Novel c.2216T > C (p.I739T) Mutationin Exon 13 and c.1481T > A (p.L494X)
Mutation in Exon 8 of MUT Gene in aFemale with Methylmalonic Acidemia
George Imataka, Osamu Sakamoto,
Hideo Yamanouchi, Shigemi Yoshihara,
Yuki Omura-Hasegawa, Go Tajima &Osamu Arisaka
Cell Biochemistry and Biophysics
ISSN 1085-9195
Cell Biochem Biophys
DOI 10.1007/s12013-013-9532-9

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Cell Biochem Biophys
DOI 10.1007/s12013-013-9532-9

TRANSLATIONAL BIOMEDICAL RESEARCH

Novel c.2216T > C (p.I739T) Mutation in Exon 13
and c.1481T > A (p.L494X) Mutation in Exon 8 of MUT Genein a Female with Methylmalonic Acidemia
George Imataka • Osamu Sakamoto • Hideo Yamanouchi •Shigemi Yoshihara • Yuki Omura-Hasegawa •Go Tajima • Osamu ArisakaÓ The Author(s) 2013. This article is published with open access at Springerlink.com

Abstract We report herein a 1.5-year-old girl withmethylmalonic acidemia (MMA) in whom two missense
mutations were found: a novel I739T mutation located inexon 13 and the L494X mutation in exon 8. The results oforganic acid test showed a pronounced increase in methylmalonate excretion with increased methylcitrate and3-OH-propionate excretion, leading to a diagnosis ofMMA, and Vitamin B12 administration was started.
Analysis of the mut gene confirmed a T-to-A substitution atnucleotide position 1481 in exon 8 and a T-to-C substitution at nucleotide position 2216 in exon 13, leading to theamino acid isoleucine at position 739 being changed tothreonine, resulting in c.2216T [ C (p.I739T). The patient

G. Imataka (&)  S. Yoshihara  O. Arisaka
Department of Pediatrics, School of Medicine, Dokkyo MedicalUniversity, 880 Kitakobayashi, Mibu, Shimotsuga,Tochigi 321-0293, Japane-mail: geo@dokkyomed.ac.jp
O. Sakamoto
Department of Pediatrics, Tohoku University Schoolof Medicine, Sendai, Miyagi, JapanH. Yamanouchi
Department of Pediatrics, Saitama Medical University,Saitama, JapanY. Omura-Hasegawa
Department of Pediatrics, Shimane University Faculty ofMedicine, Izumo, Shimane, Japan
G. Tajima
Department of Pediatrics, Graduate School of Biomedical &Health Sciences, Hiroshima University, Hiroshima, Japan

has now been on high-dose oral administration of Vitamin
B12 and carnitine therapy (900 mg of levocarnitine chloride) for 5 years without experiencing further attacks, andher cognitive and motor development is normal. Furthertests on residual enzyme activity, as well as experience
with more cases, may shed light on the relationshipbetween gene mutations and phenotypes in MMA.
Keywords Methylmalonic acidemia L-Methylmalonyl-CoA mutase  Vitamin B12Introduction
Methylmalonic acidemia (MMA; MIM# 251000) is an
autosomal recessively inherited disorder in which a defectin methylmalonyl CoA mutase (MCM, EC5.4.99.2) resultsin the build-up of methylmalonic acid and other organic
acids in the body. MMA is categorized as either apoenzyme deficiency (mut) type or coenzyme B12 metabolicdisorder (Cbl) type. The mut type is subdivided into mut0,
which results from complete deficiency, and mut-, which
results from partial deficiency. The mut0 type, which ischaracterized by apoenzyme activity of not more than
0.1 %, often presents with metabolic acidosis within
1 week after birth, frequently resulting in death in earlychildhood. The mut- type presents with repeated attacks of
ketoacidosis triggered by infection after the age of 1 year.
MCM is encoded by a single gene, mut, which is located on
6p21. Mut consists of 13 exons [1–3]. We herein report acase of a girl with MMA in which two missense mutations
were found: a novel I739T mutation located in the mostC-terminal exon 13 ever reported and the L494X mutation
located in exon 8, which is relatively common in Japanesepatients.

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Patient Report
A healthy 1.5-year-old girl went to sleep one night aftervomiting. In the morning, the parents found the girlunconscious in bed and took her to the emergency room.
The girl was 76.3-cm tall and weighed 8.4 kg with a bodytemperature of 35.2 °C and blood pressure of 77/47 mmHg.
She did not open her eyes in response to speech, and theresults of simple blood glucose test were below thedetectable limit. She regained consciousness following
administration of 20 % blood glucose, but experienced
tonic convulsions and rolling eyes for several minutes. The
convulsions were controlled with phenobarbital. Intravenous fluids were started. The patient was lucid after 3 h, andher motor functions improved after 6 h to the point where
she could walk on her own. Blood gas analysis at the time
the patient was hospitalized showed pH 7.240, pCO2
41.5 mmHg, base excess -9.2, and HCO3 -17.2 mmol,indicating metabolic acidosis. Other abnormal results werelactic acid, 35 mg/dL; NH3, 118 lg/dL; AST, 143 U/L; andALT, 46 U/L. Urine specific gravity was 1.04 with a urine
ketone level of 3?.
Organic acid test was performed on suspicion of congenital metabolic anomaly. The results showed a pronounced increase in methylmalonate excretion withincreased methylcitrate and 3-OH-propionate excretion,
leading to a diagnosis of MMA. Because the approximate
urine methylmalonic acid level at onset was 658.4 lM/mmol
creatinine, high-dose oral administration of Vitamin B12(10 mg of cobamamide) was started, resulting in a decrease
to 213.0 lM/mmol creatinine on day 7 of treatment.
HPLC quantification of succinyl-CoA production uponreaction of the patient’s lymphocyte lysates with methylmalonyl-CoA and adenosylcobalamin revealed that thepatient’s methylmalonyl-CoA activity level was 1.4 pmol
succinyl-CoA/min/106 cells, which is approximately 5 %of the level seen in healthy controls (35.9 ± 16.4 pmol
succinyl-CoA/min/106 cells).
The patient has now been on high-dose oral administration of Vitamin B12 and carnitine therapy (900 mg oflevocarnitine chloride) for 5 years. She has not experienced
further attacks; her cognitive and motor development isnormal.

mut Genetic Analysis
Analysis of the mut gene was performed for definitive
analysis of the type of MMA in this patient. A T-to-A
substitution at nucleotide position 1481 in exon 8 of themut gene was found in a heterozygous pattern (Fig. 1a).
A T-to-C substitution at nucleotide position 2216 in exon
13 was also found in a heterozygous pattern. As a result of
this base substitution, the amino acid isoleucine at position
739 was changed to threonine, resulting in c.2216T [ C(p.I739T). The child was therefore definitively diagnosed
with methylmalonic acidemia (mut) (Fig. 1b).

Discussion
In this case of MMA, we found the L494X mutation inexon 8, which is common in Japanese patients, and an
I739T mutation in exon 13, which has never previously
been reported. The patient in this case presented withconvulsions associated with hypoglycemia. The clinicalcourse after hospitalization was relatively mild for MMA.
Blood and urinalysis data suggested that this case of MMA

Fig. 1 a Mutation c.1481T [ A (p.L494X) in exon 8 of MUT gene. b Novel c.2216T [ C (p.I739T) mutation in exon 13 of MUT gene

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was an organic acid metabolic disorder, and the patient wastreated with high doses of Vitamin B12 after more detailed
analysis. Before the start of Vitamin B12 treatment, theapproximate urine methylmalonic acid levels were658.4 lM/mmol creatinine, as compared with 213.0 lM/mmol creatinine after treatment. This case was therefore
originally thought to involve Vitamin B12-reactive MMA.
However, HPLC quantification of succinyl-CoA production upon reaction of the patient’s lymphocyte lysates withmethylmalonyl-CoA and adenosylcobalamin revealed thatthe child’s enzyme activity was approximately 5 % ofnormal, which was not indicative of the B12 reactive type.
We therefore performed mut genetic analysis. Ultimately, anovel I739T mutation in exon 13 and the L494X mutationin exon 8 were found, resulting in a definitive diagnosis of
the mut- type MMA.
Of the 119 mutations of the mut gene reported previously, the novel I739T mutation in exon 13 found in thiscase is the closest to the C terminal [1–5]. The (b,a)5cobalamin (vitamin B12) binding domain occurs in
C-terminal exons 10, 11, 12, and 13 of the mut gene. An
extremely interesting point in this case is that the reaction
shown by urine methylmalonic acid levels following clinical administration of Vitamin B12 was consistent with thelocation of the genetic mutation. This warrants further
analysis, including in vitro tests on the expression ofresidual enzyme activity after the addition of Vitamin B12.
Further experience with more cases may shed light on therelationship between gene mutations and phenotypes inMMA.
Open Access This article is distributed under the terms of theCreative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the originalauthor(s) and the source are credited.

References
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