METHYLMALONIC ACIDURIAS

mut

/mut

and cblC Defects in Portuguese Population

Célia Nogueira, Marta Marques and Laura Vilarinho

Medical Genetics Center/ National Institute of Health, Praça Pedro Nunes,88, Porto, Portugal

Keywords: Methylmalonic acidurias, mut

, mut

, MUT gene, MMACHC gene.

Abstract: The methylmalonic acidurias (MMAs) are metabolic disorders resulting from deficient methylmalonyl-CoA

mutase (MCM) activity, a vitamin B12-dependent enzyme that uses adenosylcobalamin (Ado-Cbl) as a

cofactor. Several mutant genetic classes that cause MMA are known based on biochemical, enzymatic and

genetic complementation analysis. The mut

/mut

defects result from deficiency of MCM, while the cblA,

cblB and the variant 2 form of cblD complementation groups are linked to processes unique to Ado-Cbl

synthesis. The cblC, cblD and cblF complementation groups are associated with defective methyl-

cobalamin synthesis as well. Mutations in the genes associated with most of these defects have been

described. In this study we investigate at molecular level four patients with mut

/mut

MMA phenotype and

19 Portuguese patients with cblC defect. We found four different mutations already described in the

literature, in each MUT and MMACHC genes, respectively. Our data showed an evident difference in the

prevalence of these two diseases, compared with other countries worldwide.

1 INTRODUCTION

Methylmalonic Methylmalonic acidurias (MMAs)

encompass a group of genetically heterogeneous

autosomal recessive disorders of methylmalonate

and cobalamin metabolism caused by a defect in the

conversion of methylmalonyl-CoA to succinyl-CoA.

The different forms of MMAs share the biochemical

marker of increased methylmalonic acid in body

fluids. Methylmalonyl-CoA mutase (MCM)

apoenzyme deficiency is a rare metabolic disease

that may result in distinct biochemical phenotypes of

MMA, namely mut

and mut

. Patients with the mut

MMA phenotype exhibit the most severe, often life

threatening manifestation. Treatment regimens

include a protein-restricted diet, carnitine

supplementation and oral antibiotic therapy. The

MCM gene (MUT) is located in a single copy on

chromosome 6 (6p21.1) and consists of 13 exons

spanning over 35 kb (Jansen et al., 1989). The open

reading frame consists of 2.7kb, encoding 750 amino

acids, the first 32 residues of which form the

mitochondrial targeting sequence. It comprises

different functional domains (Figure 1), the N-

terminal domain (residues 1-32) followed by an

extended segment (residues 32-87) involved in the

dimerization of the two MCM monomers, the N-

terminal (β/α)

8 barrel (residues 87-416) containing

the CoA binding domain and the C-terminal

cobalamin-binding β/α

5 domain (residues 578-750).

A linker region consisting of 160 amino acids

connects the eight-stranded β/α barrel to the C-

terminal β/α domain (Fuchshuber et al., 2000).

More than 80 mutations, including small and large

scale rearrangements, truncating and missense

mutations, have been described.

Figure 1: Functional domains of MUT gene (adapted from

Acquaviva et al., 2005).

MMA with homocystinuria is an inborn error of

intracellular cobalamin metabolism resulting from

impaired conversion of dietary vitamin B12 or

cobalamin to its two metabolically active forms,

methylcobalamin (MeCbl) and adenosylcobalamin

(AdoCbl). MeCbl and AdoCbl are essential

coenzymes to methionine synthase and MCM,

respectively. The defect of these two cofactors

causes the accumulation of methylmalonic acid and

homocysteine in body fluids and a decrease of

methionine. Three genetic defects of intracellular

261

Nogueira C., Marques M. and Vilarinho L. (2010).

METHYLMALONIC ACIDURIAS - mut0/mut- and cblC Defects in Portuguese Population.

In Proceedings of the First International Conference on Bioinformatics, pages 261-263

DOI: 10.5220/0002759802610263

 SciTePress

cobalamin metabolism, cblC (MIM 277400), cblD

(MIM 277410), and cblF (MIM 277380) cause

combined MMA and homocystinuria. cblC defect is

the most frequent form and patients present with a

heterogeneous clinical picture (Rosenblatt et al,

1997). Based on the age at onset, two distinct

clinical forms have been recognized (early-onset and

late onset form). Recently the identification of the

gene responsible for cblC, MMACHC (MIM#

609831) was reported (Lerner-Ellis et al., 2006).

The gene is located in chromosome region 1p34.1

and has five exons. In the literature, forty-two

different mutations in 204 cblC individuals were

reported, including three common mutations:

c.271dupA, c.394C>T, and c.331C>T. The

c.271dupA and c.331C>T mutations were associated

with early-onset disease while the c.394C>T

mutation was associated primarily with late-onset

disease.

In the present study 19 Portuguese patients with

cblC defect and four patients with mut

/mut

MMA

phenotype were investigated. We found four

different mutations in MMACHC gene and another

four in MUT gene. We discuss the prevalence of

these diseases in our country/worldwide and the

impact that mutation identification has on routine

diagnostic procedures.

2 MATERIAL AND METHODS

2.1 Patients

In this cohort, the patients were selected after

sharing and matching our databases. The diagnosis

of mut

/mut

MMA

and cblC defect was based on the

identification of urinary and circulating metabolites

and, whenever possible, confirmed with fibroblast

studies. We studied at a molecular level four

Portuguese patients with the mut

MMA phenotype

and 19 with cblC defect diagnosed in our center, six

of them (2/4 and 5/19, respectively) detected by

extended newborn screening. The informed consent

was obtained in all studied patients.

2.2 Methods

The whole coding sequence, the flanking exon–

intron sequences of the MUT and MMACHC genes

were PCR amplified from genomic DNA as

described (Aquaviva, 2005; Lerner-Ellis, 2006).

Agarose-gel purified amplicons were directly

sequenced using the BigDye Terminator Cycle

Sequencing Version 3.1 (Applied Biosystems, Foster

City, CA), and analyzed on an ABI 3130XL DNA

Analyzer. Multiple linear regression analysis was

used to identify significant predictors of the

genotype in the entire sample, including gender, age

and clinical features. Statistical analyses were

performed using a Chi-square test with Yates

corrections (or, when appropriate, Fisher’s exact

test). Statistical significance was set at p < 0.01.

3 RESULTS AND DISCUSSION

In our center we diagnosed four cases of mut

/mut

MMA (two through newborn screening) and 19

cases of cblC defect (five from newborn screening).

The number of symptomatic cases is in agreement

with the prevalence found by expanded newborn

screening although we know that the mild forms of

cblC cannot be detected by newborn screening using

the C3 (propionylcarnitine) and C3/C2

(acetylcarnitine) and C3/C16 (palmitoilcarnitine)

ratios without the homocysteine determination. In

most populations, mainly in Europe, the mut

/mut

MMA is more prevalent than the cblC defect. In the

Mediterranean countries a few studies were carried

out and some patients were identified although this

condition is a very rare disease in middle and north

of Europe (Nogueira et al., 2008; Richard et al.,

2009).

We investigated the molecular basis of MMA in 23

unrelated patients by sequencing the entire coding

region and intron-exon boundaries of the MUT and

MMACHC gene using genomic DNA. The mutations

were homozygous in 14 patients, and compound

heterozygous in 8 patients.

All the MUT mutations have been previously

reported; one of them is a nonsense mutation

(p.R31X) and two are small deletions (p.L346del

and p.G625FsX30) (Table 1).

The four different mutations found in cblC defect

were: two missense (c.544T>C and c.565C>A), one

nonsense (c.394C>T) and a small insertion causing

frameshift (c.271dupA) (Table 2).

Our data compared with other southern-European

populations, such as Italians and Spanish, have

showed a less molecular heterogeneity (Nogueira et

al., 2008.

The recent inclusion of these conditions in the

Portuguese expanded newborn screening program

since 2004, resulted in a substantial improvement in

the ability to identify suspected cases and allows for

a more reliable determination of their incidence,

considering the total number of individuals screened

until now. By MS/MS 420,000 neonates were

BIOINFORMATICS 2010 - International Conference on Bioinformatics

262

screened and two cases of mut

/mut

(1/210.000) and

five cases of cblC (1/84.000) were identified.

The present study represents the first determination

of the incidence of MMA in Portugal, indicating that

the cblC is more frequent in our country than

worldwide. All the patients identified by newborn

screening revealed homozygosity for c.271dupA

associated with the early phenotype. As long as

more newborns will be screened, a more reliable

comparison between symptomatic versus screened

detected patients will be established.

Another prospect of molecular studies is to facilitate

projections on the clinical type and severity of a

disease. These projections may be essential to guide

a proper monitoring of patients and that is why

phenotype-genotype studies are needed.

Table 1: Genotype and clinical subgroup of patients with

mut

/mut

MMA.

Table 2: Genotype and clinical subgroup of patients with

combined methylmalonic aciduria and homocystinuria,

cblC type.

4 CONCLUSIONS

In summary, we described the genetic background of

23 Portuguese patients with MMAs (four mut

/mut

MMA and 19 MMA-cblC type. Moreover, we found

four different mutations already described in the

literature, in each MUT and MMACHC genes,

respectively. Our data showed an evident difference

in the prevalence of these two diseases, compared

with other countries worldwide. This study

corroborate the importance of a molecular testing to

confirm mut

/mut

MMA and MMA-cblC patients,

detected by extended newborn screening programs,

to offer accurate treatment and future prenatal

diagnosis to couples at high risk of having affected

children. The molecular data also contributes to

molecular epidemiology of these diseases in our

population.

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