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Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation.

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11. Ionasescu VV, Searby C, Ionasescu R, et al. Mutations of the
noncoding region of the connexin32 gene in X-linked dominant Charcot-Marie-Tooth neuropathy. Neurology 1996;47:
541–544.
12. Flagiello L, Cirigliano V, Strazzullo M, et al. Mutation in the
nerve-specific 5⬘non-coding region of Cx32 gene and absence
of specific mRNA in a CMTX1 Italian family. Mutations in
brief no. 195. Online. Hum Mutat 1998;12:361.
13. Piechocki MP, Toti RM, Fernstrom MJ, et al. Liver cellspecific transcriptional regulation of connexin32. Biochim Biophys Acta 2000;1491:107–122.
14. Kuhlbrodt K, Herbarth B, Sock E, et al. Sox10, a novel transcriptional modulator in glial cells. J Neurosci 1998;18:
237–250.
15. Inoue K, Tanabe Y, Lupski JR. Myelin deficiencies in both the
central and the peripheral nervous systems associated with a
SOX10 mutation. Ann Neurol 1999;46:313–318.
16. Warner LE, Mancias P, Butler IJ, et al. Mutations in the early
growth response 2 (EGR2) gene are associated with hereditary
myelinopathies. Nat Genet 1998;18:382–384.
17. Bondurand N, Girard M, Pingault V, et al. Human Connexin
32, a gap junction protein altered in the X-linked form of
Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10. Hum Mol Genet 2001;10:2783–2795.
18. Musso M, Balestra P, Bellone E, et al. The D355V mutation
decreases EGR2 binding to an element within the Cx32 promoter. Neurobiol Dis 2001;8:700 –706.
19. Hudder A, Werner R. Analysis of a Charcot-Marie-Tooth disease
mutation reveals an essential internal ribosome entry site element
in the connexin-32 gene. J Biol Chem 2000;275:34586 –34591.
20. Ainsworth PJ, Bolton CF, Murphy BC, et al. Genotype/
phenotype correlation in affected individuals of a family with a
deletion of the entire coding sequence of the connexin 32 gene.
Hum Genet 1998;103:242–244.
Defective Mitochondrial
Translation Caused by a
Ribosomal Protein
(MRPS16) Mutation
Chaya Miller, PhD,1 Ann Saada, PhD,1 Nava Shaul, MD,1
Naama Shabtai, MSc,1 Efrat Ben-Shalom, MD,1
Avraham Shaag, PhD,1 Eli Hershkovitz, MD,2
and Orly Elpeleg, MD1,3
The mitochondrial respiratory chain comprises 85 subunits, 13 of which are mitochondrial encoded. The synthesis of these 13 proteins requires many nuclear-encoded
proteins that participate in mitochondrial DNA replication, transcript production, and a distinctive mitochondrial translation apparatus. We report a patient with
agenesis of corpus callosum, dysmorphism, and fatal neonatal lactic acidosis with markedly decreased complex I
and IV activity in muscle and liver and a generalized mitochondrial translation defect identified in pulse-label experiments. The defect was associated with marked reduction of the 12S rRNA transcript level likely attributed to
a nonsense mutation in the MRPS16 gene. A new group
of mitochondrial respiratory chain disorders is proposed,
resulting from mutations in nuclear encoded components
of the mitochondrial translation apparatus.
Ann Neurol 2004;56:734 –738
The mitochondrial respiratory chain (MRC) comprises
85 subunits that are assembled into five enzymatic
complexes. Thirteen of the subunits are mitochondrial
encoded, and the rest are encoded by the nuclear genome. Most of the factors involved in the synthesis of
the 13 proteins, including those participating in the
synthesis and maintenance of the mitochondrial DNA
(mtDNA) and in the mitochondrial transcription and
translation processes, are encoded by the nuclear genome; only the 22 tRNAs and the 2 rRNAs are encoded by the mtDNA.
Over a period of 9 years, 604 patients were referred
to our center for muscle biopsy because of suspected
From the 1Metabolic Disease Unit, Shaare-Zedek Medical Center,
Jerusalem; 2Department of Pediatrics, Soroka Medical Center, Faculty of Medicine, Ben-Gurion University, Beer-Sheba; and 3School
of Medicine, Hebrew University, Jerusalem, Israel.
Received Jun 25, 2004, and in revised form Jul 28. Accepted for
publication Jul 29, 2004.
Published online Oct 28, 2004, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20282
Address correspondence to Dr Elpeleg, Metabolic Disease Unit,
Shaare-Zedek Medical Center, Jerusalem 91031, Israel.
E-mail: elpeleg@cc.huji.ac.il
734
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
MRC defects. Most of the patients were infants, and
the most frequent cause of referral was developmental
delay of variable degree with increased lactate level in
plasma, cerebrospinal fluid, or over the brain. Twentyone patients were found to have a unique enzymatic
profile characterized by markedly decreased activity of
several enzymatic complexes with normal complex II
activity. Because complex II is the only component of
the MRC that is solely encoded by the nuclear genome, we considered this profile to be indicative of a
defect in mitochondrial protein synthesis. Southern
blot analysis showed mtDNA depletion in muscle in 9
of 21 patients that in 4 patients was attributed to mutations in the mitochondrial thymidine kinase gene.1
In fibroblasts of the remaining 12 patients, all originating from consanguineous families, we assessed mitochondrial transcription, translation, and respiratory
chain function. The findings in one of these patients
are the subject of this report.
Case Report
The female patient was the second child born to consanguineous parents of Beduin origin. At 20 weeks of
gestation, fetal ultrasound disclosed mild dilatation of
the cerebral ventricles and agenesis of the corpus callosum. The patient was born at term and was small for
age (1,830gm). Physical examination showed dysmorphic facies with low-set ears, nonpitting edema of the
limbs, brachydactyly, and redundant skin over the
neck. The karyotype was that of a normal female. At
24 hours of age, the patient developed lethargy and
refused to feed. Generalized muscle hypotonia and
paucity of spontaneous movements were noted accompanied by severe metabolic acidosis (pH 6.90 and base
excess ⫺26), increased plasma lactate level (10.8mM),
and mildly increased liver transaminases (GGT
180U/L, GOT 213U/L). Echocardiography showed
large patent ductus arteriosus, but the myocardial walls
were of normal thickness and function. Biopsy of the
quadriceps muscle, liver, and skin for fibroblasts culture was obtained with informed consent. The patient
expired at 3 days of age because of intractable acidosis.
Results
The activity of the enzymatic complexes of the MRC
in mitochondria isolated from frozen muscle and in fibroblasts and frozen liver homogenate was determined
using standard spectrophotometric methods,2,3 and the
results, normalized for citrate synthase activity, were
compared with those of age-matched controls. The activity of complex I, II⫹III, IV, and V was markedly
decreased in muscle mitochondria, but complex II
(succinate dehydrogenase) activity was normal (Table).
In liver homogenate, complex I, II⫹III, and IV activity
was similarly decreased, and in fibroblasts, complex IV
activity was moderately reduced (see Table). The defect
in MRC function also was demonstrated by the markedly compromised survival of the patient fibroblasts,
with galactose as the sole carbohydrate source in the
medium (data not shown).
Pulse labeling of mitochondrial translation in fibroblast cultures from the patient and controls was performed as described by Chomyn4 with the following
modifications: fibroblasts were washed with
Table. The Activity of the Enzymatic Complexes of the Mitochondrial Respiratory Chain in Mitochondria Isolated From Frozen
Muscle and in Fibroblasts and Frozen Liver Homogenate
Enzymatic Complex*
Tissue
Frozen muscle mitochondria
Patient
Citrate
Synthase
670
Control (n ⫽ 27), mean ⫾ SD 1,460 ⫾ 330
(range)
(900–2,080)
Frozen liver homogenate
Patient
95
Complex I
Complex
II⫹III
Complex
II
Complex IV
Complex V
14
25%
115 ⫾ 37
(57–146)
0
0%
107 ⫾ 52
(53–249)
111
114%
207 ⫾ 55
(123–290)
141
22%
1350 ⫾ 478
(775–2,427)
63
23%
568 ⫾ 102
(174–452)
4.1
19%
22 ⫾ 9
(10–38)
ND
18
17%
93 ⫾ 31
(49–189)
ND
14
77%
18 ⫾ 4
(8–20)
Control (n ⫽ 12), mean ⫾ SD
(range)
Fibroblast homogenate
Patient
95 ⫾ 39
(48–167)
3
18%
16 ⫾ 7
(8–39)
86
ND
Control (n ⫽ 10), mean ⫾ SD
(range)
75 ⫾ 25
(41–128)
ND
ND
ND
ND
57
34%
145 ⫾ 52
(79–180)
ND
ND
ND
ND
*Values are given in nmol/min/mg protein and are expressed as a percentage of control mean normalized for citrate synthase activity.
SD ⫽ standard deviation; ND ⫽ not determined.
Miller et al: Mitochondrial Translation
735
methionine-free Dulbecco’s minimum essential medium and subsequently incubated in the same medium
for 3 hours with 0.45uCi [35S]-labeled methionine
with specific activity of 1,175Ci/mmol (NEN, Boston,
MA) in the presence of emetine (a cytoplasmic protein
synthesis inhibitor). The labeled translation products
were extracted and separated by a 15% sodium dodecyl
sulfate polyacrylamide gel electrophoresis urea gel. The
gel was treated with Amplify (Amersham, England)
and visualized by autoradiography. Using this method,
a generalized mitochondrial translation defect was evident (Fig, A). Because these findings might reflect impaired mtDNA replication, we performed Southern
blot analysis of the muscle and fibroblast DNA, according to a previously described method,1 which
showed that the mtDNA was of normal size and abundance (data not shown).
We next determined the level of mitochondrial tran-
scripts level in total RNA from fibroblasts, using labeled polymerase chain reaction products of the 12S
rRNA, 16S rRNA, ND1, COXI, ND6, and exon 3 of
the ␤-actin gene as probes. Between hybridizations, the
blot was stripped by incubation in 10mM Tris, pH
7.4/0.2% sodium dodecyl sulfate at 70°C for 1 hour.
Quantification of band intensities was performed by
PhosphorImager (Molecular Dynamics, Sunnyvale,
CA). The level of the 16S rRNA and the ND1 and
COX1 mRNA, all encoded by the heavy strand of the
mtDNA, was normal, as was the ND6 transcript,
which is encoded by the light strand. However, the
abundance of the 12S rRNA was markedly decreased
to 12% of the mean of five controls (see Fig, B). The
sequence of the muscle mitochondrial 12S rRNA gene
and the flanking tRNAPhe and tRNAVal genes was normal.
The human mitochondrial ribosome is composed of
Fig. (A) Labeling of mitochondrial encoded proteins in fibroblasts: Patient (P) and control (C) fibroblasts were labeled with radioactive methionine in the presence of emetine. Equal amounts of cellular proteins (30␮g) were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis and subjected to autoradiography. Molecular weight markers were run in the flanking slots. Translation products were annotated according to Chomyn.4 (B) RNA analysis: Northern hybridization of 5␮g RNA from fibroblasts of
the P and C, probed simultaneously for mitochondrial 12S rRNA and COX1 mRNA and successively for 16S rRNA and ND1
and ␤-actin mRNA.
736
Annals of Neurology
Vol 56
No 5
November 2004
two subunits: the small subunit (SSU), which consists
of the 12S rRNA and 29 proteins, and the large subunit, which consists of the 16S rRNA and 48 proteins.5,6 Because bacterial rRNAs are stable only if incorporated into the ribosomal subunits,7 we speculated
that the markedly reduced 12S rRNA level was the result of a defect in one of the mitochondrial SSU proteins that interferes with the subunit assembly. We
therefore have determined the sequence of the cDNA
of the 14 genes that encode evolutionarily conserved
proteins between Escherichia coli and the human mitochondrial SSU. cDNA of the mitochondrial ribosomal
protein genes S2, S5, S6, S7, S10, S11, S12, S14, S15,
S16, S17, and S21 was prepared from the patient fibroblasts, and the sequence was determined on an automatic sequencer (ABI Prism 3700; Perkin-Elmer,
Foster City, CA). This analysis showed a homozygous
c-to-t substitution at nucleotide 331 of the mitochondrial SSU protein S16 (MRPS16) cDNA (accession no.
BC021106.1), predicting a premature stop codon,
Arg111Ter. The presence of the mutation was verified
by determination of the sequence of the three exons of
MRPS16 and their exon–intron boundaries, using
primers designed on the basis of chromosome 10
sequence (f1, 74357107–74357087; r1, 74356713–
74356736; f2, 74356455–74356435; r2, 74356056 –
74356077; f3, 74355459 –74355439; r3, 74355147–
74355167). In the family, both parents and one of the
two girls born after the death of our patient were heterozygous for the mutation; the older brother and the
other sister were normal homozygotes.
Discussion
The patient reported on here presented at midgestation
with mild ventricular dilatation and agenesis of the
corpus callosum. The mother was subsequently lost to
follow-up, but at birth the newborn was dysmorphic
and small for gestational age. Low birth weight is not
uncommon among patients with MRC defects and was
found in 68 of 300 patients (23%) with various MRC
defects.8 Dysmorphism and brain anomalies are less
frequent. In the same series, only one patient had agenesis of corpus callosum, one had hypoplasia of corpus
callosum identified postnatally, and yet another patient
had enlarged ventricles detected at midgestation. Facial
dysmorphism was noted in two other patients. The rarity of these findings indicates that they are not the direct consequence of general lack of energy throughout
gestation but, rather, represent a tissue-specific effect of
some disease genes. Because the parents are consanguineous and no other affected children were known
of, it remains possible that the dysmorphic features are
a result of homozygosity for other mutations.
The human MRPS16 is a 137–amino acid protein;
it is one of the most highly conserved ribosomal proteins between mammalian and yeast mitochondria, and
the human protein is approximately 40% identical to
its bacterial homologs.5 The binding of the Thermus
thermophilus ribosomal protein S16 is an important
step in the assembly of the SSU of this organism.9 The
S16 protein is located in a narrow crevice on the SSU
and has many contacts with the rRNA, being surrounded by about five rRNA double helices. An extraribosomal function, the introduction of nicks into
supercoiled DNA molecules, has been indicated for the
ribosomal S16 protein of E. coli.10 The finding of a
premature stop codon in the human MRPS16 is predicted to result in a truncated, likely unstable, protein.
The deleterious effect of the mutation is evident by the
drastic reduction of abundance of the 12S rRNA transcript. In Drosophila, a missense mutation in the mitochondrial ribosomal protein S12 that impaired the
assembly of the protein into active ribosome resulted in
a significant reduction of the 12S rRNA transcript.11
Mutations in the mitochondrial genome that interfere with mitochondrial translation, especially in tRNA
genes but also in the 12S rRNA gene, have been extensively reported.12–14 In contrast, nuclear-encoded defects in mitochondrial translation are rarely described:
Impaired mitochondrial tRNA pseudouridinylation has
been suggested recently in patients with myopathy and
anemia,15 and in two patients with similar phenotypes,
a developmentally regulated, nuclear-encoded defect of
mitochondrial translation has been demonstrated.16 Of
604 patients referred to our center for MRC evaluation, 12 patients, including this patient, had impaired
synthesis of the mitochondrial encoded proteins but
normal size and abundance of the mtDNA. Because all
patients originated from consanguineous families, we
suggest that nuclear-encoded defects in mitochondrial
translation may cause devastating symptoms in a low
but significant number of patients. Analysis of the
mtDNA molecule, mitochondrial transcription, and
translation products is warranted in patients with decreased activity of several MRC enzymatic complexes
with normal complex II activity.
We are grateful to Dr H. Jacobs for fruitful discussions. The technical assistance of C. Belaiche and Y. Shoshani is acknowledged.
References
1. Saada A, Shaag A, Mandel H, et al. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat
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2. Hoppel C, Cooper C. An improved procedure for preparation
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treatment with digitonin. Arch Biochem Biophys 1969;135:
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3. Dooijewaard G, Slater EC. Steady state kinetics of high molecular weight (type I) NADH dehydrogenase. Biochem Biophys
Acta 1976;440:1–15.
4. Chomyn A. In vivo labeling and analysis of human mitochondrial translation products. Meth Enzymol 1996;264:196 –211.
Miller et al: Mitochondrial Translation
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5. Cavdar Koc E, Burkhart W, Blackburn K, et al. The small subunit of the mammalian mitochondrial ribosome. Identification
of the full complement of ribosomal proteins present. J Biol
Chem 2001;276:19363–19374.
6. Koc EC, Burkhart W, Blackburn K, et al. The large subunit of
the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present. J Biol Chem 2001;276:
43958 – 43969
7. Dennis PP, Young RF. Regulation of ribosomal protein synthesis in Escherichia coli B/r. J Bacteriol 1975;121:994 –999
8. von Kleist-Retzow JC, Cormier-Daire V, Viot G, et al. Antenatal manifestations of mitochondrial respiratory chain deficiency. J Pediatr 2003;143:208 –212.
9. Allard P, Rak AV, Wimberly BT, et al. Another piece of the
ribosome: solution structure of S16 and its location in the 30S
subunit. Structure Fold Des 2000;8:875– 882.
10. Bonnefoy E. The ribosomal S16 protein of Escherichia coli displaying a DNA-nicking activity binds to cruciform DNA. Eur
J Biochem 1997;247:852– 859
11. Toivonen JM, O’Dell KM, Petit N, et al. Technical knockout,
a Drosophila model of mitochondrial deafness. Genetics 2001;
159:241–254.
12. Schon EA. Mitochondrial genetics and disease. Trends Biochem
Sci 2000;25:555–560.
13. Jacobs HT. Disorders of mitochondrial protein synthesis. Hum
Mol Genet 2003;12:R293–301
14. Prezant TR, Agapian JV, Bohlman MC, et al. Mitochondrial
ribosomal RNA mutation associated with both antibioticinduced and non-syndromic deafness. Nat Genet 1993;4:
289 –294.
15. Bykhovskaya Y, Casas K, Mengesha E, et al. missense mutation
in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy and sideroblastic anemia (MLASA). Am J Hum Genet
2004;74:1303–1308.
16. Sasarman F, Karpati G, Shoubridge EA. Nuclear genetic control of mitochondrial translation in skeletal muscle revealed in
patients with mitochondrial myopathy. Hum Mol Genet 2002;
11:1669 –1681
Cardiac and Respiratory
Failure in Limb-Girdle
Muscular Dystrophy 2I
Maja Poppe, MD,1 John Bourke, MD,2
Michelle Eagle, PhD,1 Patrick Frosk, BSc,3
Klaus Wrogemann, PhD,3 Cheryl Greenberg, MD,3
Francesco Muntoni, MD,4 Thomas Voit, MD,5
Volker Straub, MD,5 David Hilton-Jones, MD,6
Cheerag Shirodaria, MD,7 and Kate Bushby MD1
Mutations in the gene encoding fukutin-related protein
cause limb-girdle muscular dystrophy 2I. In this multicenter retrospective analysis of 38 patients, 55.3% had
cardiac abnormalities, of which 24% had developed cardiac failure. Heterozygotes for the common C826A mutation developed cardiac involvement earlier than homozygotes. All patients initially improved while receiving
standard therapy. Independent of cardiac status, forced
vital capacity was below 75% in 44.4% of the patients.
There was no absolute correlation between skeletal muscle weakness and cardiomyopathy or respiratory insufficiency. These complications are a primary part of this
specific type of limb-girdle muscular dystrophy, with important implications for management.
Ann Neurol 2004;56:738 –741
The relationship between skeletal myopathy and cardiomyopathy is complex. Muscle disorders may result from
mutations in genes that independently cause cardiomyopathy,1 and cardiomyopathy forms part of the phenotype
of some muscle diseases.2 Proteins involved in muscular
dystrophies may be altered in acquired cardiac disease.1
Although the causative biochemical defects often may be
present in both skeletal and cardiac muscle, in individual
types of muscular dystrophy the relative risk of involvement of skeletal and cardiac muscle may be different, with
important implications for management.
From the 1Institute of Human Genetics and 2Department of Cardiology, University of Newcastle upon Tyne, Newcastle upon Tyne,
United Kingdom; 3Departments of Biochemistry and Medical Genetics and Pediatrics, University of Manitoba, Winnipeg, Canada;
4
Department of Paediatrics, Imperial College, Hammersmith Hospital Campus, London, United Kingdom; 5Department of Pediatrics and Paediatric Neurology, University Hospital Essen, Germany;
6
Departments of Clinical Neurology and 7Cardiology, Radcliffe Infirmary and John Radcliffe Hospital, Oxford, United Kingdom.
Received May 3, 2004, and in revised form Jul 1. Accepted for
publication Aug 5, 2004.
Published online Oct 28, 2004, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20283
Address correspondence to Dr Bushby, Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon
Tyne NE1 3BZ, United Kingdom. E-mail: kate.bushby@ncl.ac.uk
738
Annals of Neurology
Vol 56
No 5
November 2004
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