Clin Genet 2014: 85: 555561 2013 John Wiley & Sons A/S.

Printed in Singapore. All rights reserved Published by John Wiley & Sons Ltd
CLINICAL GENETICS
doi: 10.1111/cge.12227

Short Report

Retrospective study of the medium-chain

acyl-CoA dehydrogenase deficiency
in Portugal

Ventura F.V., Leandro P., Luz A., Rivera I.A., Silva M.F.B., Ramos R., F.V. Venturaa,b, , P.
Rocha H., Lopes A., Fonseca H., Gaspar A., Diogo L., Martins E., Leandroa,b, , A. Luza,b , I.A.
Leao-Teles E., Vilarinho L., Tavares de Almeida I. Retrospective study of Riveraa,b , M.F.B. Silvaa,b , R.
the medium-chain acyl-CoA dehydrogenase deficiency in Portugal. Ramosa,b , H. Rochac , A.
Clin Genet 2014: 85: 555561. John Wiley & Sons A/S. Published by Lopesc , H. Fonsecac , A.
John Wiley & Sons Ltd, 2013 Gaspard , L. Diogoe , E. Martinsf ,
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the E. Leao-Telesg , L. Vilarinhoc
commonest genetic defect of mitochondrial fatty acid -oxidation. About and I. Tavares de Almeidaa
60% of MCADD patients are homozygous for the c.985A>G a
Metabolism and Genetics Group,
(p.Lys329Glu) mutation in the ACADM gene (G985 allele). Herein, we Research Institute for Medicines and
present the first report on the molecular and biochemical spectrum of Pharmaceutical Sciences, iMed.UL,
Portuguese MCADD population. From the 109 patients studied, 83 were Faculty of Pharmacy, b Department of
diagnosed after inclusion of MCADD in the national newborn screening, 8 Biochemistry and Human Biology, Faculty
following the onset of symptoms and 18 through segregation studies. of Pharmacy, University of Lisbon,
Gypsy ancestry was identified in 85/109 patients. The G985 allele was Lisbon, Portugal, c Newborn Screening,
found in homozygosity in 102/109 patients, in compound heterozygosity in Metabolism and Genetics Unit, Genetics
Department, National Institute of Health
6/109 and was absent in one patient. Segregation studies in the Gypsy
Dr. Ricardo Jorge, Porto, Portugal,
families showed that 93/123 relatives were carriers of the G985 allele, d Department of Pediatrics, Lisbon North
suggesting its high prevalence in this ethnic group. Additionally, three new Hospital Center, Santa Maria Hospital,
substitutions c.218A>G (p.Tyr73Cys), c.503A>T (p.Asp168Val) and Lisbon, Portugal, e Childrens Hospital of
c.1205G>T (p.Gly402Val) were identified. Despite the particularity of Coimbra, Metabolic Disorders Unit,
the MCADD population investigated, the G985 allele was found in linkage Coimbra, Portugal, f Childrens Hospital
disequilibrium with H1(112) haplotype. Furthermore, two novel Maria Pia, Metabolic Diseases Unit,
haplotypes, H5(212) and H6(122) were revealed. Porto, Portugal, and g S. Joao Hospital
Center, Metabolic Diseases Pediatric
Conflict of interest Unit, Porto, Portugal

The authors report no conflict of interests.

These two authors contributed
equivalently to this work.

Key words: ACADM inborn errors of

metabolism MCAD mitochondrial
fatty acid -oxidation disorders
newborn screening

Corresponding author: Fatima V.

Ventura, Metabolism and Genetics
Group, Research Institute for Medicines
and Pharmaceutical Sciences, iMed.UL,
Faculdade de Farmacia, Universidade
de Lisboa, Av. Prof. Gama Pinto,
1649-003 Lisbon, Portugal.
Tel.: +351 21 7946400;
fax: +351 21 7946491;

555
Ventura et al.
Medium-chain acyl-CoA dehydrogenase deficiency
(MCADD, OMIM #201450) is one of the most frequent
inborn errors of metabolism (IEM) affecting North-
western European descent Caucasians. It results from
an altered activity of medium-chain acyl-CoA dehy-
drogenase (MCAD; EC 1.3.99.3), which catalyzes the
first step of mitochondrial -oxidation of C4C14 fatty
acids (1). MCADD patients present recurrent hypoke-
totic hypoglycemia, lethargy and Reye-like syndrome
but affected individuals may remain asymptomatic until
metabolically challenged. The disease long-term man-
agement mainly entails avoidance of fasting (1).
Biochemical diagnosis of MCADD is often problem-
atic, since the characteristic abnormal profiles of urinary
medium-chain dicarboxylic acids and acylglycines are
less evident or even absent between decompensation
periods. The introduction of more sensitive and specific
diagnostic tools such as the tandem mass spectrometry
(MS/MS) allowed the establishment of pathognomonic
acylcarnitine profiles in dried blood spots (DBS) and
therefore the pre-symptomatic identification of mito-
chondrial fatty acid -oxidation disorders by extended
newborn screening (NBS) programs. This has been
leading to the detection of far more MCADD cases by
NBS than by clinical onset. Moreover, NBS facilitates,
particularly through segregation studies, the identifica-
tion of carriers and asymptomatic new cases. Nonethe-
less, serious life-threatening metabolic crises have been
reported among screened individuals, indicating that
despite precociously identified, prognosis may still be
unfavorable (2).
The MCADD diagnosis through NBS also revealed
a wide ACADM gene heterogeneity (3): around 100
different mutations were reported for MCADD (Public
Human Gene Mutation Database , Cardiff, UK), being
mostly rare missense mutations correlated with milder
phenotypes or with protein folding and aggregation
defects (3).
The c.985A>G transition (G985 allele) resulting in
the p.Lys329Glu variant is the most prevalent disease-
causing ACADM mutation [5491% allele frequency
and 3080% homozygosity (4)]. Four different hap-
lotypes (H1H4), defined by the bi-allelic polymor-
phisms for BamHI, PstI and TaqI, were associated
with ACADM . The G985 allele has been found in link-
age disequilibrium with haplotype H1, supporting the
origin of this mutation from a single ancestral allele
(57).
While the MCADD molecular basis has been exten-
sively studied in several populations, the mutational
background in Portugal was so far unknown. We present
the first report on the Portuguese MCADD population.
We characterized the biochemical and molecular pro-
files of index cases, performed segregation studies and
556
e-mail: fatima.ventura@ff.ul.pt
Received 24 April 2013, revised and
accepted for publication 2 July 2013
determined the ACADM haplotype pattern among the
studied population.
Materials and methods
Population selection
This study enrolled 236 individuals, comprising all
MCADD patients diagnosed since 1994 to date, after
clinical onset or by NBS since 2004, and whenever
possible their parents and siblings (Table 1). Informed
consent was obtained from all individuals.
Metabolites analysis
Acylcarnitine profiles were analyzed by electrospray
ionization with tandem mass spectrometry (ESI-
MS/MS), in DBS collected after 36 days from birth.
Values above laboratorys cutoff (Table 2) were
repeated in duplicate using the same DBS. Urinary
organic acids and acylglycines plus plasma free
fatty acids were analyzed by GC-MS for diagnostic
confirmation.
Mutation analysis of the ACADM gene
DNA was isolated from whole blood or DBS. Muta-
tion c.985A>G was firstly screened by amplification-
created restriction site analysis (Table S1) according
to established methods (8). All ACADM exons and
adjacent intronic regions were amplified by polymerase
chain reaction (PCR) using specific oligonucleotides
(sequences available upon request), further purified and
sequenced.
Haplotypes in ACADM gene were established after
PCRRFLP analysis of the intragenic bi-allelic poly-
morphisms for BamHI, PstI and TaqI (5, 6) (Table
S1). Haplotype nomenclature was established according
to Zhang et al. (5) (Table 3).
Results
Since 1994, 109 individuals were diagnosed as
MCADD in Portugal, being 91 index cases (83 asymp-
tomatic detected by NBS and 8 after clinical onset) and
18 identified through segregation studies. Interestingly,
85/109 patients had Gypsy ancestry and 68/109 were
females. The remaining were of non-Gypsy Portuguese
(20/109), Brazilian (3/109) and Argentinean (1/109)
origin (Fig. 1, Table 1).
The G985 allele was identified in homozygosity in
102/109 patients, compound heterozygosity in 6/109
and was absent in one patient. All but one Gypsy patient
were homozygous for this allele. Amongst the Gypsy
 Retrospective study of the medium-chain acyl-CoA dehydrogenase deficiency
Table 1. Distribution of MCADD patients according to patients type, ethnicity, gender, type of diagnosis, presence of
clinical/biochemical phenotype at the time of diagnosis and age at diagnosis as function of number of patients, genotype and
ethnicity

Compound Compound
Homozygous heterozygous heterozygous
Characterization Patients (n) G985 (n) G985/mut (n) mut1/mut2 (n) Gypsies (n) Non-Gypsies (n)a

Patients type
Index case 91 84 6 1 67 24
Family member 18 18 18
Ethnicity
Gypsies 85 84 1
Non-Gypsies 24 18 5 1
Gender
Male 41 41 32 9
Female 68 61 6 1 53 15
Diagnosis
Before NBS 9 7 1 1 7 2
Clin/Biochemb 9 7 1 1 7 2
Familial studies 1c 1c 1c
After NBS 100 95 5 78 22
Index case 83 78 5 61 22
Familial studies 17 17 17
Symptomatic 9 7 1 1 7 2
Asymptomatic 100 95 5 78 22
Age at diagnosis
Newborn
<12 months 83 78 5 61 22
12 years 3 2 1 2 1
310 years 5 5 5
>11 years 5 4 1 4 1
Adults 1 1 1
Unknown 7 7 7
5 5 5

MCADD, Medium-chain acyl-CoA dehydrogenase deficiency; NBS, newborn screening; mut, mutant allele.
a Non-Gypsies patients: 1 Argentinean, 3 Brazilian, and 20 Portuguese.
b
Clin/Biochem, Clinical/Biochemical phenotype.
c
One patient diagnosed before NBS expansion through familial studies presented clinical and biochemical phenotype.

families investigated (n = 123 individuals), 93 members
were carriers for the G985 allele, while 18 (11 siblings,
2 fathers and 5 mothers), apparently asymptomatic,
were also homozygous for c.985A>G (Fig. 1b, Table
1). Interestingly, MCADD mothers had eventfulness
pregnancies and are so far asymptomatic.
Genomic DNA analysis in non-homozygous
patients for the G985 allele allowed the identifica-
tion of additional mutations (Table 4): three novel
[c.218A>G (p.Tyr73Cys), c.503A>T (p.Asp168Val),
and c.1205G>T (p.Gly402Val)]; and five previously
described [c.250C>T (p.Leu84Phe), c.351A>C
(p.Thr117Thr), c.1045C>T (p.Arg349X), c.1161A>G
(p.Val387Val) and c.1189_1190insT (p.Tyr372fs)]. The
G985 allele was found in heterozygosity with the G218,
T503, 1189_1190insT and T1205 alleles, remaining to
identify the second mutant allele in two patients. The
c.250C>T/c.1045C>T genotype was also found and
segregation studies confirmed its inheritance in trans.
In this patient, the polymorphism IVS3+10T/C and
the silent mutation c.1161A>G were also identified in
cis with c.250C>T and c.1045C>T, respectively. In
the patient with the genotype c.985A>G/c.1205G>T,
the novel mutation was transmitted by the father with
the silent mutation c.351A>C.
Haplotype characterization of ACADM gene was
performed in 50 patients and relatives (n = 107,
corresponding to 140 independent alleles). The G985
allele was found exclusively associated with H1 haplo-
type which was also the most frequent among the wild-
type alleles (51%). The novel mutation c.1205G>T was
associated with H4 haplotype. Besides the previously
described haplotypes, two novel haplotypes, H5 and H6,
were also identified in G985 carriers (Table 3).
Data on DBS acylcarnitine species with MCADD
diagnostic significance are shown in Table 2. Our results
also support the finding that C8-acylcarnitine is the
main MCADD biomarker in NBS (9). Even the low-
est detected level (0.76 mol/l) was clearly above the
laboratorys cutoff (<0.24 mol/l); C6 was only slightly
increased in 2/69 patients (<0.12 mol/l) and C10 was
below cutoff (<0.39 mol/l) in 33/69 patients. C8/C2
ratio of >0.1 and C8/C10 ratio of 7 were observed
for, respectively, 91% and 83% of MCADD patients

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Ventura et al.
Table 2. Genotype versus C6C10 acylcarnitines determined in dried blood spots (DBS) at newborn screening (NBS)

Acylcarnitines (mol/l)a
C6 C8 C10 C8/C2 C8/C10
Genotype Patients (n) Rf<0.12 Rf<0.24 Rf<0.39 Rf<0.035 Rf<2.00

c.985A>G/c.985A>G 64 0.71 4.02 0.41 0.26 9.89

(0.192.80) (0.7620.7) (0.141.34) (0.070.76) (3.823.4)
0.35 (P10) 1.83 (P10) 0.20 (P10) 0.13 (P10) 7.22 (P10)
1.13 (P90) 9.00 (P90) 0.90 (P90) 0.45 (P90) 12.97 (P90)
c.985A>G/c.1189-1190insT 1 0.75 4.95 0.53 0.3 9.42
(P57) (P61) (P62) (P61) (P43)
c.985A>G/c. 218A>G 1 0.48 1.97 0.79 0.05 2.5
(P28) (P16) (P85) (P0) (P0)
c.985A>G/c.1205G>T 1 0.48 1.72 0.31 0.14 5.4
(P28) (P9) (P31) (P13) (P6)
c.985A>G/mut1 1 0.66 4.64 0.39 0.24 11.8
(P49) (P58) (P43) (P43) (P78)
c.985A>G/mut2 1 0.51 1.68 0.54 0.08 3.12
(P33) (P7) (P66) (P4) (P1)

Acylcarnitines: C6, hexanoylcarnitine; C8, octanoylcarnitine; C10, decanoylcarnitine; C8/C2, octanoyl:acetylcarnitine ratio; C8/C10,
octanoyl:decanoylcarnitine ratio; Rf, reference value (99.5th percentile of screened population); mut, mutant allele under investigation.
a For the c.985A>G/c.985A>G genotype the values indicated are the median of acylcarnitines measured, the respective range (in

brackets) and the values corresponding to the 10th and 90th percentiles. For the other genotypes it is indicated in brackets the
percentile of the obtained value taking in account the acylcarnitines values collected for 69 index cases. Mutation nomenclature
is according to HGVS (http://www.hgvs.org/mutnomen/) with NM_000016.4 and NG_007045.1 as reference sequences for the
ACADM cDNA and genomic sequence, respectively. Only 3/69 MCADD patients revealed values close to globally used cutoff for C8
(<1 mol/l) and C8/C10 ratio (<3.5); no patient presented C8/C2 values close to globally used cutoff (<0.02). In contrast, 31/69 and
47/69 patients presented values close to globally used cutoff for C6 (<0.59 mol/l) and C10 (<0.55 mol/l), respectively.

Table 3. Haplotype determination in the ACADM locus in 50 MCADD index cases and respective families based on RFLP for BamHI,
PstI and TaqI

No. of observations
Allelesa Absolute frequency
(mutant + control alleles)
Haplotype (H) BamHI PstI TaqI (n = 140) Mutant alleles (n = 73) Control alleles (n = 67)
b
1 1 1 2 107 0.99 0.51
2 2 1 1 13 0 0.19
3 1 2 1 7 0 0.1
4 1 1 1 3 0.01c 0.05
5 1 2 2 8 0 0.12
6 2 1 2 2 0 0.03
a
Allele 1 corresponds to the most frequent allele (presence of restriction site for BamHI and TaqI and absence for PstI), while allele
2 is the least frequent allele (absence of recognition for BamHI and TaqI and presence for PstI) (6).
b Mutant allele G985 allele.
c Mutant allele T1205 allele; See Materials and Methods for more details.

homozygous for c.985A>G. Correlations between
genotype and biochemical phenotype are difficult to
establish in compound heterozygotes. However, C8,
C8/C2 and C8/C10 data suggest milder phenotypes
for c.985A>G/c.218A>G, c.985A>G/c.1205G>T and
c.985A>G/mut2 genotypes.
Urinary acylglycines were evaluated for diagnostic
confirmation (n = 24). In our GC-MS system, hex-
anoylglycine ranged between 0.7 and 140.7 mol/mmol
creatinine in patients (Fig. 2) and was never detected
in controls. Suberylglycine was virtually not detected.
Elevated levels of plasma octanoic (C8:0), cis-4-
decenoic (C10:1-n6) and dodecanoic (C12:0) acids and
the presence of urinary adipic, suberic or sebacic acids
were consistent with MCADD (data not shown).
Discussion
This study is based on a retrospective analysis
(19942013) of molecular and metabolic data of 236
individuals. To our knowledge, this group includes
all MCADD patients identified in Portugal and when
possible their families. Segregation studies contributed
to the identification of a high number of individu-
als who, despite apparently asymptomatic, also pre-
sented a MCADD compatible genotype. Currently, this
group represents 17% of the Portuguese MCADD cases,

558
 Retrospective study of the medium-chain acyl-CoA dehydrogenase deficiency
Table 4. Mutations in the ACADM gene, detected in MCADD patients identified in Portugal*

Nucleotide Predicted
changea protein change Exon Sympt. NBS Patients (n) POLYPHEN36 Align_GVGD37 SIFT38 Ref.b

c. 218A>G p.Tyr73Cys 3 N Y 1 D D+ D This study

c.250C>T p.Leu84Phe 4 Y N 1 P D D HGMD
c.351A>C p.Thr117Thr 5 N Y 1 ND ND B Ensembl
c.503A>T p.Asp168Val 7 Y N 1 D D+ D This study
c.985A>G p.Lys329Glu 11 Y/N Y/N 101 B D B HGMD
c.1045C>T p.Arg349X 11 Y N 1 ND ND ND HGMD
c.1161A>G p.Val387Val 11 Y N 1 ND ND B This study
c.1189_1190insT p.Tyr397fs 11 Y N 1 ND ND ND Ensembl
c.1205T>G p.Gly402Val 12 N Y 1 D D+ D This study

MCADD, Medium-chain acyl-CoA dehydrogenase deficiency; NBS, newborn screening.

a For mutation nomenclature see footnote of Table 2. Protein ID for the MCAD protein is P11310; Sympt., signs and symptoms

suggestive of MCADD; NBS, mutation detected after NBS; Y, yes; N, no. In silico assessment of variants pathogenic potential
performed with the softwares POLYPHEN (http://genetics.bwh.harvard.edu/pph/; Predictions: B = benign; P = possibility damaging;
D = probably damaging); Sorting Intolerant from Tolerant (SIFT; http://sift.jcvi.org/; Predictions: B = tolerated; D = damaging); and
Align_GVGD [http://agvgd.iarc.fr/agvgd_input.php; class 015 = benign (B), class 2535 = possibly damaging (P), class 4555 =
probably damaging (D); class 65 = damaging (D+)]; ND not determined.
b Reference of the first report for the mutation; references to already described mutations in the ACADM gene can be found in Public

Human Gene Mutation Database (HGMD), Cardiff, UK or Ensembl (http://www.ensembl.org/). Adapted from (17, 18).
*[Correction added on 10 December 2013, after first online publication: Table 4 has now been replaced due to errors in the values of
the earlier version.]

Fig. 2. Levels of urinary hexanoylglycine (C6 glycine) expressed as

mol/mmol creatinine in c.985A>G/c.985A>G patients diagnosed
at NBS. The dotted line represents the median value. Reference
laboratory value is below the limit of quantification. The three lowest
values correspond to 0.7, 1.5 and 1.7 mol/mmol creatinine while the
three highest values correspond to 47.8, 140.7 and 109.6 mol/mmol
creatinine.

gathered since then (>500,000 newborns) (11) show

Fig. 1. Distribution of Medium-chain acyl-CoA dehydrogenase defi-
ciency (MCADD) patients according to (a) gender, origin, and age at
diagnosis; (b) symptoms, type of patient (index or following segrega-
tion studies) and type of diagnosis [before or after newborn screening
(NBS) expansion]; NK, not known; NB(S), newborn (screening).
stressing the potential benign outcome of MCADD,
and the importance of segregation studies following an
index case identification.
Most patients (92%) were identified since 2004,
after the expansion of NBS (10). Surprisingly, the data
a MCADD birth prevalence of 1:8804, a value higher
than described worldwide (4). Comparing with birth-
prevalence of phenylketonuria reported in the same
period (1:10,000) (11), MCADD is currently the most
frequent IEM in the country.
Information concerning the MCADD prevalence in
Mediterranean countries is scarce being reported as rare
in France (12) and Spain (13), where the majority of
patients are of Gypsy origin. Our data also support
these observations, as 78% of the patients are Gypsies,
suggesting that MCADD patients in Southern-European
countries are mostly of Gypsy ancestry.

559
Ventura et al.
The c.985A>G mutation was found in homozygosity
in 94% of the patients, a much higher frequency than
reported for other populations (60%) (4). However,
it must be stressed that 82% of these patients have
Gypsy origin and 99% of those share this common
genotype. Amongst the Gypsy families, 76% of the
individuals were carriers of the c.985A>G mutation,
suggesting a high frequency of the G985 allele in this
ethnic group. The common mutation was also found in
homozygosity in 75% of the 24 non-Gypsy patients.
Curiously, this mutation was also identified in three
Brazilian descents: in homozygosity (n = 2) and in
compound heterozygosity with a novel mutation (n = 1;
c.1205G>T). This contrasts with a recent Brazilian
study, where the G985 allele showed a very low allelic
frequency without homozygotes being found (14).
Haplotype characterization of the ACADM gene
(5, 6), performed when possible in MCADD patients
and families, consistently revealed a H1 haplotype
homozygosity for the c.985A>G/c.985A>G genotype,
as previously described (5, 6), while the novel mutation
c.1205G>T was associated with H4 haplotype. Though
H1 is the most frequent haplotype among wild-type
alleles, two novel haplotypes (H5 and H6) were
also identified. These results show that the G985
allele is in linkage disequilibrium with H1 haplotype,
strongly suggesting that both in Gypsy and non-
Gypsy populations this mutation presents a common
source. The origin of the Roma people in the Indian
subcontinent (15), their migration history and the results
herein obtained, corroborate the hypothesis that Indo-
European speaking people introduced in Europe the
c.985A>G mutation (16).
Among the patients studied, 6% were non-
homozygous for the G985 allele and all but one
were compound heterozygotes holding the c.985A>G
mutation in one allele and a rare mutation in the
other. Three novel variants with unknown significance
were revealed: c.218A>G (p.Tyr73Cys), c.503A>T
(p.Asp168Val), and c.1205G>T (p.Gly402Val). In
silico analysis suggests that these novel variants may
be potentially damaging (Table 4). Recombinant forms
of these proteins are currently being characterized to
confirm their disease-causing effect (manuscript in
preparation).
Curiously, the already known mutations herein
identified are not among the commonest in MCADD,
namely the c.199C>T (p.Tyr42His), c.127G>A
(p.Glu43Lys) and c.797A>G (p.Asp266Gly) (5, 16,
17), suggesting they can be private mutations.
The C8-acylcarnitine, C8/C2 and C8/C10 ratios
and urinary hexanoylglycine have been recognized as
the most reliable MCADD biomarkers (9, 18). Our
results also support the diagnostic significance of those
findings and the discriminatory power of C8/C2 and
C8/C10 ratios face to isolated C8-acylcarnitine levels
as well as the importance of urinary hexanoylglycine
always absent in control samples.
In conclusion, this study represents the first report
on the features of the Portuguese MCADD patients.
The expansion of NBS changed the countrys disease
560
picture. MCADD is now the most frequent IEM
among screened newborns. Furthermore, our study
describes the particular situation related to the Gypsy
origin of the majority of the patients. Interestingly,
although from a closed community, the ACADM
genetic background in Gypsy patients c.985A>G
homozygosity and haplotype H1 is the same as
for North-western European MCADD patients. This
strongly supports c.985A>G as an ancient mutation
arising in a common ancestor of the first Europeans.
Supporting Information
The following Supporting information is available for this article:
Fig S1. Two novel mutations: c.503A>T (p.Asp168Val) in exon
7 (a) and c.1205G>T (p.Gly402Val) in exon 12 (b) identified by
DNA sequencing of two patients heterozygous for the c.985AG
mutation. See Materials and Methods for more details. The mutated
base is indicated by an arrow.
Table S1. Oligonucleotides used for the screening of the c.985A>G
mutation and for haplotype identification in the ACADM gene
Additional Supporting information may be found in the online
version of this article.
Acknowledgements
We are grateful to Doctor Marinus Duran and Doctor Sara
Violante for comments and critical reading of the manuscript. We
acknowledge the technical assistance of F. Louro, F. Lopes, I.
Lopes, J. Nunes, A. Serrao and M. Moedas. Part of this work
was financially supported by the Portuguese Society for Metabolic
Disorders, through a project awarded to FV Ventura and by the
Fundacao para a Ciencia e Tecnologia, Lisboa, Portugal, through
the strategic project PEst-OE/SAU/UI4013/2011 to iMed.UL.
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