An 11 base pair duplication in exon 6 of the SMN gene produces a type I spinal muscular atrophy (SMA) phenotype: further evidence for SMN as the primary SMA-determining gene
An 11 base pair duplication in exon 6 of the SMN gene produces a type I spinal muscular atrophy (SMA) phenotype: further evidence for SMN as the primary SMA-determining geneD. Williams Parsons, Patricia E. McAndrew, Umrao R. Monani1, Jerry R. Mendell2, Arthur H. M. Burghes1,2,3,* and Thomas W. Prior
Department of Pathology, Molecular Pathology Laboratory, 1Department of Molecular Genetics, College of Biological Sciences, 2Department of Neurology and 3Department of Medical Biochemistry, The Ohio State University College of Medicine, Columbus, OH 43210, USA
Received May 12, 1996;Revised and Accepted August 15, 1996
The gene for autosomal recessive spinal muscular atrophy (SMA) has been mapped to 5q12 in a region that contains repeated markers and genes. Three cDNAs that detect deletions in SMA patients have been reported. One of these, the survival motor neuron (SMN) cDNA, is encoded by two genes (SMNTand SMNC) which are distinguished by base changes in exons 7 and 8. Exon 7 of the SMNTgene is not detectable in ~95% of SMA cases, due either to deletion or sequence conversion. There is limited information on the mutations in SMA patients that have detectable SMNT: these are critical for confirmation of SMNTas the SMA gene. Using SSCP analysis of the SMN exons we screened our SMA patients that possess at least one intact SMNTallele for mutations in SMNT. We identified one type I SMA patient with an 11 bp duplication in exon 6 which causes a frameshift and premature termination of the deduced SMNT protein. Dosage and SSCP analysis of SMNTin this family indicated that the father contributed a SMNT-deleted allele to the affected child whereas the mother passed on the 11 bp exon 6 duplication SMNT allele. Analysis of RNA by RT-PCR conclusively demonstrated that the 11 bp duplication is associated with the SMNTlocus and not SMNC. This mutation provides strong support for SMN as the SMA-determining gene and indicates that disruption of SMNT on its own is sufficient to produce a severe type I SMA phenotype.
Proximal spinal muscular atrophy (SMA) is characterized by degeneration of [alpha]-motor neurons in the anterior horn of the spinal cord. SMA has an estimated incidence of 1 in 10 000 live births, with a carrier frequency of ~1 in 40 individuals (1 ). Childhood onset SMA is classified into three groups on the basis of age of onset and clinical course (2 ). Type I SMA (Werdnig-Hoffmann disease) is the most severe form, with clinical onset before the age of six months and death generally occurring within the first two years. Type II SMA is of intermediate severity: onset occurs before 18 months of age, and patients never gain the ability to walk. Type III SMA (Kugelberg-Welander disease) is the mildest form, with onset after 18 months and patients able to both stand and walk on their own.
All three forms of childhood SMA have been mapped to chromosome 5q11.2-q13.3 by linkage analysis (3 -11 ). A number of physical maps of this area have been produced, but no consensus map has been achieved due to the complex genomic organization of the region (12 -18 ), which contains numerous repeated sequences, including polymorphic markers and genes (15 -20 ). Some of these multicopy markers were used to further refine the position of the SMA gene by the demonstration of linkage disequilibrium (15 ,19 -21 ). Analysis of these markers showed specific alleles associated with SMA, loss of a copy of the marker in severe SMAchromosomes, and deletion of all copies of marker alleles on some SMAchromosomes (20 -22 ).
Three cDNAs that identify deletions in the SMA region have been reported: the survival motor neuron (SMN) gene (18 ); the neuronal apoptosis inhibitory protein (NAIP) gene (16 ); and the XS2G3 cDNA (17 ). The NAIP and XS2G3cDNAs detect the same deletion in ~50% of type I SMA patients and a smaller proportion of type II and III individuals; they also rarely detect deletions in carrier parents with no obvious clinical symptoms (16 ,17 ,22 ).
The third cDNA, survival motor neuron (SMN), is encoded by two nearly-identical genes: centromeric SMN (SMNC) and telomeric SMN (SMNT). SMNCand SMNTare both transcribed, and can be distinguished by two single nucleotide sequence variations in exons 7 and 8, neither of which alter the coding sequence (18 ). Absence of SMNTexon 7 has been reported in ~95% of SMA patients, due either to conversion of SMNT to SMNCor deletion of SMNT(18 ,23 -25 ). The presence of large deletions in a homozygous state is strongly correlated with the severe SMA phenotype (22 ). Conversion can be demonstrated in patients who have exon 8 of SMNT but lack detectable exon 7 of SMNT(18 ,25 ): in our studies these conversion events predominate in mild patients (25 ). In individuals that do not retain SMNTexon 8, conversion can be implicated on one chromosome of mild patients from both dosage (24 ) and marker studies (20 ,22 ). This would indicate that conversion (as opposed to deletion) is a common mutation on mild chromosomes. A limited number of small mutations in SMNThave been reported, including a 4 bp deletion in exon 3 (18 ,23 ). In order to confirm SMN as the SMA gene and determine whether mutations in SMNTare solely responsible for the SMA phenotype, it is extremely important to identify additional small mutations in this gene.
In the present study, we have used SSCP analysis to screen the SMN gene for mutations in our population of SMA-like individuals who retain at least one copy of SMNT. We have identified a type I spinal muscular atrophy patient who possesses one SMNT allele containing an 11 bp duplication in exon 6 and one SMNT-deleted allele. RNA studies clearly indicate that the 11 bp duplication affects the SMNTlocus and not SMNC. This finding strongly supports SMNTas the SMA-determining gene, and indicates that mutations which disrupt production of full length protein from the SMNTlocus can be solely responsible for producing a severe type I SMA phenotype.
Single strand conformation polymorphism (SSCP) analysis of SMN exons in the type I SMA patient #367 demonstrated an abnormal exon 6 SSCP pattern (Fig. 1 A). A previously reported PCR-based assay was used to confirm that the patient retained at least one copy of SMNTexons 7 and 8 (26 ); PCR amplification also demonstrated that exon 5 of NAIP was present (16 ). Sequence analysis of patient #367 exon 6 subclones revealed an 11 bp duplication of nucleotides 801-811 (TGC TGA TGC TT) of the SMN gene (Fig. 2 A and B). The duplication results in a frameshift and a premature termination codon 15 nucleotides downstream, predicting a SMN protein product containing seven altered amino acids prior to truncation. The duplication was absent in 100 unrelated healthy individuals (200 chromosomes) as well as in 60 non-SMNT deleted individuals who had been evaluated in our laboratory for a possible diagnosis of SMA (120 chromosomes).
The SMA gene is located in a region that contains many repeated sequences, including genes and markers (15 -20 ). Three cDNA clones from the SMA region detect deletions in SMA patients (16 -18 ). The cDNAs for NAIP and XS2G3 detect the same deletion in ~50% of type I patients (16 ,17 ). However, these cDNAs are also deleted in asymptomatic carriers of SMA (16 ,17 ,24 ,27 -29 ). The third cDNA, SMN, is encoded by two genes that exist in almost identical copies (SMNT and SMNC) which are only distinguishable by single base pair changes in exons 7 and 8 (18 ). In ~95% of SMA patients exon 7 of the SMNT gene is not detectable, which is usually assumed to be due to deletion of the SMNT gene (18 ,24 ,27 -29 ). However, analysis of patients who have no detectable SMNT exon 7 but do possess SMNT exon 8 indicates that conversion of SMNT to SMNC occurs (as opposed to deletion) in these cases (25 ). Therefore, the absence of SMNT can be due either to deletion or conversion of SMNT to SMNC.
Association studies with the marker AgI-CA revealed a clear correlation with phenotype: one copy of the marker on both chromosomes (or a 1,1 genotype) occurs more frequently in type I SMA, whereas a 1,2 genotype is more frequent in type II SMA (20 ,22 ). In addition, we have shown that a 1,1 genotype of AgI-CA correlates with type I SMA chromosomes that have a deletion of NAIP, indicating that large homozygous deletions are correlated with type I SMA (22 ). These results would also indicate that type II SMA patients have one chromosome which has a large deletion (single copy of AgI-CA) and one chromosome which does not (two copies of AgI-CA); this could either be due to a small deletion or conversion of SMNT to SMNC. Analysis of patients who retain exon 8 of SMNT but not exon 7 clearly indicates that conversion does occur in the SMA locus (18 ,25 ); in our studies this mutation predominates on mild chromosomes (25 ). In cases where SMNT exon 8 is not present, it is more difficult to distinguish deletion from conversion events. However, initial dosage studies using the ratio of SMNC to SMNT strongly imply that a large number of type II and III chromosomes do possess conversion alleles as opposed to deletions (24 ). We would suggest that the simplest explanation for these findings is that severe SMA is caused by the inability of the SMNT locus to produce a critical isoform (two null alleles for the critical isoform), whereas milder SMA (type II and III) results from a severe null allele for the critical isoform in combination with a mild allele. It would apppear likely that sequence converted alleles predominate in types II and III SMA (24 ,25 ). Sequence converted alleles could also occur in severe SMA; however, in this case the SMNT gene would still be nonfunctional due either to the introduction of additional mutations into the SMNT gene or an effect on its expression. In this report we show by RNA studies that in the type I patient with an 11 bp duplication in exon 6 of SMNT there is no RNA capable of producing a full length SMN protein from the SMNT locus, which is consistent with the hypothesis that type I SMA is caused by null mutation of a critical isoform from the SMNT gene. Direct proof of the hypothesis that a critical isoform of SMNT is lacking in the motor neurons of type I SMA patients must await further studies, particularly at the protein level.
Apart from sequence conversion and deletion, there is extremely limited information regarding other SMA-causing mutations. In this paper we have analyzed our patients who do not lack SMNT for mutations in SMNT. Lefebvre et al. have reported a missense mutation in exon 6 and two short deletions in the consensus splice sites of introns 6 and 7 that are assumed to cause SMA since they are not observed in normal controls (18 ). However, the most conclusive mutation to date in implicating SMNT as the true SMA-determining gene is a 4 bp deletion in exon 3 (23 ). This deletion was originally identified in a type II consanguineous family, but has also been shown to occur in one type I and two type III SMA patients. This mutation would be predicted to disrupt the reading frame of the SMNT gene and produce a severe phenotype; however, it results in all three SMA phenotypes. If we assume that the other allele in the type I patient is null for the critical isoform and in the type III patient is partially functional, then the situation for the 4 bp deletion allele is similar to that reported for cystic fibrosis (CF): the missense R117H mutation is observed in three phenotypic variants of CF, with the severity of the phenotype modulated by intron variants that affect splicing efficiency (30 ). Therefore in the type I patient, exon 3 containing the 4 bp allele would not be skipped efficiently, resulting in two null alleles for the critical isoform, whereas in the milder type II consanguineous patient, sufficient skipping occurs to produce some product.
It is extremely important to identify additional mutations that would clearly indicate that SMNT is the SMA gene. To date no duplication mutations have been reported in SMA, although they may be predicted to occur due to the repetitive nature of this region. In this paper we show that an 11 bp duplication occurs in exon 6 of the SMNT gene and causes type I SMA. The demonstration of duplication in SMA indicates that this type of mutation does occur. It is also likely that whole exon duplications occur in the SMNT gene; however, these would not be detected by our SSCP screening assay. Interestingly, whole exon mutations would not be predicted to disrupt translational reading frame and therefore could give rise to a mild phenotype. The frequency of homozygous absence of SMNT in type III patients seems to be slightly lower than in types I and II SMA(27 ). It is possible that this slightly lower frequency of detectable SMN alteration in type III SMA might be accounted for by whole exon duplications being type III SMA alleles.
Dosage analysis of SMNT exon 7 in our population of potential SMA patients that retain at least one copy of exon 7 SMNT indicates that four patients (and only one type I patient) are due to mutations at the 5q12 locus. That single type I SMA patient has an 11 bp duplication in exon 6 of the SMNT gene, which clearly disrupts the reading frame of SMNT and would appear to indicate that mutations which disrupt SMNT can cause severe SMA. This would be consistent with the hypothesis that mutations in SMNT are solely responsible for SMA: a null for critical isoform or truncated SMNT allele in the homozygous state gives rise to severe SMA without the need for deletion of additional genes. This patient does not lack exon 5 of the NAIP gene, yet has a severe SMA phenotype. We cannot rule out the possibility that this 11 bp duplication allele could be found in a patient with a milder SMA phenotype: we have only identified the mutation in a type I SMA patient. Mild (type II or III) SMA may be caused by one chromosome containing a partially functional allele. These mild alleles can probably be modified by genetic background or modifier genes, thus explaining the exceptional type II/III families where an apparently normal sibling has absence of SMNT (27 -28 ,31 -32 ).
In conclusion, we have shown that an 11 bp duplication which disrupts SMNT is a type I SMA allele. This would indicate that mutations in SMNT are solely responsible for SMA, and that it is likely that null mutations for a critical isoform produced by the SMNT locus give rise to severe SMA. Confirmation of the lack of a critical isoform in motor neurons from the SMNT locus must await further studies at the protein level.
The patients selected for this study were from two groups: families that were collected for genetic studies (whom we have described previously) and patients sent to our laboratory for diagnostic purposes. The first group of patients fulfilled the criteria for SMA defined by the international SMA consortium (2 ). Patient #367 is from this first group of patients and has the classical features of type I SMA: confirmatory EMG and muscle pathology, with death at the age of 6 months. Sixty patients that had at least one copy of exon 7 of the SMNT gene were analysed for SMN alterations in exons other than exon 7. However, dosage assays for SMNT exon 7 are only consistent with four of these patients being SMA caused by mutations at the 5q12 locus (most of these SMA patients would be expected to be compound heterozygotes).
SSCP analysis. SSCP analysis was performed according to the procedure of Orita et al. (33 ) with minor modifications. PCR primers used for amplification of SMNT exons were based on intron/exon boundary DNA sequences reported by Burglen et al. (34 ). The sequences of PCR primers 541C618 (5'-CTC CCA TAT GTC CAG ATT CTC TTG-3') and EX63 (5'-AAG AGT AAT TTA AGC CTC AGA CAG-3') used for amplification of SMN exon 6 were kindly provided by Dr Judith Melki (Hopital des Enfants Malades, Paris, France). DNA from peripheral blood leukocytes (100 ng) was amplified by PCR in a 25 [mu]l reaction mixture containing 200 [mu]M dNTPs, 1 U Taq polymerase (Perkin Elmer), 30 ng of each primer, 2.5 mM MgCl2, 2.5 [mu]l 10* PCR buffer (670 mM Tris, 100 mM [beta]-mercaptoethanol, 166 mM ammonium sulfate, 67 [mu]M EDTA, 0.5 [mu]g/ml BSA), and 0.1 [mu]l [[alpha]-32P]ATP (10 [mu]Ci/[mu]l; Amersham). The PCR reaction consisted of an initial denaturation step at 94oC for 2 min followed by 35 cycles of 30 s at 94oC, 30 s at 58oC and 30 s at 72oC. There was no final extension step. Four [mu]l of PCR product was added to an equal volume of loading dye (95% formamide, 10 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol). The samples were denatured at 95oC for at least 5 min and placed on ice until loading on a 0.5* MDE gel (AT Biochem). The gels were run at 6 W for 15 h at 4oC, then transferred onto blotting paper (Alhstrom), dried and exposed to Hyperfilm-MP (Amersham) for 12-24 h at -70oC.PCR amplification of SMN. DNA was extracted from peripheral blood and skeletal muscle using a salting out protocol (35 ). SMN exon 6 was PCR amplified using 30 ng each of primers 541C618 and EX63 (as for SSCP analysis of exon 6) in a PCR reaction mixture of 50 [mu]l containing 1 U Taq polymerase (Perkin Elmer), 0.5 mM dNTPs, 3 mM MgCl2 and 5 [mu]l 10* PCR buffer. The PCR reaction consisted of an initial denaturation step at 95oC for 5 min followed by 35 cycles of 1 min at 95oC, 2 min at 55oC and 3 min at 72oC. A final extension of 8 min at 72oC was performed. The PCR products were visualized on ethidium bromide-stained 8% polyacrylamide gels: the mutant exon 6 allele migrated more slowly than the smaller normal allele, allowing the two to be easily distinguished (a heteroduplex band was also seen on the gel when the mutant allele was present). The presence of SMNT exons 7 and 8 was confirmed using a previously-described protocol (26 ).PCR amplification of NAIP. PCR amplification of NAIP exon 5 was performed using primers described by Roy et al. (16 ). Reactions were carried out in 2.5 mM MgCl2 buffer, with an initial denaturation step of 3 min at 94oC, followed by 35 cycles of 94oC for 30 s, 58oC for 30 s and 72oC for 30 s. There was no final extension step. The PCR products were visualized on ethidium bromide-stained 2% agarose gels and scored for the presence or absence of exon 5.Dosage analysis of SMNT and SMNC. PCR amplification of SMN exon 7 was performed using primers R111 and X7-DraI described by Lefebvre et al. (18 ) and van der Steege et al. (26 ). PCR amplification of CFTR exon 4 was performed using primers CF621-F and CF621-R (36 ). R111 and CF621-F primers were end-labelled with [[gamma]-32P]ATP (10 [mu]Ci/[mu]l; Amersham). DNA from peripheral blood leukocytes (200 ng) was amplified by PCR in a 25 [mu]l reaction mixture containing 200 [mu]M dNTPs, 0.5 U Taq polymerase (United States Biochemicals), 30 ng of each primer, 3 mM MgCl2 and 2.5 [mu]l 10* PCR buffer (United States Biochemicals). The PCR reaction consisted of an initial denaturation step at 95oC for 5 min followed by 16 cycles of 1 min at 95oC, 2 min at 55oC and 3 min at 72oC. Eight [mu]l of PCR product were digested overnight at 37oC using 20 U DraI (New England Biolabs) and 1 [mu]l of the buffer supplied by the manufacturer. The digestion products were combined with 5 [mu]l loading dye and electrophoresed on a 6% denaturing polyacrylamide gel. The gel was dried and exposed to Hyperfilm-MP (Amersham) for 16 h.
RT-PCR. Total RNA was isolated from ~200 mg of skeletal muscle using TRIzol Reagent (Gibco BRL), then first-strand cDNA synthesis was carried out from 2 [mu]g RNA with oligo(dT) and Superscript II Rnase H- Reverse Transcriptase (200 U/[mu]l; Gibco BRL), according to the manufacturer's instructions. The single-stranded cDNAs were PCR amplified using 30 ng each of forward primer (541C618) within exon 6 and reverse primer (541C1120; 5'-CTA CAA CAC CCT TCT CAC AG-3') within exon 8, with reaction mixture components and PCR conditions identical to those used for PCR amplification of SMN exon 6 (as above).Restriction enzyme analysis. PCR amplification of SMN exons 6-8 from single-stranded cDNA was performed as above, using 30 ng of forward primer (541C618) end-labelled with [[gamma]-32P]ATP (10 [mu]Ci/[mu]l; Amersham). Eight [mu]l of PCR product was digested overnight at 37oC with 10 U DdeI (New England Biolabs) and 1 [mu]l of the buffer supplied by the manufacturer. The digestion products were combined with 5 [mu]l loading dye, and 3 [mu]l of the mixture was electrophoresed on a 5% denaturing polyacrylamide gel. For reference, 3 [mu]l of undigested PCR product was also electrophoresed on the same gel. The gels were run for 12 000 V-h, then transferred onto blotting paper (Alhstrom), dried and exposed to Hyperfilm-MP (Amersham) for 3-6 h at room temperature.
PCR and RT-PCR amplification products to be sequenced were subcloned into the TA cloning vector (Invitrogen), according to the manufacturer's instructions. Sequencing of miniprep DNA that had been purified from the subclones using Wizard Minipreps (Promega) was performed with the dsDNA Cycle Sequencing System (Gibco BRL). Sequencing reaction products were analyzed using a 5% denaturing polyacrylamide gel. The gels were transferred onto blotting paper, dried and exposed to Hyperfilm-MP (Amersham) for 12-24 h at -70oC. The mutation was sequenced on both DNA strands, and multiple subclones were analyzed.
We are grateful to all SMA families for their kind cooperation and to all clinicians for their help in providing both blood samples and clinical details of patients. This research was funded by grants from the Muscular Dystrophy Association (MDA) and Families of SMA. The authors thankfully acknowledge the SMA consortium and the funding provided by MDA and ENMC to support these meetings. We are also grateful to Dr Judith Melki for making primer sequences for amplification of SMN exons available to us prior to publication, and for the photographic assistance of Arthur Weeks in preparation of this manuscript.
1 Pearn, J. (1980) Classification of spinal muscular atrophies. Lancet, i, 919-922.
2 Munsat T.L. and Davies K.E. (1992) Meeting report: International SMA Consortium meeting. Neuromusc. Disord., 2, 423-428.
3 Brzustowicz, L.M., Lehner, T., Castilla, L.H., Penchaszadeh, G.K., Wilhelmsen, K.C., Daniels, R., Davies, K.E., Leppert, M., Ziter, F., Wood, D., Dubowitz, V., Zerres, K., Hausmanowa-Petrusewicz, I., Ott, J., Munsat, T.L. and Gilliam, T.C. (1990) Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3. Nature, 344, 540-541.MEDLINE Abstract
4 Melki, J., Abdelhak, S., Sheth, P., Bachelot, M.F., Burlet, P., Marcadet, A., Aicardi, J., Barois, A., Carriere, J.P., Fardeau, M., Fontan, D., Ponsot, G., Billette, T., Angelini, C., Barbosa, C., Ferriere, G., Lanzi, G., Ottolini, A., Babron, M.C., Cohen, D., Hanauer, A., Clerget-Darpoux, F., Lathrop, M., Munnich, A. and Frezal, J. (1990) Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature, 344, 767-768.MEDLINE Abstract
5 Simard, L.R., Vanasse, M., Rochette, C., Morgan, K., Lemieux, B., Melancon, S.B. and Labuda, D. (1992) Linkage study of chronic childhood-onset spinal muscular atrophy (SMA): confirmation of close linkage to D5S39 in French Canadian families. Genomics, 14, 188-190.MEDLINE Abstract
6 MacKenzie, A., Roy, N., Besner, A., Mettler, G., Jacob, P., Korneluk, R. and Surh, L. (1993) Genetic linkage analysis of Canadian spinal muscular atrophy kindreds using flanking microsatellite 5q13.3 polymorphisms. Hum. Genet., 90, 501-504.MEDLINE Abstract
8 Daniels, R.J., Thomas, N.H., MacKinnon, R.N., Lehner, T., Ott, J., Flint, T.J., Dubowitz, V., Ignatius, J., Donner, M., Zerres, K., Rietschel, M., Cookson, W.O.C., Brzustowicz, L.M., Gilliam, T.C. and Davies, K.E. (1992) Linkage analysis of spinal muscular atrophy. Genomics, 12, 335-339.MEDLINE Abstract
9 Wirth, B., Pick, E., Leutner, A., Dadze, A., Voosen, B., Knapp, M., Piechaczek-Wappenschmidt, B., Rudnik-Schoneborn, S., Schonling, J., Cox, S., Spurr, N.K. and Zerres, K. (1994) Large linkage analysis in 100 families with autosomal recessive spinal muscular atrophy (SMA) and 11 CEPH families using 15 polymorphic loci in the region 5q11.2-q13.3. Genomics, 20, 84-93.MEDLINE Abstract
10 Brahe, C., Velona, I., van der Steege G, Zappata, S., van de Veen, A.Y., Osinga, J., Tops, C.M.J., Fodde, R., Khan, P.M., Buys, C.H.C.M. and Neri, G. (1994) Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis. Hum. Genet., 93, 494-501.MEDLINE Abstract
11 Wirth, B., El-Agwany, A., Baasner, A., Burghes, A., Koch, A., Dadze, A., Piechaczeck-Wappenschmidt, B., Rudnik-Schoneborn, S., Zerres, K. and Schonling, J. (1995) Mapping of the spinal muscular atrophy (SMA) gene to a 750 kb interval flanked by two microsatellites. Eur. J. Hum. Genet., 3, 56-60.MEDLINE Abstract
12 Kleyn, P.W., Wang, C.H., Lien, L.L., Vitale, E., Pan, J., Ross, B.M., Grunn, A., Palmer, D.A., Warburton, D., Brzustowicz, L.M., Kunkel, L.M. and Gilliam, T.C. (1993) Construction of a yeast artifical chromosome contig spanning the spinal muscular atrophy disease gene region. Proc. Natl Acad. Sci. USA, 90, 6801-6805.MEDLINE Abstract
13 Francis, M.J., Morrison, K.E., Campbell, L., Grewal, P.K., Christodoulou, Z., Daniels, R.J., Monaco, A.P., Frischauf, A.-M., McPherson, J., Wasmuth, J. and Davies K.E. (1993) A contig of non-chimaeric YACs containing the spinal muscular atrophy gene in 5q13. Hum. Mol. Genet.2, 1161-1167.MEDLINE Abstract
14 Carpten, J.D., DiDonato, C.J., Ingraham, S.E., Wagner-McPherson, C., Nieuwenhuijsen, B.W., Wasmuth, J.J. and Burghes, A.H.M. (1994) A YAC contig of the region containing the spinal muscular atrophy gene (SMA): Identification of an unstable region. Genomics, 24, 351-356.MEDLINE Abstract
15 Melki, J., Lefebvre, S., Burglen, L., Burlet, P., Clermont, O., Millasseau, P., Reboullet S., Benichou, B., Zeviani, M., Le Paslier, D., Cohen, D., Weissenbach, J. and Munnich, A. (1994) De novo and inherited deletions of the 5q13 region in spinal muscular atrophies. Science, 264, 1474-1477.MEDLINE Abstract
16 Roy, N., Mahadevan, M.S., McLean, M., Shutler, G., Yaraghi, Z., Farahani, R., Baird, S., Besner-Johnston, A., Lefebvre, C., Kang, X., Salih, M., Aubry, H., Tamai, K., Guan, X., Ioannou, P., Crawford, T.O., de Jong, P.J., Surh, L., Ikeda, J.-E., Korneluk, R.G. and MacKenzie, A. (1995) The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell, 80, 167-178.MEDLINE Abstract
17 Thompson, T.G., DiDonato, C.J., Simard, L.R., Ingraham, S.E., Burhges, A.H.M., Crawford, T.O., Rochette, C., Mendell, J.R. and Wasmuth, J.J. (1995) A novel cDNA detects homozygous microdeletions in greater than 50% of type I spinal muscular atrophy patients. Nature Genet., 9, 56-62.MEDLINE Abstract
18 Lefebvre, S., Burglen, L., Reboullet, S., Clermont, O., Burlet, P., Viollet, L., Benichou, B., Cruaud, C., Millasseau, P., Zeviani, M., Le Paslier, D., Frezal, J., Cohen, D., Weissenbach, J., Munnich, A. and Melki, J. (1995) Identification and characterization of a spinal muscular atrophy-determining gene. Cell, 80, 155-165.MEDLINE Abstract
19 Burghes, A.H.M., Ingraham, S.E., McLean, M., Thompson, T.G., McPherson, J.D., Kote-Jarai, Z., Carpenten, J.D., DiDonato, C.J., Ikeda, J.-E., Surh, L., Wirth, B., Sargent, C.A., Ferguson-Smith, M.A., Fuerst, P., Moyzis, R.K., Grady, D.L., Zerres, K., Korneluk, R., MacKenzie, A. and Wasmuth, J.J. (1994) A multicopy dinucleotide marker that maps close to the spinal muscular atrophy gene. Genomics, 21, 394-402.
20 DiDonato, C.J., Morgan, K., Carpten, J.D., Fuerst, P., Ingraham, S.E., Prescott, G., McPherson, J.D., Wirth, B., Zerres, K., Hurko, O., Wasmuth, J.J., Mendell, J.R., Burghes, A.H.M. and Simard, L.R. (1994) Association between AgI-CA alleles and severity of autosomal recessive proximal spinal muscular atrophy. Am. J. Hum. Genet., 55, 1218-1229.MEDLINE Abstract
21 McLean, M.D., Roy, N., MacKenzie, A.E., Salih, M., Burghes, A.H.M., Simard, L., Korneluk, R.G., Ikeda, J-E. and Surh, L. (1994) Two 5q13 simple tandem repeat loci are in linkage disequilibrium with type I spinal muscular atrophy. Hum. Mol. Genet., 3, 1951-1956.MEDLINE Abstract
22 Wirth, B., Hahnen, E., Morgan, K., DiDonato, C.J., Dadze, A., Rudnik-Schoneborn, S., Simard, L.R., Zerres, K. and Burghes, A.H.M. (1995) Allelic association and deletions in autosomal recessive proximal spinal muscular atrophy: association of marker genotype with disease severity and candidate cDNAs. Hum. Mol. Genet., 4, 1273-1284.MEDLINE Abstract
23 Bussaglia, E., Clermont, O., Tizzano, E., Lefebvre, S., Burglen, L., Cruaud, C., Urtizberea, J.A., Colomer, J., Munnich, A., Baiget, M. and Melki, J. (1995) A frame-shift deletion in the survival motor neuron gene in Spanish spinal muscular atrophy patients. Nature Genet., 11, 335-337.MEDLINE Abstract
24 Velasco, E., Valero, C., Valero, A., Moreno, F., Hernandez-Chico, C. (1996) Molecular analysis of the SMN and NAIP genes in Spanish spinal muscular atrophy (SMA) families and correlation between number of copies of cBCD541 and SMA phenotype. Hum. Mol. Genet., 5, 257-263.MEDLINE Abstract
25 DiDonato, C.J., Ingraham, S.E., Mendell, J.R., Prior, T.W., Linard, S., Moxley, R.T., Florence, J. and Burghes, A.H.M. (1996) Deletion and conversion in SMA patients: Is there a relationship to severity? Ann. Neurol. in press.
26 van der Steege, G., Grootscholten, P.M., van der Vlies, P., Draaijers, T.G., Osinga, J., Cobben, J.M., Scheffer, H. and Buys, C.H.C.M (1995) PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet, 345, 985-986.MEDLINE Abstract
27 Hahnen, E., Forkert, R., Merke, C., Rudnik-Schoneborn, S., Schonling, J., Zerres, K. and Wirth, B. (1995) Molecular analysis of candidate genes on chromosome 5q13 in autosomal recessive spinal muscular atrophy: evidence for homozygous deletions of the SMN gene in unaffected individuals. Hum. Mol. Genet., 4, 1927-1933.MEDLINE Abstract
28 Cobben, J.M., van der Steege, G., Grootscholten, P., de Visser, M., Scheffer, H. and Buys, C.H.C.M. (1995) Deletions of the survival motor neuron gene in unaffected siblings of patients with spinal muscular atrophy. Am. J. Hum. Genet., 57, 805-808.MEDLINE Abstract
29 Rodrigues, N.R., Owen, N., Talbot, K., Patel, S., Muntoni, F., Ignatius, J., Dubowitz, V. and Davies, K.E. (1996) Gene deletions in spinal muscular atrophy. J. Med. Genet., 33, 93-96.
30 Kiesewetter, S., Macek Jr., M., Davis, C., Curristin, S.M., Chu, C.-S., Graham, C., Shrimpton, A.E., Cashman, S.M., Tsui, L.-C., Mickle, J., Amos, J., Highsmith, W.E., Shuber, A., Witt, D.R., Crystal, R.G. and Cutting, G.R. (1993) A mutation in CFTR produces different phenotypes depending on chromosomal background. Nature Genet., 5, 274-278.MEDLINE Abstract
31 Brahe, C., Servidei, S., Zappata, S., Ricci, E., Tonali, P. and Neri, G. (1996) Genetic homogeneity between childhood-onset and adult-onset autosomal recessive spinal muscular atrophy. Lancet, 346, 741-742.
32 Wang, C.H., Xu, J., Carter, T.A., Ross, B.M., Dominski, M.K., Bellcross, C.A., Penchaszadeh, G.K., Munsat, T.L. and Gilliam, T.C. (1996) Characterization of survival motor neuron (SMNT) gene deletions in asymptomatic carriers of spinal muscular atrophy. Hum. Mol. Genet., 5, 359-365.MEDLINE Abstract
33 Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics, 5, 874-879.MEDLINE Abstract
34 Burglen, L., Lefebvre, S., Clermont, O., Burlet, P., Viollet, L., Cruaud, C., Munnich, A. and Melki, J. (1996) Structure and organization of the human survival motor neuron (SMN) gene. Genomics, 32, 479-482.MEDLINE Abstract
35 Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16, 1215.MEDLINE Abstract
36 Zielenski, J., Bozon, D., Kerem, B., Markiewicz, D., Durie, P., Rommens, J.M. and Tsui, L.C. (1991) Identification of mutations in exons 1 through 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genomics, 10, 229-235.MEDLINE Abstract
*To whom correspondence should be addressed at: Department of Neurology, 452 Means Hall, 1654 Upham Drive, Columbus, OH 43210, USA
This page is maintained by OUP admin. Last updated Thu Oct 31 15:28:51 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
