Genetic analysis of three genes causing isolated methylmalonic acidemia: identification of 21 novel allelic variants

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Abstract

Isolated methylmalonic aciduria (MMA) is an inborn error of metabolism due to the impaired isomerization of l-methylmalonyl-CoA to succinyl-CoA. This reaction is catalyzed by the mitochondrial protein methylmalonyl-CoA mutase (MCM, EC 5.4.99.2), an adenosylcobalamin-dependent enzyme. Four different forms of isolated MMA have been described: mut MMA associated with defects in the MCM apoenzyme, and phenotypically divided into two subtypes mut and mut0 MMA, and three different defects involved in the synthesis of the active form of the cofactor adenosylcobalamin, termed cbl MMA, and classified into three different complementation groups cblA, cblB, and cblH associated with defects in the MMAA and MMAB genes and with an unidentified protein, respectively. In this work we describe the genetic analysis of 25 MMA patients, mainly from Spain. Using biochemical and cellular approaches our patients have been classified, identifying 13 mut MMA, 7 cblA, 2 cblB, and 3 noncblA, noncblB deficient patients. cDNA and genomic DNA sequence analysis of the MUT, MMAA, and MMAB genes have allowed us to identify 27 different changes, 21 novel ones. Among the missense mutations identified in the MUT gene only one, the c.970G > A (p.A324T) variant located in the substrate binding domain is likely a mut mutation. The remaining missense mutations c.326A > G (p.Q109R), c.983T > C (p.L328P), c.1846C > T (p.R616C), and c.1850T > G (p.L617R) are probably mut0. In the MMAA patients analyzed, frameshift mutations are prevalent. We have explored the genotype–phenotype correlation for this clinically heterogeneous disease.

Introduction

Methylmalonic aciduria (MMA) is an inborn error of metabolism due to the impaired isomerization of l-methymalonyl-CoA to succinyl CoA during the oxidation of propionate towards the Krebs cycle. The intramolecular rearrangement is catalyzed by a cobalamin-dependent mitochondrial enzyme named l-methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) using adenosylcobalamin (Adocbl) as cofactor. In mammals the enzyme is required for the degradation of odd-chain fatty acids, the amino acids valine, isoleucine, methionine, threonine, and cholesterol. Biochemical and somatic cell studies have delineated four different forms associated with isolated MMA deficiency. Disorders associated with defects in the MCM apoenzyme are designed mut MMA (OMIM 251000) and the defects in enzymes required for mitochondrial synthesis of the active form of Adocbl are termed cbl MMA. Three different complementation groups, namely cblA (OMIM 607,481), cblB (OMIM 607,568), and cblH (OMIM 606,169) have been identified, in all of them the synthesis of AdoCbl is blocked. Patients with mut MMA have been divided into two subtypes, mut0, with no MCM activity in fibroblasts and mutpatients with MCM residual activity stimulated by high levels of cobalamin in the culture medium. Several mut0 cells lines have low levels of mutant mRNA, including those with missense mutations [1], [2]. The enzyme from mut patients shows an increase in the apparent Km for AdoCbl of 100–1000 times compared to normal [3], [4].

The mature MCM is a homodimer of 718 amino acids, member of a small family of enzymes that use AdoCbl as cofactor. In humans, MCM is encoded by a single gene (MUT) mapped to chromosome 6p21 [5], which consists of 13 exons spread over 35 kb [6] and which expresses a 2.7 kb mRNA transcript [7]. The comparison of different B12-dependent enzymes from prokaryotic as well as eukaryotic organisms reveals the existence of three different domains [8], [9]. The flavodoxin-like carboxy-terminal domain of the enzyme (residues 578–750) comprises the cobalamin-binding domain, including the invariant histidine 627 residue conserved in all studied cobalamin-utilizing enzymes [10]. A second important region is an (α/β)8 barrel (residues 191–416) to which the substrate methylmalonyl-CoA is bound. The amino-terminus domain shows less homology with other enzymes and may play a role in the dimerization of the human enzyme. Moreover, a marked similarity exists between human MCM and the α-subunit of homologous MCM from the prokaryote Propionibacterium shermanii, with 65% identity between the two proteins. This protein has been crystallized and can be used as three-dimensional model of the human MCM protein [9], [11]. Availability of this model provides insight on the structural and functional effects of most of the MCM mutations [12].

Recently, the MMAA and MMAB genes (OMIM 607481 and 607568) responsible for the cblA and cblB disorders, respectively, have been identified [13], [14]. The seven exons of the MMAA gene are located on chromosome 4q31.1–2 and encode a predicted protein of 418 amino acids. Two different functions for the MMAA gene product have been proposed, based on the annotation of homologous prokaryotic proteins, a role in the transport of vitamin B12 into the mitochondria [14] and the involvement in the protection or reactivation of MCM [15]. In this latter work, the authors provide evidence that the homologous MeaB protein is involved in the protection of MCM from suicide inactivation possibly by a stabilization function. The gene responsible for the cblB complementation group, MMAB, encodes a small protein of 250 amino acids with high similarity to other cob(I)adenosyltransferases. Recently, the crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434) has been elucidated and several mutations have been mapped to residues highly conserved located in the proposed active site located in the junction between subunits [16]. Up to now the gene responsible for the cblH complementation group has not been identified [17].

The clinical presentation of isolated MMA may vary from a fatal neonatal presentation to an infantile form. The clinical symptoms include recurrent vomiting, failure to thrive, hypotonia, and in the most severe cases, infant death. Patients with the mut0 phenotype exhibit the most severe form of the disease with the poorest prognosis. The clinical outcome of mut patients is less severe but quite variable. Both are B12 nonresponsive patients. Patients with cblA, cblB, and cblH disorders may have a B12-responsive MMA and cblA patients have the best outcome [18].

Cloning and expression of MCM cDNA has facilitated the identification and characterization of close to 60 different mutations associated with mut0 and mut phenotypic manifestations [19], [20], [21], [22], [23], [24], [25], [26]. Until now few allelic variants have been identified in the MMAA and MMAB genes [13], [14]. This paper reports an extensive genetic analysis of 25 patients with isolated MMA, mainly from Spain, with defects in the characterized MUT, MMAA, and MMAB genes. After the identification of the affected gene by enzymatic determinations and/or genetic complementation we have performed genetic analysis. We have identified 27 different allelic variants, 21 are novel ones, affecting one of the three genes. This study represents the first analysis of the molecular basis of MMA in Spain.

Section snippets

Patients and methods

The study includes 25 unrelated patients with isolated MMA and their families. The relevant patients’ data including origin are summarized in Table 1. Incorporation of 14C from [1-14C]propionate into acid-precipitable material was assayed in intact fibroblasts grown in basal medium and in 1 μg/ml hydroxocobalamin (OHCbl) supplemented medium as described previously in [27]. MCM activity in fibroblasts homogenates was determined by the permanganate oxidation assay as described by [28], using 36 

Results

Of the 25 unrelated patients 13 were mut, 7 cblA, and 2 cblB forms. The remaining three cell lines showed deficient propionate incorporation, normal mutase activity, and complemented with cblA and cblB tester cells, indicating a probable cblH defect. All mut patients but one, whose cells were not available, presented undetectable MCM activity in cultured fibroblasts (<0.05 versus 0.94 ± 0.40 nmol/min/mg protein in controls). Although most mut cell lines showed no effect on the defective [14

Discussion

In the present study, we report the mutational spectrum of isolated MMA patients from the Spanish population and historically related ones. In this study of 44 different mutant chromosomes from mut, cblA, and cblB affected patients, 27 different changes, 21 novel ones, accounting for 20 different genotypes have been detected. We have not found any prevalent mutation. In the mut MMA patients only three mutations are present in more than one allele (p.R228X, p.A324T, and p.V227fs), one of them is

Acknowledgments

The authors thank the patients, their parents, and the following doctors and physicians for sending the samples and phenotype data: K. Aldamiz, J. Campos, V. Cornejo, R. Lama, C. Marchante, M. Martínez-Pardo, C. Pedrón, and L. Peña y M Pérez. We also thank C. Hernandez, R. Navarrete, and A. Sanchez for their excellent technical assistance. This work received support from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo (REDEMETH, G03/054, RECGEN C03/07) and from Fundación

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    These contributed equally to this work.

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