense 5′-gactgagtcgacggcatgaggcctgctca-3′ (bp 2290-2316).

The 1276 nucleotide product (bp 1040-2316 of the α-subunit mRNA) encompassed exons 11-20 (C. K. Powell, H. F. Sims, and A. W. Strauss, unpublished data)(8). This DNA product was applied to a nylon membrane by vacuum blotting and was probed with oligonucleotides containing the normal and mutant sequences in exons 15 and 16 using conditions previously described(13).

RNA and protein analysis. These were performed using standard methods(13).

RESULTS

Biochemical characterization of trifunctional protein deficiency in this family with AFLP. We evaluated members of the family described in the “Case History” (see “Methods”) with maternal AFLP and transient, hypoglycemic coma at 4 mo of age in the son. 3-Hydroxyacyl-CoA dehydrogenase assays with protein extracts of cultured fibroblasts from the affected infant revealed marked deficiency of activity versus long chain substrate (3-ketopalmitoyl-CoA), but normal activity with short chain(acetoacetyl-CoA) substrate. Thus, compared with normal controls, fibroblasts from this infant had a profound defect in LCHAD activity(Table 1). The short chain 3-hydroxyacyl-CoA dehydrogenase, a soluble matrix protein, has some activity versus C16 substrate, suggesting that the residual activity in the affected child may be due to this enzyme. Assay of long chain 3-ketoacyl-CoA thiolase activity, which is encoded by the TFP β-subunit, also revealed markedly reduced activity (29% of control). This is consistent with TFP deficiency in this child(1, 11). Extracts of fibroblasts isolated from the parents and two siblings also showed significant reduction in LCHAD activity, consistent with heterozygosity. Thus, the clinical presentation, metabolic profile, and enzymatic assays revealed TFP deficiency in the affected child.

Table 1 LCHAD activities in fibroblasts from family members
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Delineation of the molecular defect in exon 16 of the LCHAD region of the TFP α- subunit. Using the humanα-subunit genomic sequence we previously characterized (C. K. Powell, H. F. Sims, and A. W. Strauss, unpublished results)(14), we designed oligonucleotides to amplify each of the 20 α-subunit exons. Genomic DNA from all family members was amplified with all 20 sets of oligonucleotides, and the products derived from each exon were analyzed by SSCV and compared with amplified exons from a normal individual.

Amplified exon 16 DNA from the affected child and his mother with AFLP showed differences from the normal pattern upon SSCV analysis (Fig. 1). The exon 16 DNA from these two individuals also shared some bands with normal, suggesting heterozygosity. Exon 16 conformers from the father and two male siblings were identical to the normal control. Sequence analysis of amplified exon 16 genomic DNA from the mother and affected child revealed that a single mutation, C to T, corresponding to nucleotide 1678 of the TFP α-subunit mRNA, was present in half of the subclones (Fig. 2). This mutation (R524Stop) changes the arginine codon (CGA) at amino acid residue 524 of the mature trifunctional protein α-subunit to a termination codon (TGA). These results prove heterozygosity for this C1678T mutation in the mother and her clinically affected son.

Figure 1
figure 1

SSCV analysis of exon 16 amplified genomic DNA from all family members. Genomic DNA from all five family members and a normal control was amplified by PCR and the radiolabeled products analyzed for SSCV after gel electrophoresis and autoradiography. The two bands (labeled WT) which characterize the normal allele are seen in all individuals. The mother with AFLP and the affected child have an additional band (labeled M for mutant), consistent with heterozygosity for a mutant allele. The family is depicted above the autoradiograph with the standard symbols for males (□) and females (○). The half-filled symbols indicate the presence of a mutation on one allele in that individual, i.e. the mother and affected child.

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Figure 2
figure 2

Nucleotide sequences of two subclones of the affected child's exon 16 amplified genomic DNA. After amplification of genomic DNA from the affected child with oligonucleotides flanking exon 16 (see“Methods”), digestion with SacI, and placement into pGEM3Z vector, two subclones were subjected to nucleotide sequence analysis by the dideoxy chain termination method. The clone shown on the right has the normal sequence, with a C (indicated by the ←C) at bp 1678. The sequence on the left reveals the mutation, a T at position 1678 (indicated by the T→), which creates a termination codon.

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Confirmation of the C1678T mutation by ASO hybridization. To verify the presence of the C1678T mutation, oligonucleotides corresponding to the normal sequence or the mutant allele were used to probe PCR-amplified, exon 16, genomic DNA from all family members and 10 normal controls (Fig. 3). Only the affected infant and his mother showed hybridization with the mutant probe (Fig. 3,bottom). Amplified DNA from all family members, including the mother and affected child, and all normal controls showed hybridization to the normal probe. This confirms that the affected infant and his mother with AFLP are heterozygous for the R524Stop mutation. Moreover, this nucleotide difference is not frequent in the normal population.

Figure 3
figure 3

ASO analysis of amplified exon 16 genomic DNA isolated from family members and normal individuals. Equal aliquots of amplified exon 16 genomic DNA from all family members (indicated by the symbols above the autoradiogram as in Fig. 1) and 10 normal control individuals (numbered 1-10 above the autoradiogram) was blotted on to a nylon membrane and probed with labeled oligonucleotides (see“Methods”) containing either the normal exon 16 sequence (upper row of samples labeled Wild-Type) or the R524Stop mutation (lower row labeled Mutant). After washing, the membrane was subjected to autoradiography.

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Delineation of the molecular defect in exon 15 of the LCHAD region of the TFP α -subunit. SSCV analysis revealed aberrant bands in the exon 15, PCR-amplified products derived from the affected child, his father, and two male siblings (data not shown). Other conformers in exon 15 DNA from these individuals were shared with the normal control. This SSCV banding pattern was identical to that derived from exon 15 genomic DNA of a known heterozygous individual with the mutation G1528C(8). Sequence analysis of exon 15 genomic DNA demonstrated this G1528C mutation in one-half of the subclones from the affected infant, his father, and two siblings (data not shown). This mutation changes the glutamic acid codon at residue 474 of mature α-subunit to glutamine(E474Q) and has previously been shown result in a protein with severely reduced LCHAD activity(8). The mother had a normal pattern by SSCV and a normal sequence in 5 subclones. Thus, the father, two clinically normal siblings, and the affected child are heterozygous for the E474Q mutation in the LCHAD domain of the TFP α-subunit.

Confirmation of the E474Q mutation by ASO hybridization. To verify the presence of the E474Q mutation in these four family members, oligonucleotides were created that corresponded to the normal sequence or mutant allele and were used to probe PCR-amplified exon 15 DNA from all family members, a normal control, and known heterozygous and homozygous individuals with the E474Q mutation(8). Amplified DNA from all individuals except the known homozygous mutant patient showed hybridization to the normal probe. Amplified DNA from the affected child, his father, his two siblings, the known heterozygous mutant mutant individual, and the homozygous mutant patient showed hybridization to the mutant probe (results not shown).

We conclude that the affected child is a compound heterozygote inheriting the paternal, E474Q, and the maternal, R524Stop, mutations in the LCHADα-subunit gene. All other family members are simple heterozygotes.

Quantification of allele-specific mutant mRNA expression. Previous reports have demonstrated that mRNA transcripts which contain premature termination codons may be present in the cytoplasm at greatly reduced levels(15–18). We postulated that the α-subunit termination codon mutation, R524Stop, in this family might similarly result in low level mutant mRNA expression in the cytoplasm. To test this prediction, we used RT-PCR amplification of mRNA derived from cultured fibroblasts of all family members to quantify expression ofα-subunit alleles (Fig. 4). Total cellular RNA was used as a template for RT-PCR amplification with nested oligonucleotide primers encompassing the LCHAD region of the α-subunit (Fig. 4A). The product of this amplification includes the sites (nucleotide 1528 in exon 15 and nucleotide 1678 in exon 16) of both mutations. Equal aliquots of the amplified products from all family members and a normal individual were analyzed separately by hybridization to four, allele-specific, labeled oligonucleotides (Fig. 4B) corresponding to 1) the normal sequence in exon 15; 2) the exon 15, G1528C mutation; 3) the normal sequence in exon 16; and 4) the exon 16, C1678T mutation.

Figure 4
figure 4

ASO analysis of RT-PCR amplified mRNA isolated from cultured fibroblasts of a normal individual and all family members. Total cellular RNA was amplified by RT-PCR with nested oligonucleotides and the scheme outlined in panel A. The α-subunit mRNA is depicted to scale with the exons indicated by alternating clear and hatched boxes and numbered, 1-20. The higher box is the coding region, the lower boxes show the 3′- and 5′- untranslated regions. The upper section demonstrates the position of the R524Stop (C1678T) maternal mutation in exon 16. The lower section depicts the E474Q (G1528C) paternal mutation in exon 15. The central section indicates with arrows the locations of the oligonucleotide primers used for the two RT-PCR reactions, with S for sense-orientation and AS for antisense orientation. In panel B, four equal aliquots of the amplified DNA from each individual (as described by the labels and symbols above the autoradiogram) were applied to a nylon membrane and separately hybridized with the four probes corresponding to the normal exon 15 or 16 sequences (labeled Wild-Type) or containing either of the two mutant sequences (labeled Mutant) in exons 15 and 16. After washing, the membrane was subjected to autoradiography.

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The results show that mRNA transcripts derived from the R524Stop (C1678T) mutant allele are present in fibroblasts from the affected child and his mother in barely detectable amounts. This is consistent with rapid degradation of this mutant mRNA. On the other hand, α-subunit mRNA derived from the E474Q mutant allele in the patient, his father, and two normal siblings is expressed at similar levels to normal α-subunit mRNA. This is consistent with stable expression of LCHAD mRNA containing this missense mutation.

DISCUSSION

Our results demonstrate autosomal recessive inheritance of LCHAD and TFP deficiency in a family with maternal AFLP occurring during the pregnancy with the male fetus who at age 4 mo developed severe, but reversible, nonketotic, hypoglycemic coma. Molecular characterization revealed that the affected child is a compound heterozygote, having inherited a paternal E474Q mutation and a maternal R524Stop mutation.

In our original description of AFLP and LCHAD deficiency in three families(8), we suggested that the E474Q mutation might be common because five of the six alleles in the affected children contained this mutation. IJlst et al.(19) reported that this same mutation was highly prevalent in children with isolated LCHAD deficiency. In their original study, using RT-PCR amplification of α-subunit mRNA, IJlst et al.(19) reported apparent homozygosity in 24 of the 26 LCHAD-deficient patients, with heterozygosity in the other two. Updated results among 34 patients with isolated LCHAD deficiency by this group (I. IJlst and R. J. A. Wanders, personal communication), with genomic DNA or familial studies, reveal that the E474Q mutation occurs on 87% of the abnormal alleles. With the results presented here, we have documented that 6 of the 8 mutant alleles in families with AFLP are the E474Q mutation and that the other two mutations create premature termination codons. Thus, this mutation is, indeed, a frequent mutation in families with isolated LCHAD deficiency or with pediatric LCHAD deficiency associated with maternal AFLP.

However, it is also apparent that other TFP α-subunit mutations occur. For example, we have described a TFP-deficient individual with two different splice site mutations resulting in complete absence ofα-subunit(20). Moreover, among 26 patients with enzymatic evidence of LCHAD deficiency (J. Ibdah, M. J. Bennett, and A. W. Strauss, unpublished results), only 3 are homozygous for the E474Q mutation and 12 others are heterozygous for this mutation. We have defined six additional mutations including a short deletion and several missense mutations, demonstrating significant heterogeneity in α-subunit mutations.

Our results show that the R524Stop mutation in the affected child and his mother with AFLP results in a low abundance mRNA transcript. Premature termination codons leading to a decreased cytoplasmic mRNA pool have been described in association with a variety of disorders and reduce mRNA levels by several mechanisms. These include an increased rate of mRNA decay inβ0-thalassemia(18), nonsense codon-mediated effects on nuclear RNA processing in β-39 thalassemia(16), alterations in the secondary structure of the pre-mRNA causing missplicing(15), and mutation-induced exon skipping. The mechanism responsible for the decreased level of the R524Stop transcript in our family is uncertain. mRNA derived from the E474Q mutant, paternal allele is expressed well; but results in the production of a mature α-subunit with greatly decreased LCHAD activity(8).

Our results with the family reported here emphasize the limitations of sole reliance upon an RT-PCR-based approach, with mRNA as the template, for molecular diagnosis. mRNA amplification without examination of the genomic DNA sequence (see Fig. 4) would not have detected the R524Stop mutation in this severely affected child or his mother and led to an incorrect diagnosis of homozygosity for the E474Q mutation because the R524Stop transcript was undetectable. Thus, any mutations resulting in low level expression of α-subunit mRNA derived from the mutant allele would be missed by the RT-PCR approach.

Although recent reports suggested the association of pediatric LCHAD deficiency(5–7) with maternal AFLP, this and our previous report(8) prove this relationship at the molecular level. We have delineated three fetal genotypes in four pregnancies complicated by maternal AFLP: 1) homozygosity for the E474Q mutation in fetuses of two pregnancies and 2) fetal compound heterozygosity with one E474Q mutant and different premature termination codon mutations as the second mutation. In these four cases, the affected infants demonstrated severely decreased LCHAD activity. In one of these families, another child was the product of a pregnancy complicated by AFLP and died suddenly at 5 mo of age with acute hypoglycemia and fatty infiltration of the liver, consistent with LCHAD deficiency. Among the other six pregnancies in these four families, clinically apparent maternal AFLP did not occur when the delivered fetuses were clinically normal and had a normal genotype or heterozygosity for the E474Q mutation.

The development of maternal AFLP during the final weeks of pregnancy in these patients may be precipitated by physiologic stresses which accompany normal pregnancy. Decreased maternal mitochondrial β-oxidation of fatty acids had been demonstrated during the third trimester of pregnancy(21). In addition, maternal serum levels of FFA, the substrate for mitochondrial β-oxidation, increase steadily throughout gestation and peak late in the third trimester(22). This combination of decreased enzymatic capacity due to genetic and gestational factors, combined with the increased production of fatty acids during late gestation may lead to intrahepatic accumulation of long chain fatty acids. These metabolites are known inhibitors of mitochondrial enzymes, and their intrahepatic accumulation could cause a further decrement in maternalβ-oxidation activity and, ultimately, the development of AFLP.

Elucidation of the underlying genetic defects(8) in some women with AFLP provides insight into this life-threatening disease of previously unknown etiology. More importantly, recognition of the association between AFLP and pediatric LCHAD or TFP deficiency demonstrates the need for screening infants at risk and provides the opportunity for prospective, preventive treatment of affected infants. This is important, because in 50% of affected children, the initial presentation is sudden death and because dietary management reduces or eliminates the occurrence of acute metabolic crises, morbidity, and mortality(23). Therefore, evaluation for LCHAD or TFP deficiency of all infants of pregnancies complicated by AFLP should be done. Genetic screening for LCHAD mutations in women with a history of previous AFLP would provide important information which may influence decisions regarding future pregnancy. Finally, our results emphasize that molecular heterogeneity in α-subunit mutations exists, even among families with AFLP and LCHAD deficiency.