Identification of 16 new disease-causing mutations in the CPT2 gene resulting in carnitine palmitoyltransferase II deficiency

Molecular Genetics and Metabolism 89 (2006) 323–331 www.elsevier.com/locate/ymgme

IdentiWcation of 16 new disease-causing mutations in the CPT2 gene resulting in carnitine palmitoyltransferase II deWciency Paul J. Isackson a, Michael J. Bennett b, Georgirene D. Vladutiu a,¤ a

Department of Pediatrics, School of Medicine and Biomedical Sciences, State University of New York at BuValo, 936 Delaware Ave., BuValo, NY 14209, USA b Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine and Children’s Hospital of Philadelphia, Philadelphia, PA, USA Received 3 June 2006; received in revised form 9 August 2006; accepted 9 August 2006 Available online 22 September 2006

Abstract The exonic regions of the carnitine palmitoyltransferase 2 (CPT2) gene were characterized from 101 patients with deWned clinical and biochemical evidence for the adult onset form of CPT II deWciency and in 2 patients detected as newborns with abnormal acylcarnitine proWles. Twenty-seven disease-causing mutations within the CPT2 gene were identiWed in this cohort, 16 of which were novel. A total of 60 disease-causing mutations have been identiWed to date in CPT2 and 41 of these are predicted to produce amino acid substitution/deletions. The implications of these mutations are described in light of recent advances in our understanding of the molecular structure of members of the carnitine acyltransferase family. © 2006 Elsevier Inc. All rights reserved. Keywords: Fatty acid oxidation; Mutation; Sequence; Skeletal muscle; Carnitine palmitoyltransferase deWciency

Introduction Carnitine palmitoyltransferase II (CPT II) (EC 2.3.1.21) deWciency is one of the most common defects of oxidative lipid metabolism in humans [1]. CPT II deWciency has three distinct clinical phenotypes: a common adult onset myopathy (MIM255110) characterized by episodes of muscle pain, cramps, elevated serum creatine kinase levels and myoglobinuria triggered by prolonged exercise or fasting [2]; a severe late infantile form with hypoketotic hypoglycemia and multiple organ system involvement including liver failure, cardiomyopathy, and peripheral myopathy; and a rare lethal neonatal form (MIM600649) with dysmorphic features, renal cysts, and additional symptoms of the late-infantile form [3]. To date, 44 mutations have been identiWed in the CPT2 gene encoding CPT II responsible for this disorder [1,4]. One *

Corresponding author. Fax: +1 716 888 1371. E-mail address: [email protected] (G.D. Vladutiu).

1096-7192/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2006.08.004

mutation, c.338C > T (p.Ser113Leu), has been found in greater than 50% of adult cases [5]. CPT II is structurally similar to other members of the carnitine acyltransferase family: carnitine palmitoyltransferase I (CPT I), carnitine octanoyltransferase (CrOT) and carnitine acetyltransferase (CrAT). CPT I and CPT II speciWcally utilize long chain acyl-CoA substrates, while CrOT and CrAT are speciWc for medium and short chain acyl-CoAs, respectively [6]. These family members are diVerentially localized throughout the cell and have fundamental roles in fatty acid oxidation and the regulation of acyl-CoA pools. CPT II, CPT I, and carnitine/acylcarnitine translocase (CACT) are required for the transport of long chain fatty acids into mitochondria with subsequent -oxidation [7]. CPT I is bound to the outer mitochondrial membrane, CACT is bound to the inner mitochondrial membrane and CPT II is associated with the inner mitochondrial membrane. CPT I converts long chain acyl-CoAs to acylcarnitine which is transported across the mitochondrial membrane and converted back to acylCoA by CPT II allowing processing by -oxidation [8].

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Structural information has recently become available relevant to understanding CPT2 sequence alterations from the crystal structure of rat CPT II [9,10] as well as two other members of the carnitine acyltransferase family [11–14]. All three of these enzymes were found to be monomeric structures each containing structurally similar N- and C-terminal domains [9–11,13,14]. His372 of CPT II is absolutely conserved in all members of the carnitine acyltransferase family and has been implicated in playing a fundamental role in catalysis by chemical modiWcation, mutagenesis, and crystal structure analyses [6,8,13]. This active site histidine residue is located in the center of the enzyme at the interface of the N- and C-terminal domains and can be approached from either side via tunnels that form binding sites for carnitine and CoA [13].

In this report, we describe the genetic characterization of a large cohort of CPT II deWcient patients with adult onset symptoms as well as two cases obtained through expanded newborn screening predicted to be adult-onset cases [15]. Sixteen novel mutations are described in light of this newly available structural information and the severity of individual mutations is discussed in relation to the ratio of CPT to citrate synthase activities. Materials and methods Subjects DNA from 103 patients was examined for sequence mutations causing the adult onset form of CPT II deWciency (Table 1). This group included:

Table 1 IdentiWed mutations in patients with adult onset CPTII deWciency Patient

Nucleotide sequence

Predicted amino

Muscle enzyme activity

N

Variation

Acid change

CPT

CS

1–20 21–30 31–32

c.[338C > T]+[338C > T] c.[338C > T]+[1238_1239delAG] c.[149C > A]+[338C > T]

p.[Ser113Leu]+[Ser113Leu] p.[Ser113Leu]+[Gln413fs] p.[Pro50His]+[Ser113Leu]

17.5 § 10.5 (n D 8) 11.7 § 4.0 (n D 6) 17.7 (n D 1)

19.3 § 6.6 17.4 § 6.4 15

0.92 § 0.44 0.68 § 0.18 1.20

10.2 § 4.5 (n D 8)

15.4 § 6.1

0.66 § 0.24

26.0 § 8.6 (n D 9)

10.9 § 2.8

2.41 § 0.78

14.1 § 1.9 (n D 6)

10.5 § 5.0

1.62 § 0.61

38.6 § 8.6 (n D 31)

12.8 § 4.4

3.17 § 1.07

All other compound heterozygotes 33 c.[250T > C]+[338C > T] 34 c.[256_257delAG]+[338C > T] 35 c.[338C > T]+[1273_1274delAC] 36 c.[338C > T]+[1634_1636delAAG] 37 c.[338C > T]+[1816_1817delGT] 38 c.[38delG]+[338C > T] 39 c.[36_43dupGGGCCCCG]+[338C > T] 40 c.[338C > T]+[534_558delinsT] 41 c.[338C > T]+[691A > T] 42 c.[338C > T]+[1444_1447delACAG] 43 c.[338C > T]+[1511C > T] 44 c.[338C > T]+[1646G > A] c.[338C > T]+[1369A> T] 45b 46b c.[338C > T]+[1569_1570delCA] 47 c.[149C > A]+[1646G > A] 48 c.[370C > T]+[1891C > T] 49 c.[113_114dupGC]+[1810C > T] 50 c.[359A > G]+[534_558delinsT]

p.[Cys84Arg]+[Ser113Leu] p.[Ser86fs]+[Ser113Leu] p.[Ser113Leu]+[Thr425fs] p.[Ser113Leu]+[Glu545del] p.[Ser113Leu]+[Val606fs] p.[Gly13fs]+[Ser113Leu] p.[Ala15fs]+[Ser113Leu] p.[Ser113Leu]+[Leu178_Ile186delinsPhe] p.[Ser113Leu]+[Arg231Trp] p.[Ser113Leu]+[Thr482fs] p.[Ser113Leu]+[Pro504Leu] p.[Ser113Leu]+[Gly549Asp] p.[Ser113Leu]+[Lys457X] p.[Ser113Leu]+[His523fs] p.[Pro50His]+[Gly549Asp] p.[Arg124X]+[Arg631Cys] p.[Ser38fs]+[Pro604Ser] p.[Tyr120Cys]+[Leu178_Ile186delinsPhe]

Heterozygotes 51–55 c.[338C > T]+[ D ] 56 c.[149C > A]+[ D ] 57 c.[149C > A]+[1645+5G > A]c 58 c.[302C > T]+[ D ] 59 c.[1438G > A]+[ D ] 60 c.[1763C > G]+[ D ] 61 c.[1679G > A]+[ D ] 62–64 c.[1055T > G]+[ D ] 65 c.[1646G > A]+[ D ]

p.[Ser113Leu]+[ D ] p.[Pro50His]+[ D ] p.[Pro50His]+[ D ] p.[Ala101Val]+[ D ] p.[Gly480Arg]+[ D ] p.[Ser588Cys]+[ D ] p.[Arg560Gln]+[ D ] p.[Phe352Cys]+[ D ] p.[Gly549Asp]+[ D ]

Residual CPT activity in aVected range 66 c.[930C > T]c+[ D ] 67–71 c.[ D ]+[ D ]

p.[ D ]+[ D ] p.[ D ]+[ D ]

Residual CPT activity in carrier range 72–103 c.[ D ]+[ D ]

p.[ D ]+[ D ]

a

CPT/CSa

Muscle enzyme activities were available for a limited number of patients as indicated. Patients identiWed by expanded newborn screening. c DNA sequence alterations, c.1645+5G > A and c.930C > T, do not alter the predicted amino acid sequence but may aVect RNA splicing or stability as discussed in the text. b

P.J. Isackson et al. / Molecular Genetics and Metabolism 89 (2006) 323–331 23 muscle biopsies with deWciencies of CPT II enzyme activity and ratios of CPT to citrate synthase predicting the presence of 2 mutations (11.88 § 4.82 nmol/min/g and 0.71 § 0.26, respectively); 2 muscle biopsies with CPT deWciency but inadequate data for calculating a ratio; 26 muscle biopsies with ratios of CPT to citrate synthase within carrier range and 20 biopsies with higher ratios. Six genomic DNA specimens were derived from cultured Wbroblasts of CPT II-deWcient patients as determined in Dr. Bennett’s laboratory and genomic DNA was derived from 24 whole blood specimens of patients referred to rule out CPT II deWciency. Histories were provided with 18 of the blood samples and of these, 72% reported rhabdomyolysis and/or elevated serum CK with exercise intolerance and 22% reported abnormal elevations in long-chain acylcarnitines. In addition, genomic DNA from blood was sequenced from two patients determined to have elevated long-chain acylcarnitine levels through expanded newborn screening [15]. The mean and standard deviations for CPT activity and for ratios of CPT to citrate synthase were calculated retrospectively from 23 individuals with 2 mutations and known CPT and citrate synthase activities in muscle and 8 individuals with 1 mutation, known CPT and citrate synthase activities, and not likely to have a second mutation in the CPT2 gene as per earlier studies [16]. The ratio is only helpful in cases where muscle biopsy is performed for biochemical analysis and mutation status is unknown. The ratio serves to distinguish heterozygotes from those more likely have 2 mutations and generally leads to the recommendation of further molecular characterization. It also helps to normalize equivocal data in which both citrate synthase and CPT may be elevated due to increased mitochondrial content. A biochemical diagnosis could be missed in these cases without the ratio relationship. In cases where only a blood specimen is provided for mutation analysis, physicians must rely on supportive clinical information to justify the rationale for analysis since plasma acylcarnitine levels in adults may be normal. Genomic DNA was isolated from whole blood collected in EDTA or from frozen skeletal muscle biopsies with the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN).

Assay of enzymatic activity CPT II activity was assayed in extracts of frozen skeletal muscle tissue by measuring the conversion of [C14]palmitoyl-CoA to [C14]palmitoylcarnitine by the method of Norum [17]. In most cases, citrate synthase levels were also measured as a control for mitochondrial content [16] by the method of Srere [18]. Additional assays of CPT I and II activities were quantiWed in Wbroblasts or muscle homogenates by the method of McGarry et al. [8].

Allele-speciWc assays for common CPT2 mutations Six of the relatively common disease-causing CPT2 mutations, c.149C > A (p.Pro50His), c.338C > T (p.Ser113Leu), c.1238_1239delAG (p.Gln413fs), c.1507C > T (p.Arg503Cys), c.1646G > A (p.Gly549Asp), and c.1891C > T (p.Arg631Cys), were assayed by PCR ampliWcation of genomic DNA with allele-speciWc primers followed by resolution of products by polyacrylamide gel electrophoresis. The ampliWcation primers are described in the Supplementary material section.

DNA sequencing Each of the Wve CPT2 exons and immediate Xanking regions were PCR ampliWed with primers shown in Table S1 (Supplementary material). PCR reactions contained 100 ng genomic DNA, 1£ AmpliTaq Gold PCR buVer, 2.5 mM MgCl2, 0.25 mM each dNTP, 5% glycerol, 0.2 M forward and reverse primers, and 2 U AmpliTaq Gold (ABI) in 100 l total volume. Following denaturation for 5 min at 96 °C, 40 cycles of 30 s at 96 °C denaturation, 30 s at 62 °C annealing, and 1 min at 72 °C extensions, followed by a 10 min extension at 72 °C were performed with a PTC-200 thermal cycler (MJ Research, Waltham, MA). PCR products were puriWed with QIAquick columns (Qiagen, Valencia, CA) and sequenced with the primers used for ampliWcation. PCR products containing exons 1, 4, and 5,

325

which were greater than 1 kb, were also sequenced with internal primers. All identiWed mutations were conWrmed by DNA sequence of both strands. Sequencing was performed with the Big Dye Terminator v3.1 Cycle Sequencing kit (ABI) on an ABI 3100 Genetic Analyzer by the Roswell Park Cancer Institute Biopolymer Facility (BuValo, NY).

Results and discussion Mutation analysis of muscle DNA from patients with CPT II activity and ratios of CPT to citrate synthase diagnostic for CPT II deWciency [16] revealed 16 of 23 patients (70%) had 2 mutations in the CPT2 gene among 6 evaluated routinely in our laboratory by allele-speciWc ampliWcation: c.149C >A (p.Pro50His), c.338C>T (p.Ser113Leu), c.1238_1239delAG (p.Gln413fs), c.1507C>T (p.Arg503Cys), c.1646G >A (p.Gly549Asp), and c.1891C>T (p.Arg631Cys). DNA from the remaining 7 muscle specimens was sequenced for the identiWcation of additional mutations. Five novel mutations were identiWed increasing the diagnostic yield to 21 of 23 (91%) muscle DNA samples. Among 26 muscle biopsies with CPT activity in the carrier range as well as CPT to citrate synthase ratios predictive of carriers, 8 (31%) specimens had mutations in the CPT2 gene including 5 novel mutations. No additional mutations were found among muscle specimens with ratios of CPT to citrate synthase >3.2. Two of the 6 routinely assayed mutations in the CPT2 gene were found in 14 of 26 (54%) leukocyte DNAs. Sequence analysis revealed an additional 6 mutations increasing the diagnostic yield among leukocyte DNAs to 20 of 26 (77%). Among the 6 cultured Wbroblast DNAs analyzed, 2 were homozygotes (p.[Ser113Leu]+ [Ser113Leu]), 2 were compound heterozygotes (p.[Ser113Leu] +[Gly13fs] and p.[Tyr120Cys] +[Leu178_Il e186delinsPhe]), and 2 contained no mutations in the CPT2 gene coding regions. Of the 50 patients with two mutations, 20 (40%) were homozygous for the common c.338C> T (p.Ser113Leu) mutation and 10 (20%) were compound heterozygotes for the p.Ser113Leu and p.Gln413fs mutations. The p.Ser113Leu mutation accounted for 66% of the mutant alleles identiWed in all 50 patients while the p.Gln413fs mutation, only found in compound heterozygosity with the p.Ser113Leu mutation, accounted for 10% of mutant alleles. Among all variant alleles identiWed among individuals with only one mutation, the p.Ser113Leu mutation accounted for 33%. None of the patients in this study were related, however, seven families were examined with various combinations of CPT2 gene mutations including p.Ser113Leu, p.Arg124X, p.Gln413fs, p.Lys457X, p.Thr482fs, p.Gly549Asp, p.Arg631Cys, and in all cases, transmission of these mutations followed Mendelian inheritance. Two of the DNA samples evaluated were from two healthy newborn patients in which tandem mass spectrometry from blood spots had identiWed elevated levels of long chain acylcarnitines. One patient was heterozygous for p.Ser113Leu and p.Lys457X mutations and the other was heterozygous for p.Ser113Leu and p.His523fs mutations (Table 1). The p.Lys457X and p.His523fs mutations have not been previously identiWed and would lead to the production of truncated, inactive forms of CPT II. Because

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Fig. 1. Comparison of hCPT2 (Genbank accession number NM_000098), hCPT1A (NM_001876), hCROT (NM_021151) and hCrAT (NM_000755) amino acid sequences using the T-CoVee multiple sequence alignment program http://www.ch.embnet.org/software/TCoVee.html. Amino acid residues identiWed with substitution mutations are indicated with arrow symbols. Underlined residues of the CPT II sequence indicate deleted residues in the p.Leu178_Ile186delinsPhe and p.Glu545del mutations. -helical (H) and -strand (E) regions are numbered based on the crystal structure of rat CPT II [10].

of the presence of the p.Ser113Leu mutation in both cases, these patients will be at risk for developing the adult onset form of CPT II deWciency. Eight novel sequence mutations were identiWed in this study that are predicted to result in amino acid substitution/deletions; c.250T > C (p.Cys84Arg), c.302C > T (p.Ala101Val), c.691A > T (p.Arg231Trp), c.1438G > A (p.Gly480Arg), c.1511C > T (p.Pro504Leu), c.1634_1636delAAG (p.Glu545del), c.1679G > A (p.Arg560Gln), and c.1763C > G (p.Ser588Cys) (Fig. 2A). This brings the total number of amino acid substitution/deletion alterations identiWed to 43, including the 2 common non-disease-causing polymorphisms p.Val368Ile and p.Met647Val [4,5,19– 32]. The p.Gly480Arg and p.Ser588Cys mutations aVect residues that are conserved between all members of the carnitine acyltransferase family (Fig. 1). Eight new mutations were identiWed in this study that are predicted to result in

truncated forms of CPT II; c.38delG (p.Gly13fs), c.36_43dupGGGCCCCG (p.Ala15fs), c.256_257delAG (p.Ser86fs), c.1273_1274delAC (p.Thr425fs), c.1369A > T (p.Lys457X), c.1444_1447delACAG (p.Thr482fs), c.1569_1570delCA (p.His523fs), and c.1816_1817delGT (p.Val606fs). A total of 17 mutations within the coding regions of CPT2 predicted to result in truncated protein products have been identiWed [20,22,26,27,33–35]. Two additional mutations aVecting the splice sites near exons 2 (c.234-1G > A) and 3 (c.340+5G > A), resulting in mRNA degradation or alternatively spliced mRNA have also been reported [36,37] and this results in a total of 60 potentially disease-causing mutations currently identiWed in the CPT2 gene. This includes the p.Phe352Cys variation that we have identiWed as the sole sequence variant in three cases with myopathic symptoms and clearly reduced residual skeletal muscle CPT II activity. By our criteria, this would qualify

P.J. Isackson et al. / Molecular Genetics and Metabolism 89 (2006) 323–331

327

Fig. 1 (continued)

as a disease-causing mutation, as it was originally reported [19]. However, studies in the Japanese population revealed that this alteration is present at the carrier level in 20% of that population [19] and recombinant expression of CPT II containing the p.Phe352Cys alteration resulted in an enzyme with 70% wild-type activity [19]. This may be further evidence of contributing genetic factors and suggests that Japanese carriers may have other compensating genetic polymorphisms. Two sequence variants were identiWed in this study that may inXuence mRNA stability or splicing, c.930C > T and c.1645+5G > A. The eVects of these alterations have not been examined at the mRNA level. However, one of these, c.930C > T (patient 66, Table 1), results in a silent change at amino acid residue Gly310 that has not been found in any other patient examined. Silent sequence variations have been found to result in altered mRNA expression in other genes [38] and the CPT/CS activity ratio of 2.3 of patient 66 is consistent with one disease-causing sequence mutation. The variant in intron 4, c.1645+5G > A (patient 57), was found in two brothers with myopathic symptoms. This sequence alteration has also not been identiWed in any other patients and is identical in nature to the sequence alteration reported by Deschauer et al. [37], c.340+5G > A in intron 3, which results in a splicing defect. It is also noteworthy that identiWed sequence variations that are predicted to cause amino acid substitutions in the relatively common diseasecausing mutations, p.Pro50His, p.Ser113Leu and p.Gly549Asp, are the result of nucleotide base changes within 4 bp of splicing sites. Although these mutations have been extensively examined and discussed in terms of possible resultant protein structural changes, their potential

inXuence on mRNA expression levels and splicing Wdelity has not been carefully examined. Of the 43 sequence alterations that are predicted to result in amino acid substitutions or deletions, 37 (86%) alter residues that are absolutely conserved in an interspecies comparison of CPT II amino acid sequences including human, rat, mouse, dog, chicken, zebraWsh, and puVerWsh. The two common non-disease-causing polymorphisms, (p.Val368Ile, p.Met647Val) [5], as well as the mutations, p.Cys84Arg, p.Ser113Leu, p.Gly480Arg, and p.Ile502Thr, do not aVect residues that are absolutely conserved in the interspecies comparison. Thirteen of the 60 (22%) diseasecausing CPT2 mutations have been identiWed more than once in unrelated individuals, c.113_114dupGC (p.Ser38fs) [20] (this study), c.149C > A (p.Pro50His) [21,22], c.338C > T (p.Ser113Leu) [5], c.359A > G (p.Tyr120Cys) [20] (this study), c.370C > T (p.Arg124X) [33] (this study), c.534_558delinsT (p.Leu178_Ile186delinsPhe) [1,23] (this study), c.1055T > G (p.Phe352Cys) [19], c.1238_1239delAG (p.Gln413fs) [22,39], c.1507C > T (p.Arg503Cys) [22,40], c.1646G > A (p.Gly549Asp) [22] (this study), c.1810C > T (p.Pro604Ser) [23] (this study), c.1883A > C (Tyr628Ser) [24,41], and c.1891C > T (p.Arg631Cys) [25] (this study). These sequence alterations should be included in an expanded mutation screen for diagnostic purposes. No mutations were identiWed within the coding regions of CPT2 or immediate Xanking regions in 37 patients with signiWcant partial CPT II deWciencies and clinical symptoms of muscle pain and exercise intolerance. In particular, no mutations were found in 2 patients with CPT II/CS activity ratios of 0.8 and 1.0, usually suggestive of homozygosity, or in an additional 28 patients with ratios between

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1.8 and 3.0, indicative of carrier status [16]. It is possible that sequence variations exist in these patients within intronic regions or transcriptional regulatory elements that were not examined in this sequence study. Sequence variations within genes encoding regulatory proteins inXuencing the transcription of CPT2 or the activity of the enzyme could also be responsible. The common polymorphic variants, p.Val368Ile and p.Met647Val, have been suggested to have possible roles in altering CPT II activity under certain conditions or in combination with other disease-causing mutations [5]. However, the residual enzymatic levels reported for these common variants are not signiWcantly diVerent from wild-type to reasonably be expected to result in disease symptoms. There was no signiWcant over-representation of these common polymorphisms in our group of manifesting patients with no disease-causing mutations, 62% p.Val368Ile and 28% p.Met647Val compared to 51% p.Val368Ile and 25% p.Met647Val reported in the general population [21]. The fact that symptomatic CPT II deWcient patients and presumed carriers with no detectable disease-causing mutations exist, suggests that CPT II activity is down-regulated at either the RNA or protein level and is likely to be regulated at both levels. In rat skeletal muscle, CPT II mRNA levels have been found to increase Wvefold following fasting [42]. Peroxisome proliferator activated receptor (PPAR) agonists up-regulate CPT II mRNA expression and CPT II activity in cultured cells [43]. Regulation of CPT II activity by post-translational modiWcation or regulatory interactions with other proteins has not been examined. However, there are several reasons to expect that CPT II is regulated at the protein level. The human CPT II amino acid sequence contains consensus sequences for three N-glycosylation sites, one glycosaminoglycan site, one cAMP phosphorylation site, eleven protein kinase C phosphorylation sites, six casein kinase phosphorylation sites, and six Nmyristoylation sites. The activity of the homologous CPT IA has been shown to be regulated by phosphorylation at a casein kinase II site [44]. The homologous enzyme, ChAT, is regulated at the transcriptional level and by post-translational phosphorylation by protein kinase C and calcium/ calmodulin-dependent protein kinase [45]. Attempts to make genotype/phenotype correlations between diVerent disease-causing sequence mutations have been diYcult. Analysis of the CPT2 gene containing various mutations transfected into COS-1 cells has indicated that the p.Phe352Cys mutation reduces enzyme activity levels to 70% of wild-type [19], the p.Ser113Leu alteration reduces activity to 34% of wild-type [5], and more severe mutations result in activities 5-10% of wild-type [19]. From these results, there seems to be a correlation between the enzyme activities observed with individual mutations and the severity of the clinical manifestations. However, no signiWcant correlations were apparent between speciWc mutations and residual enzyme activities observed in muscle biopsy tissue from patients with the adult form of CPT II deWciency (Table 1). Among the adult patients examined,

the presence of two of any of the identiWed disease-causing mutations resulted in a CPT/CS ratio of 0.78 § 0.35 and one allelic variation resulted in a CPT/CS ratio of 2.45 § 0.36, consistent with previous results [16]. Interestingly, the mean ratio among homozygotes for the reportedly mild S113L was 0.92 § 0.44. A single patient who was a compound heterozygote for 2 mild mutations (P50H and S113L) also had a higher CPT/CS ratio (1.2). The ratio was lower (0.68 § 0.18) in patients who were compound heterozygotes for the S113L and the more severe Gln413fs mutation. In fact, patients with one of two mutant alleles representing a frameshift, deletion or truncation mutation other than Gln413fs also had overall lower ratios (0.58 § 0.16) than that of the S113L homozygotes. The one factor that clearly distinguishes between the diVerent mutations in deWning the severity of their eVect is whether homozygotes manifest as severe infantile or the milder, adult onset form of the disease. Five mild mutations, p.Pro50His, p.Ser113Leu, p.Arg161Trp, p.Glu174Lys, and p.Ile502Thr, have been identiWed in the homozygous state in patients with the adult onset form of the disease [4]. Seven mutations have been associated with the infantile/ prenatal presentation of the CPT II deWciency disorder in the homozygous state, p.Arg151Gln, p.Pro227Leu, p.Asp328Gly, p.Arg382Lys, p.Phe383Tyr, p.Gln413fs, and p.Tyr628Ser [4]. The remaining mutations have not been identiWed in homozygotes. The 18 known truncation mutations would be expected to produce completely inactive products and to result in an infantile hepatocardiopathic or the lethal infantile presentation. Examinations of disease-causing mutations in CPT IA [46] and ChAT [45] have found mutations spread throughout the enzymes that either have direct eVects on the catalytic site or substrate binding sites or are more distant from the active site and appear to inXuence activity by structural perturbations or improper folding. Most of the amino acid substitution mutations in CPT I that have been examined in recombinant expression systems have resulted in reduced solubility due to improper folding [46]. This may also be the case with many of these mutations present in the CPT2 gene. The reported thermolability of CPT II containing mutations p.Ser113Leu [47] and p.Phe352Cys [48] suggest that these mutations have structural destabilizing eVects. It can be seen from the location of aVected amino acids in the structure of rat CPT II (Fig. 2A) that the identiWed amino acid substitution mutations are spread throughout the molecule as found with the homologous enzymes, CPT IA [46] and ChAT [45], as well as disease-causing mutations in other fatty acid oxidation enzymes such as very long chain acyl-CoA dehydrogenase [49]. Analyses of the crystal structures of CPT II [9,10] have identiWed a hydrophobic segment unique to CPT II relative to other members of the carnitine acyltransferase family from residues 176–206 that contains two helices (6⬘ and 6⬙). This segment may play a role in regulatory structure–function relationships unique to CPT II and has been proposed to interact with the inner mitochondrial membrane [9,10]. A deletion mutation has

P.J. Isackson et al. / Molecular Genetics and Metabolism 89 (2006) 323–331

Fig. 2. Location of amino acid substitution mutations in the structure of rat CPT II. (A) Location of the eight amino acid substitution mutations identiWed in this study. (B) The N-terminal hydrophobic segment unique to CPT II of amino acid residues 176–206. Residues of the mutation p.Leu178_Ile186delinsPhe are blue and the remainder of the segment is red. (C) Location of Tyr120 (blue) within the active site of CPT II (residues Tyr116, Asp372, His376, and Arg498 of the active site are red). Figures were prepared with PyMOL (http://www.pymol.org) based on the crystal structure of rat CPT II kindly provided by Dr. Liang Tong [10].

been identiWed within this segment, p.Leu178_Ile186delinsPhe, by Yang et al. [23], Sigauke et al. [1] and in two unrelated patients in this study. The deleted amino acids predicted to result from this mutation are located in a loop region between 1⬘ and 6⬘ (Fig. 2B) and would clearly be expected to inXuence membrane or protein interactions of this hydrophobic region. Two disease-causing mutations in CPT2 that are likely to directly aVect carnitine binding are the p.Tyr120Cys [20] (this study) and p.Ser588Cys (this study) mutations. The Tyr120 residue is absolutely conserved among the

329

carnitine acyltransferases (Fig. 1) and has been found in crystal structures of CrAT [12,13], CrOT [14], and CPT II [9,10] to form a hydrogen bond network along with Tyr116, Asp376, Arg498 and a tightly associated water molecule in the carnitine binding pocket (Fig. 2C). The p.Ser588Cys alteration aVects the Wrst serine residue of the STS motif that is absolutely conserved among members of the carnitine acyltransferase family. Possible roles of these residues in catalysis have been previously examined by mutagenesis studies due to the fact that they are the only conserved serine residues and speculation that a serine residue could participate in catalysis [50]. Mutation of these residues did not support a direct role in catalysis, however they do appear to be important for carnitine binding and transition state stabilization. Mutation of Ser588 to alanine in bovine CrOT increased the KM for carnitine 15-fold and had no eVect on kcat, suggesting that Ser588 is involved in carnitine binding [50]. This study identiWed 15 cases of symptomatic CPT II deWcient patients with one disease-causing mutation. CPT II deWciency is generally considered to be an autosomal recessive disease, however, cases of symptomatic carriers are well known [5,22,27]. The possibility of dominant-negative mutations has been suggested as one explanation for the existence of symptomatic carriers [22,27]. The possibility that certain mutant forms of CPT II are capable of forming complexes with wild-type subunits and negatively inXuence overall CPT II activity relies on the reported homotetrameric structure of CPT II [51,52]. However, the existence of a homotetrameric complex is not well established [6]. DiVerent molecular weight complexes have been observed with diVerent detergents [53]. If CPT II exists as a homotetramer, it would diVer in this respect from other members of the family, CrAT, CrOT, and ChAT, which exist as monomers based on gel Wltration and crystallographic evidence [13,14,45]. In the recent expression and crystallographic studies of rat CPT II [9,10], the enzyme expressed in Escherichia coli crystallized in both studies as an asymmetric dimer without strong interchain interactions suggesting a monomeric structure. Recombinant CPT II behaved as a monomer in gel Wltration and analytical centrifugation experiments performed by Rufer et al. [9]. However, Hsiao et al. [10], who did not use a detergent during the puriWcation, reported evidence of oligomers (hexamer or octamers) of recombinant CPT II. The inXuence of a multimeric structure on CPT II activity has not been investigated. However, analysis of the mutations detected in symptomatic carriers in this study is indeed supportive of the possibility of dominant-negative mutations in that all of the identiWed mutations in manifesting carriers, p.Pro50His, p.Ala101Val, p.Ser113Leu, p.Phe352Cys, p.Gly480Arg, p.Gly549Asp, p.Arg560Gln, and p.Ser588Cys, are predicted to cause amino acid substitutions that are likely to be capable of folding properly and forming multimeric complexes. In particular, it is noteworthy that 19/100 disease-causing mutations found in the 50 homozygous or compound heterozygous

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patients in this study were truncation mutations, whereas no truncation mutations were found in the 15 manifesting carriers, consistent with the possibility that truncations or severely mis-folded proteins are incapable of forming complexes and therefore incapable of inXuencing wildtype CPT II produced from the other allele through multimeric interactions. Acknowledgments This work was supported by grants from the Muscular Dystrophy Association (G.D.V.), the John R. Oishei Foundation (G.D.V.) and The Children’s Guild of BuValo. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ymgme. 2006.08.004. References [1] E. Sigauke, D. Rakheja, K. Kitson, M.J. Bennett, Carnitine palmitoyltransferase II deWciency: a clinical, biochemical, and molecular review, Lab. Invest. 83 (2003) 1543–1554. [2] S. DiMauro, P.M.M. DiMauro, Muscle carnitine palmitoyltransferase deWciency and myoglobinuria, Science 182 (1973) 929–931. [3] F. Taroni, G. Uziel, Fatty acid mitochondrial beta-oxidation and hypoglycaemia in children, Curr. Opin. Neurol. 9 (1996) 348–354. [4] L. Thuillier, H. Rostane, V. Droin, F. Demaugre, M. Brivet, N. Kadhom, C. Prip-Buus, S. Gobin, J.M. Saudubray, J.P. Bonnefont, Correlation between genotype, metabolic data, and clinical presentation in carnitine palmitoyltransferase 2 (CPT2) deWciency, Hum. Mutat. 21 (2003) 493–501. [5] F. Taroni, E. Verderio, F. Dworzak, P.J. Willems, P. Cavadini, S. DiDonato, IdentiWcation of a common mutation in the carnitine palmitoyltransferase II gene in familial recurrent myoglobinuria patients, Nat. Genet. 4 (1993) 314–320. [6] R.R. Ramsey, R.D. Gandour, F.R. van der Leij, Molecular enzymology of carnitine transfer and transport, Biochim. Biophys. Acta 1546 (2001) 21–43. [7] L.L. Bieber, Carnitine, Annu. Rev. Biochem. 57 (1988) 261–283. [8] J.D. McGarry, K.F. Woeltje, M. Kuwajima, D.W. Foster, The regulation of ketogenesis and the renaissance of carnitine palmitoyltransferase, Diabetes Metab. Rev. 5 (1989) 271–284. [9] A.C. Rufer, R. Thoma, J. Benz, M. Stihle, B. Gsell, E. De Roo, D.W. Banner, F. Mueller, O. Chomienne, M. Hennig, The crystal structure of carnitine palmitoyltransferase 2 and implications for diabetes treatment, Structure 14 (2006) 713–723. [10] Y.-S. Hsiao, G. Jogl, L. Tong, Crystal structure of rat carnitine palmitoyltransferase II (CPT-II), Biochem. Biophys. Res. Commun. 346 (2006) 974–980. [11] D. Wu, L. Govindasamy, W. Lian, Y. Gu, T. Kukar, M. Agbandje-McKenna, R. McKenna, Structure of human carnitine acetyltransferase. Molecular basis for fatty acyl transfer, J. Biol. Chem. 278 (2003) 13159–13165. [12] L. Govindasamy, T. Kukar, W. Lian, B. Pedersen, Y. Gu, M. Agbandje-McKenna, S. Jin, R. McKenna, D. Wu, Structural and mutational characterization of L-carnitine binding to human carnitine acetyltransferase, J. Struct. Biol. 146 (2004) 416–424. [13] G. Jogl, L. Tong, Crystal structure of carnitine acetyltransferase and implications for the catalytic mechanism and fatty acid transport, Cell 112 (2003) 113–122. [14] G. Jogl, Y.-S. Hsiao, L. Tong, Crystal structure of mouse carnitine octanoyltranferase and molecular determinants of substrate selectivity, J. Biol. Chem. 280 (2005) 738–744.

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