Identification of a Novel Mutation in Patients with Medium-Chain Acyl

Molecular Genetics and Metabolism 69, 259–262 (2000) doi:10.1006/mgme.2000.2978, available online at http://www.idealibrary.com on

BRIEF COMMUNICATION Identiﬁcation of a Novel Mutation in Patients with Medium-Chain Acyl-CoA Dehydrogenase Deﬁciency

of age. There are few reports of first symptoms after A novel mutation was identified in two unrelated age 4 years. In the present study, we report two patients with medium-chain acyl-CoA dehydrogenase deficiency. First, a 19-year-old Caucasian fe- patients with MCAD deficiency, including an un- male presented with a devastating illness, resulting usual adult patient, and their molecular defects. in sudden death in adulthood which is unusual. The second patient, now a 3.5-year-old male, presented SUBJECTS AND METHODS at 17 months of age with a hypoglycemic seizure and dehydration. Sequence analysis revealed a Patients novel mutation G617T in exon 8 resulting in an arginine to leucine substitution at codon 206 (R206L). Patient 1. The proband, a 19-year-old previously Both patients were compound heterozygous for this healthy Caucasian female, is the daughter of G617T and the common mutation A985G. © 2000 healthy nonconsanguineous parents. She presented Academic Press to a hospital for alteration in consciousness. One day Key Words: MCAD deficiency; mitochondrial ␤-ox- prior to presentation she developed nausea, vomit- idation; mutation. ing, and drowsiness after camping for a day with her friends and drinking alcoholic beverages. She had Mitochondrial ␤-oxidation plays a major role in stable vital signs after IV hydration. On neurologic energy production, especially during fasting periods. examination, she was oriented only to place and Inherited mitochondrial fatty acid oxidation disor- person and did not follow any commands. Cranial ders consist of 17 autosomal recessive defects, nerves were intact. She responded to painful stimu- of which medium-chain acyl-CoA dehydrogenase lation. Deep tendon reflexes were normal. Normal (MCAD) deficiency (MIM 201450) is the most com- laboratory data included glucose, blood alcohol level, mon disorder with a carrier frequency of approxi- and kidney and liver function tests. Her WBC count mately 1:80 in most Caucasian populations (1,2). and serum uric acid levels were mildly elevated. MCAD deficiency is characterized by episodes of ill- Electrolytes revealed mild metabolic acidosis and ness in early childhood associated with fasting re- mild ketonuria. Brain computerized axial tomogra- sembling Reye’s syndrome: coma, hypoglycemia, hy- phy scan was normal. A few hours after admission perammonemia, and fatty liver. A common mutation the patient was found pulseless and unresponsive. A985G has been identified among patients with Resuscitation efforts were unsuccessful. On autopsy, MCAD deficiency, which account for 88.9% of the liver pathology revealed macrovesicular steatosis. mutant alleles (3–7). There is significant phenotypic Plasma acylcarnitine profile showed highly elevated heterogeneity in MCAD deficiency, even within the octanoyl canitine, indicating MCAD deficiency. The same family. The affected children may have only family later volunteered a history of Reye-like at- one episode of illness or multiple recurrences. In tack at 18 months of age. some cases, the first episode can be devastating, Patient 2. The proband, now a 3.5-year-old male, resulting in sudden death. On the other hand, they is the only child of a healthy Caucasian mother and may appear to be asymptomatic (8). Most children an African-American father with apparent Noonan’s present with acute illness between 3 and 15 months syndrome. The patient presented for the first time,

259 1096-7192/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 260 BRIEF COMMUNICATION at 17 months of age, with vomiting and a hypoglycemic seizure. He also had microcephaly and a facial profile similar to his father’s, suggesting dominant transmission of Noonan’s syndrome. Urine organic acids showed increased quantities of both hexanoyl- glycine and suberylglycine and the presence of pheno- barbital. Plasma acylcarnitine profile showed elevated C6, C8, and C10:1 consistent with MCAD deficiency.

Preparation of Genomic DNA Genomic DNA was isolated from postmortem for- malin-fixed liver from patient 1 by standard methods using the Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN) according to the manufacture’s procedure. Genomic DNA was also isolated from peripheral blood obtained from patient 2, the parents of patients, and normal controls. DNA FIG. 1. Genomic DNA sequence analysis. Direct sequencing was prepared by a standard method using the of MCAD gene cloned from a patient with MCAD deficiency (top) and from a normal individual (bottom) was performed in an ABI QIAamp blood kit (Qiagen, Inc. Valencia, CA). 377 automated sequencer. A. The novel G617T mutation. Partial DNA sequence of patient 1 with MCAD deficiency show the over- Sequence Analysis of MCAD lapping black and blue peaks, indicating the presence of a heterozygous, G-and-T nucleotide. The arrows denote nucleotide po- All 12 MCAD exons were amplified from genomic sition 617 in the MCAD cDNA. The DNA sequence of a normal DNA by the use of specific intronic primers, derived control individual is shown on the bottom for comparison. B. The from the 5Ј and 3Ј intronic sequences and AmpliTaq common A985G mutation. Partial DNA sequence of patient 1 (PE Applied Biosystems). The DNA fragments were with MCAD deficiency show the overlapping black and green ␮ peaks, indicating the presence of a heterozygous, A-and-G nucle- amplified using 50 ng of genomic DNA in a 50- l otide. The arrow indicates the position of the A 985-to-G transi- reaction volume consisting of 1ϫ PCR buffer (Per- tion. (Bottom) Normal control. kin–Elmer), 2.5 units of Taq polymerase, 0.25 mM dNTPs, and 20 pmol of each relevant primer. PCR cycling conditions were as follows: after heating the The sense primer was a modified primer in which reactions for 1 min at 94°C, 30 cycles were per- G-613 was artificially substituted with A. The am- formed at 94°C for 15 s, 56°C for 15 s, and 72°C for plified fragments were then digested with an endo- 30 s, followed by a final extension at 72°C for 10 min. nuclease AflIII, subjected to electrophoresis on 12% The corresponding PCR products were purified by polyacrylamide gel, stained with 0.5 mg/ml ethidium agarose gel electrophoresis and QIAEX II gel extrac- bromide, and visualized with UV light. tion kit (Qiagen). Direct sequencing of PCR products was performed with dRhodamine terminator cycle RESULTS sequencing kit (PE Applied Biosystems) on an ABI Mutation Analysis by Sequencing Prism 377 DNA sequencer (PE Applied Biosystems). Patient 1 was a late-onset MCAD deficiency with Restriction-Endonuclease Analysis of Mutations a common mutation A985G that was previously de- tected by multiple primer extension assay, which Genomic DNA was isolated and PCR was carried was developed in our laboratory (data not shown). out in the manner described above. Because the To screen for the second mutation within the MCAD MCAD G617T mutation did not create or eliminate a gene, all 12 exons and their flanking intronic se- restriction site for a suitable endonuclease, PCR am- quences were amplified from genomic DNA. The plification was performed with mismatched primers. PCR products were purified and sequenced. Sense primer: 5Ј TGTATCTCTTAGGTATTTT- Sequence analysis revealedaGtoTsubstitution TTATTGACAC 3Ј; at nucleotide position 617 (Fig. 1A), resulting in an Antisense primer: 5Ј AGGCTTTATTAGCAGGAG- arginine to leucine substitution at codon 206 CTTTA 3Ј. (R206L). BRIEF COMMUNICATION 261

Patient 2 was a 17-month-old male. The exons of consists of two ␤-sheets (13). The two packed the MCAD gene, including their flanking intronic ␤-sheets are encoded by exons 6–8. The mutation of sequences, were amplified and sequenced as de- G617T which changes the polar residue arginine- scribed above. The G617T mutation as well as the 206 to the hydrophobic residue leucine may affect A985G mutation (data not shown) was also identi- the folding of the ␤-sheet domain. This region where fied indicating that patient 2, like patient 1, was a arginine-206 is located is also important for the compound heterozygote. The presence of the G617T FAD–polypeptide interaction. The flavin ring is mutation was identified on one allele from the pa- locked in the crevice between the ␤-domain and the tient’s father. He did not carry the A985G mutation. C-terminal domain of one subunit and the C-termi- DNA from the patient’s mother was not available. nal domain of a neighboring subunit. Fourier map- ping also showed tubular electron densities near the Mutation Test by PCR/Restriction-Endonuclease FAD ring and between the ␤-sheet domain and the Analysis two ␣-helix domains (13,14). Therefore, this region is believed to be important for the electron transfer To confirm the G617T mutation by PCR/restric- from FAD bound to MCAD to ETF. tion–AflIII analysis, a 68-bp fragment was amplified by PCR with mismatched primers. The G617T mu- ACKNOWLEDGMENT tant allele results in the loss of an AflIII restriction site. The amplified fragments were digested with This work was supported in part by the Courtwright–Summers AflIII and then subjected to electrophoresis on 12% Metabolic Disease Fund. polyacrylamide gel. The PCR/restriction enzyme di- gestion results were consistent with the sequencing REFERENCES results (data not shown). The G617T mutation was not identified in any control individuals, suggesting 1. Gregersen N, Winter V, Curtis D, Deufel T, Willems PJ, that it does reflect a pathological variation. Ponzone A, Ding JH, Yang BZ, Iafolla AK, Chen YT, Roe CR, Kolvrra S, Scheiderman K, Andresen BS, Bross P, Bolund L. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: DISCUSSION The prevalent mutation G985 (K304E) is subject to a strong founder effect from northwestern Europe. Hum Hered 43: MCAD is one of the most frequently inherited 342–350, 1993. metabolic disorders among Caucasians. In most 2. Tanaka K, Gregersen N, Ribes A, Kim J, Kolvraa S, Winter cases, acute illness presents between 3 and 15 V, Eiberg H, Martinez G, Deufel T, Leifert B, Santer R, months of age. Once the diagnosis is established, the Francois B, Pronicka E, Laszlo A, Kmoch S, Kremensky I, Kalaydjicva L, Ozalp I, Ito M. A survey of the newborn treatment and appropriate management are effec- populations in Belgium, Germany, Poland, Czech Republic, tive. In contrast, without diagnosis, most children Hungary, Bulgaria, Spain, Turkey, and Japan for the G985 with MCAD deficiency are at significant risk of sud- variant allele with haplotype analysis at the medium chain den death with either the initial or a later episode. Acyl-CoA dehydrogenase gene locus: Clinical and evolution- MCAD deficiency is found almost exclusively among ary consideration. Pediatr Res 41:201–209, 1997. Caucasians, particularly those of Northwestern Eu- 3. Matsubara Y, Narisawa K, Miyabayashi S, Tada K, Coates PM. Molecular lesion in patients with medium-chain acyl- ropean origin (9–12). There are isolated cases of CoA dehydrogenase deficiency. Lancet 335:1589, 1990. Southern European and North African origin. Inter- 4. Matsubara Y, Narisawa K, Miyabayashi S, Tada K, Coates estingly, the father of patient 2, who is a G617T PM, Bachmann C, Elsas LJ 2d, Pollitt RJ, Rhead WJ, Roe carrier, is African-American. In addition this is the CR. Identification of a common mutation in patients with first reported mutation located in exon 8, which is medium-chain acyl-CoA dehydrogenase deficiency. Biochem the region believed to be important for FAD– Biophys Res Commun 171:498–505, 1990. polypeptide interaction and for the electron transfer 5. Ding JH, Roe CR, Iafolla AK, Chen YT. Medium-chain acyl- from FAD bound to MCAD to electron transferring coenzyme A dehydrogenase deficiency and sudden infant death. N Engl J Med 325:61–62, 1991. flavoprotein (ETF) (13). MCAD is a homotetrameric enzyme located in the 6. Tanaka K, Yokota I, Coates PM, Strauss AW, Kelly DP, Zhang Z, Gregersen N, Andresen BS, Matsubara Y, Curtis mitochondrial matrix. The MCAD monomer is com- D, et al.. Mutations in the medium chain acyl-CoA dehydro- posed of three domains of approximately equal size. genase (MCAD) gene. Hum Mutat 1:271–279, 1992. The N-terminal and the C-terminal domains are 7. Workshop on Molecular Aspects of MCAD Deficiency. Mu- ␣-helices packed together and the middle domain tations causing medium-chain acyl-CoA dehydrogenase de- 262 BRIEF COMMUNICATION

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