Characterization of survival motor neuron (SMNT) gene deletions in asymptomatic carriers of spinal muscular atrophy
Characterization of survival motor neuron (SMN T ) gene deletions in asymptomatic carriers of spinal muscular atrophyChing H. Wang1,2, Jin Xu1, Todd A. Carter1, Barbara M. Ross1, Mary K. Dominski3, Cecelia A. Bellcross3, Graciela K. Penchaszadeh1, Theodore L. Munsat4 and T. Conrad Gilliam1,*
1Departments of Genetics and Development and Psychiatry, College of Physicians and Surgeons at Columbia University and New York State Psychiatric Institute, New York, NY 10032, USA, 2Departments of Pediatrics and Neurology, College of Physicians and Surgeons at Columbia University, New York, NY 10032, USA, 3Dean Medical Center, Madison, WI, USA and4Department of Neurology, Tufts University and New England Medical Center, Boston, MA, USA
Received October 18, 1995;Revised and Accepted December 11, 1995
Previous reports have established that the telomeric copy of the survival motor neuron (SMNT) gene and the intact copy of the neuronal apoptosis inhibitory protein (NAIP) gene are preferentially deleted in patients with spinal muscular atrophy (SMA). Although deletions or mutations in the SMNT gene are most highly correlated with SMA, it is not clear to what extent NAIP or other genes influence the SMA phenotype, or whether a small fraction of SMA patients actually have functional copies of both SMNT and NAIP. To evaluate further the part of SMNT in the development of SMA, we analyzed 280 asymptomatic SMA family members for the presence or absence of SMNT exons 7 and 8. We report the following observations: (i) 4% of the sample harbored a polymorphic variant of SMNT exon 7 that looks like a homozygous deletion; (ii) approximately 1% of the parents are homozygously deleted for both exons 7 and 8; (iii) one asymptomatic parent lacking both copies of SMNT exons 7 and 8 displays a `subclinical phenotype' characterized by mild neurogenic pathology; (iv) another asymptomatic parent lacking both SMNT exons showed no signs of motor neuron disorder by clinical and neurodiagnostic analyses. The demonstration of polymorphic variants of exon 7 that masquerade as homozygous nulls, and the identification of SMA parents who harbor two disease alleles, serve as a caution to those conducting prenatal tests with these markers.
Spinal muscular atrophy (SMA) is a motor neuron disease characterized by degeneration of spinal cord anterior horn cells and muscular atrophy. The autosomal recessive form of this disorder affects about 1:10 000 live births with a carrier frequency of 1/40-1/60. It is the most common fatal genetic disorder of infancy and the second most common childhood neuromuscular disease following Duchenne muscular dystrophy. Childhood-onset SMA is classified into three clinical subtypes: (i) SMA type I, or Werdnig-Hoffmann disease-the most severe phenotype, usually leading to early infant death; (ii) SMA type II, or the intermediate subtype-onset in early childhood often resulting in early impairment of walking; and (iii) SMA type III, or Kugelberg-Welander disease-onset in late childhood, with considerable clinical heterogeneity and the risk of wheelchair dependence in adult years. The gene for the three types of childhood-onset SMA has been mapped to a single locus on chromosome 5q13 by linkage analysis (1 -3 ). Further studies using recombinant mapping methods identified a genetic interval of 1-2 cM flanked by new microsatellite markers (4 -7 ,13 ). Several YAC contigs spanning the SMA gene region have been constructed by different research groups (5 ,8 ,9 ,11 ). Further refinement of the region was complicated by low-copy repeat sequences mapping in and around the SMA region (8 ,11 -13 ). Gene-coding sequences likewise tend to exist in multiple copies within the disease gene region. In some cases pseudogenes map to the SMA region while the intact, presumably functional, copies map elsewhere in the genome (14 ).
Recently, independent laboratories reported two candidate genes in the SMA region: the survival motor neuron (SMN) gene and the neuronal apoptosis inhibitory protein (NAIP) (15 ,16 ). The survival motor neuron (SMN) gene exists as two highly homologous copies, a centromeric (SMNC) and a telomeric form (SMNT) which differ by only 2 bp in the entire 1.5 kb coding sequence. The SMNT copy is homozygously missing in over 90% of SMA patients, whereas at least one copy of SMNT is present in the vast majority of control DNA samples (15 ,17 -19 ). SMNC is not preferentially deleted in SMA patients, but is homozygously deleted in about 5% of samples tested. Expression of the SMNC gene appears to differ in SMA versus control samples. In samples lacking a copy of SMNT, there is a noticeable increase in the expression of an alternatively spliced SMNC RNA transcript (15 ). Thus, while SMNC does not appear to be a target for SMA mutations, its pattern of expression may contribute to the pathophysiology of the disorder.
The second candidate gene, NAIP, maps in the near vicinity of SMNT. NAIP likewise exists in multiple, highly homologous copies, although it appears that only one copy contains the full complement of exons. The intact copy of NAIP is homozygously deleted in about 40% of SMA type I cases and to a lesser extent in SMA II and III individuals (16 ,19 ). In the minority of SMA samples where no evidence was found for deletion of either SMNT or NAIP, several small insertion/deletion mutations have been found at the splice-site junctions of SMNT (15 ).
In summary, three types of evidence implicate SMN as the most critical gene influencing the onset of SMA: first, deletion of the 3'-end of SMNT is most highly correlated with incidence of SMA; second, a minority of SMA patients reveal small insertion/deletion mutations at splice-site junctions in SMNT whereas no disease-related point mutations have been reported for NAIP; and third, homozygous deletion of intact NAIP has been reported in asymptomatic parents of SMA patients (16 ), while recent evidence shows that SMNT is likewise missing in a small fraction of asymptomatic SMA siblings and parents (18 ,19 ; this manuscript).
From the sum of evidence available to date, it is still not clear whether all cases of SMA arise from deletions or alteration of the SMNT gene. Five to 10% of SMA samples reveal at least one SMNT gene with intact exons 7 and 8. As current single strand conformation polymorphism (SSCP) assays cannot distinguish the 5'-ends of the SMNT and SMNC genes, it is very difficult to screen for deletions specific for this region of the gene. Furthermore, the promoter sequences for SMNT have not yet been identified, nor has the full intronic DNA sequence been reported. Thus, it is not known whether the remaining 5-10% of SMA cases actually harbor deletions including the 5'-end of SMNT, or deletions or point mutations in the promoter or intronic sequences. If they do not, then a small proportion of SMA cases presumably arise from the inactivation of other genes, including but not necessarily limited to NAIP.
To evaluate further the part of SMNT in the etiology of SMA, we performed clinical assessment of individuals who are homozygous null for SMNT exons 7 and 8. The identification of such asymptomatic individuals might indicate that deletion of SMNT (exons 7 and 8) alone is insufficient to cause SMA symptoms, or alternatively, that compensating factors can, in rare cases, override the deletion and lead to a `normal' phenotype. We screened 280 asymptomatic family members (214 parents and 66 siblings) of SMA patients for homozygous deletion of SMN exons 7 and 8. Two asymptomatic parents from different families were identified who are missing both exons 7 and 8. One parent showed neurogenic abnormalities in electromyography (EMG) and muscle biopsy, consistent with subclinical SMA. The other parent, who has a normal EMG and muscle biopsy, was from a family that appeared to be unlinked to the chromosome 5q13 DNA markers (2 ,20 ). We now show that this family is actually consistent with chromosome 5q13 linkage and that the parent transmits different mutant chromosomes to two affected offsprings.
. Incidence of SMNT homozygous nulls among asymptomatic SMA family members
Type I
Type II
Type III
Total
(n = 100)
(n = 112)
(n = 68)
(n = 280)
Exon 7 only
2
7
3
12 (4%)
Exon 8 only
0
0
0
0
Exons 7+8
0
1
1
2 (0.7%)
SSCP analysis was perfomed to detect the presence or homozygous absence of SMNT exons 7 and 8 (15 ). This assay does not distinguish between the presence of one versus two copies of either exon. The sample consists of 214 parents and 66 siblings. The `type' of SMA (I = Werdnig Hoffmann disease/severe SMA; II = intermediate type; III = Kugelberg Welander disease/mild SMA) is indicated at the top of the Table. n = the number of asymptomatic family members analyzed. The numbers in the three columns indicate the number of individuals missing both copies of exon 7, exon 8 or exons 7 and 8. Twelve aysmptomatic family members (eight parents and four siblings, 4% of the total sample tested) are homozygous null for exon 7 only. Two asymptomatic parents from two SMA families (representing 0.7% of the total samples, but 0.9% of the parents tested) are homozygous null for both exons 7 and 8.
Table 1 shows the results of screening 280 asymptomatic family members of SMA patients for the presence or absence of SMNT exons 7 and 8. The sample consisted of unaffected parents and siblings. All SMA families meet diagnostic criteria established by the `International SMA Consortium' (22 ). Twelve individuals were lacking both copies (homozygous null) of SMNT exon 7 only, and two individuals were homozygous null for both SMNT exons 7 and 8. Figure 1 shows three families in which unaffected family members are apparently missing both copies of the SMNT exon 7 band. In each of these families, the SMNT exon 7 band (lower closed arrow in the upper panel) is present in one parent (open symbol), missing in the affected proband (black symbol), and missing in one asymptomatic parent and one sibling (stippled symbol). Analysis of SMNT exon 8 in these families (see upper closed arrow in the lower panel) shows that only the affected proband is homozygously missing this exon. Closer inspection of the exon 7 profiles suggests the presence of two additional bands labeled `a' and `b' in the asymptomatic family members with apparent deletions. Band `a' was separated from the overlapping SMNC exon 7 band on a longer gel and its DNA sequence was determined. Band `a' was shown to contain a `C' nucleotide at base pair position 873 denoting the SMNT locus (data not shown). The same band contains an A to G polymorphism in the intronic sequence 96 bp 5' to the start of exon 7. This polymorphism alters the band migration such that the SSCP pattern mimics the pattern of a homozygous null for SMNT exon 7. The band `b' sequence is the exact complement of band `a', verifying that bands `a' and `b' are the two complementary strands of the same locus. Haplotype analysis of families 2532 and 7322using CA-repeat microsatellite markers flanking the SMN gene locus (13 )are shown in Figure 2 . The combination of SMN and microsatellite genotypes shows that affected probands inherit two SMNT null alleles while the healthy siblings inherit a null allele from one parent along with a polymorphic variant of SMNT exon 7 from the other parent. Thus, the asymptomatic siblings appear to be homozygous nulls by SSCP analysis, but actually inherit a normal, presumably functional copy of SMNT exon 7. Analysis of the remaining six asymptomatic family members (five parents and one sibling) with apparent exon 7 deletions likewise revealed polymorphic variants of the SMNT specific exon.
We describe two individuals with homozygous null deletion of telomeric SMN exons 7 and 8 who demonstrate no clinical symptoms of SMA. In one case, an SMA type III brother and his asymptomatic sister appear to inherit identical `disease alleles', one of which is transmitted to the son with type II SMA. EMG and muscle biopsy show that the sister has mild neurogenic pathology consistent with a mild, `asymptomatic' manifestation of SMA. Barring an undetected de novo mutation in the affected brother, this result suggests that other factors, genetic or environmental, may compensate for the loss of SMNT function. If genetic in nature, such factors presumably map outside the immediate disease gene region. The second individual is an asymptomatic father of SMA children who is homozygous null for SMNT exons 7 and 8. In contrast to the aforementioned asymptomatic carrier, this individual shows no sign of SMA either by clinical examination or by neurodiagnostic studies.Interestingly, either of his homologues, in combination with the maternal SMA homologue, leads to typical SMA in the offspring.
The data from both families are consistent, therefore, with the notion that deletion of SMNT exons 7 and 8 per se is not sufficient to account for the clinical symptoms typical of SMA. Several explanations could account for this observation. First, homozygous deletion of SMNT exons 7 and 8 is normally sufficient to produce SMA symptoms, but in rare cases, compensating factors protect against the manifestation of disease symptoms. Such factors might include a quantitative or qualitative alteration of the SMNC gene; a third SMN homologue; or an unlinked, background gene product. Second, the majority of SMA homologues are likely to be homozygous null for the entire SMNT coding sequence. In rare circumstances, deletion, gene conversion or unequal recombination may produce a partial SMNT gene which is truncated, converted or recombined with the 3' portion of SMNC. This variant SMN protein may retain sufficient function to allay the onset of symptoms. It is noteworthy that no groups have successfully identified deletion breakpoints within the SMNT gene. Third, the majority of SMA mutations may include critical DNA sequence (which is not deleted in these two asymptomatic parents) in addition to the SMNT and NAIP gene sequences. This interpretation implicates yet another gene product in the development of SMA.
The identification of polymorphic variants of SMNT exon 7 that masquerade as null alleles provides a cautionary note to the interpretation of prenatal and diagnostic tests. In those cases where only SMNT exon 7 is missing, it will be important to distinguish null alleles from polymorphic alleles. This is particularly important when the assay is used in prenatal diagnosis where false positives may lead to unnecessary termination of pregnancy. Direct sequence analysis of the SSCP bands may be necessary for verification of the exon subforms, particularly when novel bands are present. Alternatively, a PCR-enzyme digestion method can be used to confirm the SSCP results (21 ).
Two research groups have recently reported deletion of the SMNT gene in unaffected family members of SMA patients (18 ,19 ). In the study of Cobben et al. (18 ), four unaffected siblings from two SMA families were found to harbor homozygous deletions of SMNT exons 7 and 8.In the study of Hahnen et al. (19 ), one mother and six unaffected siblings from four SMA families showed homozygous deletions of exons 7 and 8 of the SMNT gene. All of these unaffected siblings display the same microsatellite haplotypes as their respective probands. Our data are consistent with these recent findings and extend the observations by describing one homozygous null SMNT (exons 7 and 8) individual with a subclinical SMA phenotype, and another `null' individual whose neurodiagnostic studies showed no pattern of motor neuron disease. Our data likewise indicate that misinterpretation of an SMNT exon 7 `null' genotype is possible due to the existence of polymorphic variation in this region. Cobben et al. used a PCR-enzyme digestion method to screen for SMNT deletions (21 ); therefore, misinterpretation due to polymorphic variation is unlikely. The nonradioactive SSCP method used in the study of Hahnen et al., however, would be susceptible to the type misinterpretation we have described due to polymorphic variation. Therefore, careful examination of SSCP bands and direct sequencing may be needed to verify that these individuals are actually homozygous null for exon 7.
Our data indicate that about 1% of SMA parents harbor two disease alleles and that some of these asymptomatic parents are likely to have a mild neurogenic pathology similar to that seen in SMA patients. The occurrence of homozygous disease alleles among asymptomatic parents should alert medical professionals involved in the genetic counseling of SMA families. The small fraction of SMA families that appear to be unlinked to chromosome 5q13 DNA markers will likely be enriched for SMA parents with this genotype. Family 1116 (Fig. 6 ) illustrates this occurrence. This family as well as five others in our collection initially appeared to be unlinked to chromosome 5q13 DNA markers. With one exception, we have found SMNT deletions in all these families (data not shown). The exception proved to be a case of misdiagnosis.
SMA families were obtained through Muscular Dystrophy Association clinics in the United States and through an international group of collaborators. All cases of SMA were diagnosed according to consensus criteria established by an international SMA consortium (22 ). Family 6376 was referred by a local genetic counselor. The 3 year old proband was diagnosed with SMA type II at 18 months old. The maternal uncle is 32 years old and diagnosed with SMA type III (Kugelberg-Welander disease) at 14 years of age. The mother is 33 years old and clinically asymptomatic. She lives an active life-style including a demanding professional occupation and regular aerobic exercise. Her muscle biopsy shows mild neurogenic atrophy and the EMG shows presence of fasciculation consistent with SMA. Family 1116 includes two affected children diagnosed at ages 13 and 11 with SMA type III. All cases of SMA were confirmed by EMG and muscle biopsy. The asymptomatic father had clubfeet at birth but exhibits no sign of motor disability at age 60. His electrophysiological studies show mild motor conduction slowing and reduced sensory amplitude consistent with axonal sensory-motor peripheral neuropathy. His EMG shows no active denervation. His muscle biopsy shows normal fiber size (no atrophy or hypertrophy) and no fiber type grouping. These studies indicate no sign of motor neuron disorder.
Microsatellite markers D5S1411, D5S1413, D5S1414 and D5S1408 were used to genotype SMA families 6376 and 1116 according to a previously published procedure(13 ).
PCR primers for exon 7 (R111 and 540C770) and exon 8 (541C960 and 541C1120) were derived from previously published sequences(15 ). One hundred ng of genomic DNA was added to 25 µl of reaction mix containing 25 µM of primers, 2.5 mM MgCl2 , 1 U of Taq polymerase (Perkin-Elmer), and 2.5 µCi of [[alpha]-32P]dCTP (New England Nuclear). The PCR reaction includes an initial 11 cycles of 30 s at 90oC, 30 s at 65-55oC with 1oC decrement per cycle, and 30 s at 72oC. This was followed by an additional 24 cycles of 30 s at 90oC, 55oC and 72oC each and a final step of 5 min at 72oC. Four µl of the PCR product were added to an equal volume of denaturing dye containing 96% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol. The mixtures were denatured at 95oC for 10 min then chilled on ice and loaded on to a 0.5*MDE gel (MDETM, AT Biochem, PA). The gel was run at 4 W for 18-20 h in 4oC followed by autoradiography.
To verify the exon subforms of the SSCP bands, we excised the bands directly from the SSCP gel. The single-stranded DNA retained in the gel was eluted by soaking in 20 µl of TE buffer (10 mM Tris and 1 mM EDTA) for 2 h in room temperature. Five µl of the eluent was used for PCR amplification. The purified PCR products were then DNA sequenced using the original oligonucleotide primers and a DyeDeoxy terminator cycle sequencing kit (Applied Biosystem).
This work is supported by the Families of SMA (Chicago, IL), Andrew's Buddies, Inc., the Muscular Dystrophy Association of America, and the National Institute of Health Grant NS28877. CHW is a recipient of an NIH Clinical Investigator Award, NSO1576, and receives support from the Colleen Giblin Foundation for Pediatric Neurology Research.
4 Brzustowicz,L.M., Kleyn,P.W., Boyce,F.M., Lien,L.L., Monaco,A.P., Penchaszadeh, G.K., Das,K., Wang,C.H., Munsat,T.L., Ott,J., Kunkel,L.M. and Gilliam,T.C. (1992) Fine-mapping of the spinal muscular atrophy locus to a region flanked by MAP1B and D5S6. Genomics, 13, 991-998.MEDLINE Abstract
5 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
6 Soares,V.M., Brzustowicz,L.M., Kleyn,P.W., Knowles,J.A., Palmer,D.A., Asokan,S., Penchaszadeh, G.K., Munsat,T.L. and Gilliam,T.C. (1993) Refinement of the spinal muscular atrophy locus to the interval between D5S435 and MAP1B. Genomics, 15, 365-371.MEDLINE Abstract
7 Clermont,O., Burlet,P., Burglen,L., Lefebvre,S., Pascal,F., McPherson,J., Wasmuth,J., Cohen,D., Le Paslier,D., Weissenbach,J., Lathrop,M., Munnich,A., and Melki,J. (1994) Use of genetic and physical mapping to locate the spinal muscular atrophy locus between two new highly polymorphic DNA markers. Am. J. Hum. Genet., 54, 687-694.
8 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 artificial chromosome contig spanning the spinal muscular atrophy disease gene region. Proc. Natl Acad. Sci. USA, 90, 6801-6805.MEDLINE Abstract
9 Carpten,J.D., DiDonato,C.J., Ingraham,S.E., Wagner-McPherson,C., Nieuwnhuijsen,B.W., Wasmuth,J.J. and Burghes, A.H.M. (1994) A YAC contig of the region containing the spinal muscular atrophy gene: identification of an unstable region. Genomics,24, 351-356.MEDLINE Abstract
10 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
11 Burghes,A.H.M., Ingraham,S.E., McLean,M., Thompson,T.G., McPherson,J.D., Kote-Jarai,Z., Carpten,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.
12 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
13 Wang,C.H., Kleyn,P.W., Vitale,E., Ross,B.M., Lien,L., Xu.J., Carter,T.A., Brzustowicz.L.M., Obici,S., Selig,S., Pavone,L., Parano,E., Penchaszadeh,G.K., Munsat,T., Kunkel,L.M. and Gilliam,T.C. (1995) Refinement of the spinal muscular atrophy locus by genetic and physical mapping. Am. J. Hum. Genet., 56, 202-209.MEDLINE Abstract
14 Selig,S., Bruno,S., Scharf,J.M., Wang,C.H., Vitale,E., Gilliam, T.C. and Kunkel,L. (1995) Expressed cadherin pseudogenes are localized to the critical region of the spinal muscular atrophy gene. Proc. Natl Acad. Sci. USA, 92, 3702-3706.MEDLINE Abstract
15 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
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 Rodrigues,N.R., Owen,N., Talbot,K., Ignatius,J., Dubowitz,V. and Davies,K. (1995) Deletions in the survival motor neuron gene on 5q13 in autosomal recessive spinal muscular atrophy. Hum. Mol. Genet., 4, 631-634.MEDLINE Abstract
18 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
19 Hahen,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 of homozygous deletions of the SMN gene in unaffected individuals. Hum. Mol. Genet. (in press).
20 Brzustowicz,L.M., Merette,C., Kleyn,P.W., Lehner,T., Penchaszadeh,G.K., Das,K., Munsat,T., Ott,J. and Gilliam,T.C. (1993) Assessment of nonallelic genetic heterogeneity of chronic (type II and III) spinal muscular atrophy. Hum. Hered., 43, 380-387.MEDLINE Abstract
21 van der Steeg,G., Grootschholten,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 the clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet, 345, 985-986.
22 Munsat,T.L. and Davies,K.E. (1992) Workshop report: Internaational SMA Consortium meeting. Neuromuscul. Disord., 2, 423-428.MEDLINE Abstract
23 Gilliam,T.C., Freimer,N.B., Kaufmann,C.A., Powchik,P.P., Bassett,A.S., Bengtsson,U. and Wasmuth,J.J. (1989) Deletion mapping of DNA markers to a region of chromosome 5 that cosegregates with schizophrenia. Genomics, 5, 940-944.MEDLINE Abstract
*To whom correspondence should be addressed at: 722 West 168th Street, Unit 23, New York, NY 10032, USA
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