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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
0
/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
0
/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
0
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
0
/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
0
/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
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