From the Laboratoire CERTO, Faculté de
Médecine Necker, 156 rue de Vaugirard, 75015 Paris, France,
§ Service de Neuropédiatrie, Hôpital St. Vincent
de Paul, 79 avenue Denfert Rochereau, 75014 Paris, France, ¶ CNRS
UPR1524, 9 rue de Hetzel, 92190 Meudon, France,
Instituto
Nazionale Neurologico "Carlo Besta," via Celoria 11, 20133 Milano,
Italy, and ** INSERM U30, Hôpital Necker, 149 rue de Sèvres,
75015 Paris, France
Received for publication, January 16, 2001
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ABSTRACT |
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The gene SURF1 encodes a
factor involved in the biogenesis of cytochrome c oxidase,
the last complex in the respiratory chain. Mutations of the
SURF1 gene result in Leigh syndrome and severe cytochrome
c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh
syndrome revealed different SURF1 mutations in four of
them. Only these four cases had associated demyelinating neuropathy.
Three mutations were novel splicing-site mutations that lead to the
excision of exon 6. Two different novel heterozygous mutations were
found at the same guanine residue at the donor splice site of intron 6;
one was a deletion, whereas the other was a transition
[588+1G>A]. The third novel splicing-site mutation was a
homozygous [516-2_516-1delAG] in intron 5. One patient only had a
homozygous polymorphism in the middle of the intron 8 [835+25C>T].
Western blot analysis showed that Surf1 protein was absent in all four
patients harboring mutations. Our studies confirm that the
SURF1 gene is an important nuclear gene involved in the
cytochrome c oxidase deficiency. We also show that Surf1
protein is not implicated in the assembly of other respiratory chain
complexes or the pyruvate dehydrogenase complex.
Leigh syndrome (LS),1 or
subacute necrotizing encephalomyelopathy (MIM 256000), is a
progressive and often fatal neurological disorder in young children,
characterized by bilaterally symmetrical necrotic lesions in the brain
stem and basal ganglia (1). A common associated biological feature is
hyperlactatemia. It is a genetically heterogeneous disease, caused by
defects in the enzymes normally involved in the respiratory chain and
in mitochondrial energetic metabolism (e.g.
pyruvate dehydrogenase). One of the most common enzymatic defects is
the deficiency of cytochrome c oxidase (COX), complex IV of
the mitochondrial respiratory chain. Recently, mutations in the nuclear
SURF1 gene, which encodes a factor involved in COX
biogenesis, have been identified in patients with LS (2, 3). This gene
is located on chromosome 9q34, consists of nine exons, and encodes a
protein of 300 amino acids. We have studied 58 patients who were
thought to have LS based on their clinical features and on magnetic
resonance imaging (MRI), which showed lesions on the basal ganglia and
the brain stem. 15 of these patients presented cytochrome
c oxidase deficiency, seven were typical LS, but only four
had a severe COX deficiency (LScox). 15 other patients were
defective in pyruvate dehydrogenase with mutations in the E1 Patients--
Patient 1 was the first child of
non-consanguineous parents. At nine months, she presented delayed
growth, difficulties in swallowing, severe hypotonia, trunk ataxia,
nystagmus. She had optic atrophy and a demyelinating peripheral
neuropathy. Brain MRI showed symmetric lesions of the basal ganglia and
brain stem. She had hyperlactatemia (4.9 mmol/liter; normal
value is below 2 mmol/liter). She died at 4 years.
Patient 2 was born to non-consanguineous parents. She developed a mild
psychomotor delay with progressive ataxia, ophthalmoplegia and
retinopathy, and a demyelinating neuropathy. Recurrent vomiting attacks are associated with increasing ptosis and ataxia. At 2 years, brain MRI showed lesions in the brain stem and thalamus and
cerebellar atrophy. High levels of lactate were found in blood (4.14 mmol/liter). She is still alive at 14 years of age.
Patient 3 was the third child of non-consanguineous parents. The mother
had two miscarriages. A sister died at 2 months of age with the
diagnosis of sudden death. At 1 year of age the patient had a severe
growth delay, a trunk ataxia, pyramidal signs, dystonia, and abnormal
ocular movements. She had difficulties in swallowing and presented
recurrent respiratory distress. She had optic atrophy, demyelinating
neuropathy, and hyperlactatemia (4.75 mmol/liter). MRI showed
bilateral, symmetrical lesions of the basal ganglia and the brain stem.
She died at the age of 2 years.
Patient 4 was born to consanguineous parents and had a cousin affected
by the same condition. She had a metabolic encephalopathy, which was
revealed at 3 months of age by severe hypotonia, hyperlactatemia, and
nystagmus. The MRI exhibited bilateral lesions in the caudate nuclei.
Patient 5 was a Lybian Arab male baby born from first cousin parents.
Persistent vomiting, profound developmental delay, and severe hypotonia
were the main clinical features leading to his death at 18 months. The
MRI showed lesions typical of LS.
Patient 6 was the only child of consanguineous parents (common
grandmother). Growth retardation and hypotonia were observed at 4 months. At the age of 6 years he presented a progressive encephalomyelopathy, with cortical atrophy and necrosis of the basal
ganglia and brain stem. The lactate level in the blood was 3.2 mM and 5.4 mM in the cerebrospinal fluid.
Patient 7, a girl, was born to consanguineous parents (first cousin
parents) and had a brother affected by the same disorder (developmental
delay and hyperlactatemia). Both showed typical Leigh syndrome features
confirmed by cerebral tomography scan examination. MRI detected
lesions in the basal ganglia. They are still alive at the age of 10 and
7 years, respectively.
Biochemical Studies--
Enzymatic assays were carried out on
mitochondria isolated from muscle or lymphoblasts (4) of all patients
except patient 5, for whom assays were performed in muscle homogenate.
The activities of the respiratory chain complexes were measured
spectrophotometrically as described previously (5). Pyruvate
dehydrogenase complex (PDHC) activity was measured in lymphoblastoid
cell lines by the release of [14C]CO2 from
[1-14C]pyruvic acid (6).
For Western blot analysis, proteins derived from lymphoblastoid cell
lines (patients 1, 2, 3, 4, and 7) or fibroblasts (patient 5) were
separated by 10% SDS polyacrylamide gel electrophoresis under reducing
conditions and transferred onto nitrocellulose membrane (7). The blots
were probed with polyclonal antibodies directed against the PDH
holoenzyme or against the C terminus of the Surf1 protein
(peptide-Y-13-V) (3).
DNA Genomic Studies from Cultured Lymphoblasts--
Genomic DNA
was purified by use of the DNAzol Reagent (Life Technologies, Inc.) as
described by the manufacturer. The nine exons of the SURF1
gene were amplified as described by Tiranti et al. (3) and
sequenced in a commercially available, automated sequencer (Genome
Express, Grenoble, France).
RNA Extraction and RT-PCR--
Total RNA was extracted from
cultured lymphoblasts with an RNA-plusTM kit (Quantum
Bioprobe, Quebec, Canada). Each RNA sample (5 µg) was
reverse-transcribed using d(T)18 primers and a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech) according to
the manufacturer's instructions. The coding sequence from exon 4 to exon 9 was amplified by PCR as described by Poyau et al.
(8). We also used three new primer pairs; the first pair was chosen to
study patient 3. For161 (5'-CAAAAGCGGAAGATGACTCC-3') and Rev558 (5'-GGTTCGTTCCCAGGAAGAAA-3') amplified the cDNA region from base 181 to base 538. For RT-PCR of patient 5, a first PCR was done using
the primers For-9 (5'-CCGGGTGCGATGGCGGCGGTGGCTGCGTTGCAG-3') and
Rev955 (5'-CAGTAGCACATGATCCAGCATAAAG-3'). A "nested" PCR was then
carried out on this first product with For435
(5'-GGCCCGGGAGGGCGGCCTC-3') and Rev927
(5'-CAGTCTTGAAATACTGCATTATCCAGGG-3'). The PCR products were analyzed by
agarose gel electrophoresis (2%), stained with ethidium bromide (0.5 µg/ml), and visualized by UV transillumination. cDNA fragments
containing putative deletions in the gene were purified from PCR
products by use of a Qiaquick PCR extraction kit (Qiagen, Courtaboeuf,
France) and sequenced as above.
We studied seven unrelated patients with typical LS. Four of them
(patients 1, 2, 3, and 5) had dramatically reduced COX activity. This decrease was obvious in mitochondria purified from fresh muscle
biopsy (Table I) and
lymphoblastoid cell lines (Table II).
Patients 4, 6, and 7 presented only a slight decrease of COX activity
in muscle (Table I).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-PDH
and Hs-PDX1 genes, 11 were found to have a complex I
deficiency, and four had the clinical features of the maternal
inheritance Leigh syndrome with a neuropathy ataxia retinitis
pigmentosa mutation in the ATPase6 gene of the mitochondrial DNA. We
identified six mutations, including three new splicing-site mutations,
in the SURF1 gene in the four patients with typical LS and
severe COX deficiency.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
COX activity in muscle mitochondria (from controls and patients 1, 2, 3, and 4) and muscle homogenate (controls and patient 5), expressed in
milliUnits/mg of protein and in COX/citrate synthase ratio
COX activity of mitochondria purified from lymphoblastoid cell lines
(control, 1, 2, and 3)
No band corresponding to the Surf1 protein was found by Western blot
analysis of the four typically LScox patients 1, 2, 3, and
5 (Fig. 1). Conversely, a Surf1-specific band was detected in patients 4 and 7. The intensity of the band was
similar to that found in the controls. A nonspecific band just below
the Surf1 protein was equally intense in all samples, suggesting that
the quantity of total proteins was approximately the same.
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These results prompted us to look for mutations in the SURF1
gene by directly sequencing PCR fragments obtained with primers described previously (3). Six different mutations were found in the
four patients who lacked the Surf1 protein (patients 1, 2, 3, and 5)
(Fig. 2). Three of these mutations have
already been described. The most frequent mutation reported in
SURF1 gene, the [312_321del311_312insAT], was found in
patients 1 and 2 (2, 3); the [737T>C] mutation was detected in
patient 2 (8), and a mutation in the splicing donor site of intron 3 [240+1G>T] was found in patient 3 (9).
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Three new mutations were found, two of which involved the first base of intron 6: a deletion of the G in position 588+1 was detected in patient 1, and a transition [588+1G>A] was found in patient 3. Patient 5 harbored an AG deletion in the acceptor site of intron 5, [516-2_516-1delAG]. Patients 1, 2, and 3 were compound heterozygotes, whereas patient 5 was homozygous for the mutation.
We carried out RT-PCR to study the consequences of these mutations on
the RNA sequence on patients 1, 2, 3, and 5 (Fig.
3A). A single band of 730 bp
was obtained in samples from patient 2 and a control. However, using the same primers we obtained two bands
from the samples of patients 1 and 3. The additional band was ~60 bp
smaller than the control band in both cases (660 bp).
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The sequencing of these RT-PCR products of patients 1 and 3 permitted us to conclude that the two mutations in the first base of the donor splice site of intron 6 (point mutation or deletion) led to the complete excision of exon 6. The second mutation [240+1G>T] in patient 3 has been described before (9), but its consequence on the RNA sequence was unknown. RT-PCR with the forward primer used to amplify exons 4 to 9 could not detect this mutation, because the mutation was within the primer 4 region. Thus, we used two new primers (For261 and Rev558) that annealed on either side of intron 3. The PCR product (460 bp) obtained with the new primers was 80 bp longer than in the control (380 bp) (Fig. 3B), and the additional sequence corresponded with that of intron 3. For patient 5 we performed nested PCR with two other primer pairs that normally give a 520-bp fragment, and we obtained a unique band of ~450 bp (Fig. 3C). The sequencing of this RT product revealed that the [516-2_516-1delAG] mutation at the acceptor site of intron 5 led to the complete excision of the 73 bp of exon 6, as in patients 1 and 3.
We sequenced the genomic DNA PCR products of patients 4, 6, and 7. We found no mutations in DNAs from patients 6 or 7, but we could detect a homozygous polymorphism in intron 8, [835+25C>T] in the DNA of patient 4.
The common mitochondrial DNA mutations (myoclonic epilepsy and ragged red fibers, mitochondrial encephalopathy lactic acidosis stroke-like episodes, neuropathy ataxia retinitis pigmentosa, and deletions) associated with LS were ruled out in all patients by restriction fragment length polymorphisms and Southern blot analysis.
To determine whether the Surf1 protein is implicated in the stability
of other mitochondrial complexes, we carried out enzymatic assays on
the respiratory chain complexes and the pyruvate dehydrogenase complex
(PDHC) in patients 1, 2, and 3 (who lack the Surf1 protein). Complex II
and PDHC activity (Table II), as well as the activities of complexes I
and III (data not shown), were comparable with those of controls.
Western blot analysis with an antibody directed against PDHC showed
subunit-specific bands of similar size and intensity in samples from
patients and controls (data not shown).
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DISCUSSION |
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In this study we found that LScox was associated with SURF1 mutations in four of the seven patients (patients 1, 2, 3, and 5). Their clinical presentations were similar to those previously reported, but the incidence of mutations in our study (57%) was between that found by the studies done by Tiranti et al. (9) (75%) and Sue et al. (10) (25%). These differences can be explained by the biochemical and genetic heterogeneity of LS. Of the 58 cases of suspected LS that we studied, 15 had a deficiency in COX activity, 15 had pyruvate dehydrogenase deficiency, 11 were suspected of having complex I defects, and four had neuropathy ataxia retinitis pigmentosa mutations. However, we can confirm that the four cases of typical Leigh syndrome were associated with severe cytochrome oxidase deficiency and have SURF1 mutations (patients 1, 2, 3, and 5).
Patients 4, 6, and 7 were also considered to be typical LS cases based on the stringent inclusion criteria, which include the presence of a progressive neurological disease, elevated levels of cerebrospinal lactic acid, and typical MRI abnormalities involving the basal ganglia and brain stem. However, they only had a partial decrease of COX activity, and patients 6 and 7 had no mutations in the SURF1 gene. Patient 4 was homozygous for the [835+25C>T] mutation. Interestingly, we found a partial COX defect in the muscles of her cousin; this cousin had the same symptoms and was heterozygous for the same mutation. This change is probably a polymorphism with no pathological significance.
We did not notice any phenotypic differences between the three patients (1, 3, and 5) with splicing-site mutations in the SURF1 gene. They had early onset severely delayed growth, optic atrophy, neuropathy, and died very early (between 18 months and 4 years). Residual COX activity was always less than 25% of normal. This can be explained by the nature of the mutations and their effect on the protein; patient 1 had both the [312_321del311_312insAT] in exon 4 (2, 3), which leads to a truncated protein (with a stop codon at position 105 in the protein), and a deletion in the first base of intron 6, [588+1delG], which results in the production of an RNA in which the skipping of exon 6 predicts the synthesis of a truncated protein (with a stop codon at position 183). Likewise, patient 3 had two splicing mutations, a mutation in the first base of intron 3, [240+1G>T], which results in an mRNA that contains the entire intron 3, with a stop codon after amino acid 89. The second mutation is located in the first base of intron 6; this mutation [588+1G>A] has the same result on the RNA and on the protein as the [588+1delG] mutation described for patient 1. The amplification of only one abnormal band, corresponding to an mRNA retaining intron 3 (Fig. 3B), may be because of the extreme instability of the abnormal RNA that lacks exon 6. Indeed, we obtained a very faint band corresponding to this allele when the first primer pair was used for RT-PCR analysis (Fig. 3A).
As patient 5 was homozygous for the [516-2_516-1delAG] mutation in intron 5, he had only one species of RNA in which exon 6 was excised, giving a truncated protein with a stop codon at position 183.
Three of the six splicing-site mutations have never been described before, two of them are in intron 6 of patients 1 and 3, and the third is the homozygous mutation in intron 5 of patient 5. All of these mutations lead to the excision of exon 6 in mRNA.
Paradoxically although the first base of the 5' splice site is mutated in both the splicing-site mutations in patient 3, the consequences seem to be different. One leads to the retention of the mutated intron 3, whereas the other leads to the excision of the preceding exon, exon 6. The most common effect of a 5' splice-site mutation is the excision of the preceding exon as observed in the latter case. However, inclusion of the mutant intron is rare. We can explain the retention of intron 3, because intron 3 is generally the last intron to be excised. As a result, the spliceosome machinery does not find any adjacent normal 5' splicing site, and no splicing occurs (11).
Patient 2, with no splicing-site mutation, had a different phenotype to the other patients (patients 1, 3, and 5). Even though she had typical features of Leigh symptom, she was still alive at 14 years of age. This is probably because of the fact that she had a less severe COX deficiency. We found about 40% residual COX activity in both muscle and lymphoblastoid mitochondria. Patient 2 had the [312_321del311_312insAT] mutation, which results in a truncated protein, and also carried the missense [737T>C] mutation, which transforms Ile246 (ATT) to Thr (ACT). The protein is probably abnormal and too unstable to be detected by Western blot. It is an interesting case, because it is the first described in the literature with a phenotype of a late Leigh syndrome associated with two SURF1 mutations.
Our results suggest that respiratory chain complexes (other than complex IV) and PDHC, are normally assembled in patients in which the Surf1 protein is either lacking or abnormal. The Surf1 protein is currently only known to be involved in complex IV assembly.
Patients 4, 6, and 7 had all the clinical features of LScox, but no mutation in the SURF1 gene was found. This suggests that although most cases of LScox can be explained by homozygous or compound heterozygous mutations in the SURF1 gene, mutations in other genes may lead to LScox.
In this paper, we revealed new splicing-site mutations that are well
correlated with the severity of the illness and the dramatic decrease
in COX activity. Further studies are necessary to characterize the COX
deficiency affecting patients 4, 6, and 7. However, we have confirmed
that SURF1 mutations are responsible for Leigh syndrome
associated with severe COX deficiency (12) and found a new phenotype of
late Leigh syndrome associated with SURF1 mutations never
described previously (2, 3, 8, 13).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. G. Ponsot for helpful collaboration and C. Galimberti for technical assistance. We thank Association Retina France and Université René-Descartes.
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FOOTNOTES |
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* This work was supported in part by Contract QLG2-CT-1999-00660 from the European Union. M. O. P. is supported by a grant from the Ministère Français de la Recherche et de l'Enseignement Superieur, and R. D. is supported by a EC postdoctoral grant.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratoire
CERTO, Faculté de Médecine Necker, 156 rue de Vaugirard,
75015 Paris, France. Tel.: 1 40 61 53 55; Fax: 1 40 61 54 74; E-mail:
marsac@necker.fr.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M100388200
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ABBREVIATIONS |
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The abbreviations used are: LS, Leigh syndrome; COX, cytochrome c oxidase; LScox, Leigh syndrome associated with highly reduced COX activity; MRI, magnetic resonance imaging; PDH, pyruvate dehydrogenase; PDHC, pyruvate dehydrogenase complex; RT-PCR, reverse transcriptase polymerase chain reaction; bp, base pair.
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REFERENCES |
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1. | Leigh, D. (1951) J. Neurol. Neurosurg. Psychiatry 14, 216-221 |
2. | Zhu, Z., Yao, J., Johns, T., Fu, K., De Bie, I., Macmillan, C., Cuthbert, A. P., Newbold, R. F., Wang, J., Chevrette, M., Brown, G. K., Brown, R. M., and Shoubridge, E. A. (1998) Nat. Genet. 20, 337-343[CrossRef][Medline] [Order article via Infotrieve] |
3. | Tiranti, V., Hoertnagel, K., Carrozzo, R., Galimberti, C., Munaro, M., Granatiero, M., Zelante, L., Gasparini, P., Marzella, R., Rocchi, M., Bayona-Bafaluy, M. P., Enriquez, J. A., Uziel, G., Bertini, E., Dionisi-Vici, C., Franco, B., Meitinger, T., and Zeviani, M. (1998) Am. J. Hum. Genet. 63, 1609-1621[CrossRef][Medline] [Order article via Infotrieve] |
4. | Trounce, I. A., Kim, Y. L., Jun, A. S., and Wallace, D. C. (1996) Methods Enzymol. 264, 484-509[Medline] [Order article via Infotrieve] |
5. | Degoul, F., Nelson, I., Lestienne, P., Francois, D., Romero, N., Duboc, D., Eymard, B., Fardeau, M., Ponsot, G., Paturneau-Jouas, M., Chaussain, M., Leroux, J. P., and Marsac, C. (1991) J. Neurol. Sci. 101, 168-177[CrossRef][Medline] [Order article via Infotrieve] |
6. | Bonne, G., Benelli, C., De Meirleir, L., Lissens, W., Chaussain, M., Diry, M., Clot, J. P., Ponsot, G., Geoffroy, V., M., Leroux, J. P., and Marsac, C. (1993) Pediatr. Res. 33, 284-288[Abstract] |
7. | Geoffroy, V., Poggi, F., Saudubray, J. M., Fouque, F., Lissens, W., Lindsay, G., Sanderson, S. J., De Merleir, L., Marsac, C., and Benelli, C. (1996) Pediatrics 97, 267-272[Medline] [Order article via Infotrieve] |
8. | Poyau, A., Buchet, A., Bouzidi, M. F., Zabot, M. T., Echenne, B., Yao, J., Shoubridge, E. A., and Godinot, C. (2000) Hum. Genet. 106, 194-205[CrossRef][Medline] [Order article via Infotrieve] |
9. | Tiranti, V., Jaksch, M., Hofmann, S., Galimberti, C., Hoertnagel, K., Lulli, L., Freisinger, P., Bindoff, L., Gerbitz, K. D., Comi, G. P., Uziel, G., Zeviani, M., and Meitinger, T. (1999) Ann. Neurol. 46, 161-166[CrossRef][Medline] [Order article via Infotrieve] |
10. | Sue, C. M., Karadimas, C., Checcarilli, N., Tanji, K., Papadopoulou, L. C., Pallotti, F., Guo, F. L., Shanske, S., Hirano, N., De Vivo, D. C., Van Coster, R., Kaplan, P., Bonilla, E., and Di Mauro, S. (2000) Ann. Neurol. 47, 589-595[CrossRef][Medline] [Order article via Infotrieve] |
11. | Lewin, B. (1997) Gene VI , pp. 889-920, Oxford University Press, Oxford |
12. | Péquignot, M. O., Dey, R., Zeviani, M., Tiranti, V., Godinot, C., Poyau, A., Sue, C., Di Mauro, S., Abitbol, M., and Marsac, C. (2001) Hum. Mutat., in press |
13. | Teraoka, M., Yokoyama, Y., Ninomiya, S., and Seino, Y. (1999) Hum. Genet. 105, 560-563[CrossRef][Medline] [Order article via Infotrieve] |