Purpose: This study describes SMN1 deletion frequency, carrier studies, and the effect of the modifying SMN2 gene on the spinal muscular atrophy (SMA) phenotype. A novel allele-specific intragenic mutation panel increases the sensitivity of SMN1 testing.
Methods: From 1995 to 2001, 610 patients were tested for SMN1 deletions and 399 relatives of probands have been tested for carrier status. SMN2 copy number was compared between 52 type I and 90 type III patients, and between type I and type III patients with chimeric SMN genes. A fluorescent allele-specific polymerase chain reaction (PCR) -based strategy detected intragenic mutations in potential compound heterozygotes and was used on 366 patients.
Results: Less than half of the patients tested were homozygously deleted for SMN1. A PCR-based panel detected the seven most common intragenic mutations. SMN2 copy number was significantly different between mild and severely affected patients.
Conclusions: SMN1 molecular testing is essential for the diagnosis of SMA and allows for accurate carrier testing. Screening for intragenic mutations in SMN1 increases the sensitivity of diagnostic testing. Finally, SMN2 copy number is conclusively shown to ameliorate the phenotype and provide valuable prognostic information.
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by homozygous mutation of the SMN1 gene1 on chromosome 5q13.2–12 5q-SMA has an incidence of 1/10,000 with an estimated carrier frequency of 1/50.13–17 Deletion and gene conversion events result in a 95% to 98% rate of homozygous loss of the SMN1 gene in patients with classic SMA.1,18–31 A further 1% to 3% of well-characterized SMA cases are the result of compound heterozygosity with an intragenic mutation and a deletion.31,32 Clinically, SMA patients are classified as type I, II, or III, based on the severity of the disease and the age of onset.33,34 SMA type I, also known as Werdnig-Hoffmann disease,35,36 is the most severe presentation. Type I infants are hypotonic and have severe proximal muscle wasting due to a lack of α-motor neurons in the anterior horn of the spinal cord. These patients are diagnosed prenatally or within 6 months after birth. They never sit unaided and usually die within the first 2 years, most often due to respiratory muscle weakness. Patients with intermediate SMA type II develop the disease before 18 months of age. They are able to sit unaided, but do not stand or walk. SMA type III, also called Kugelberg-Welander disease, is less severe.37,38 Type III patients are diagnosed after 18 months, are able to walk, can have children, and often live at least into the second or third decade.39
This study presents the clinical experience of 6 years of SMA testing in the Molecular Pathology Laboratory at The Ohio State University. Despite the fact that more than 95% of 5q-linked SMA patients lack any intact SMN1, it has been our experience that <43% of patients referred for diagnostic testing have the common SMN1 deletion. Although the remaining patients share some of the common features of SMA patients, including hypotonia, progressive muscle weakness, and loss of ambulation, they do not have chromosome 5q-linked proximal spinal muscular atrophy. For this reason, the molecular diagnosis is absolutely necessary for the clinical diagnosis of SMA and for accurate genetic counseling.
In an attempt to increase diagnostic sensitivity, we describe an efficient and inexpensive fluorescent allele-specific polymerase chain reaction (PCR) panel that allows for the rapid identification of the seven most common intragenic mutations found in compound heterozygotic SMA patients. This panel can easily be expanded or altered to meet the mutational profile of a specific population.
For autosomal recessive disorders, it is expected that parents will be obligate carriers of a mutant allele. However, there are two factors which complicate SMA carrier testing. One is the presence of de novo mutations, which occurs in approximately 2% of probands.40 The other factor is the presence of single chromosomes with two copies of SMN1 confounding all dosage-based assays for SMN1 copy number.17,31,41 We present the results of the carrier testing of 399 familial relations of SMA probands and describe the population most interested in being tested. Approximately 5% of parents of homozygously deleted patients had two nondeleted copies of the SMN1 gene.
SMN2 is a copy of the SMN1 gene present in a duplicated region of chromosome 5q. Two exonic base changes differentiate SMN2 from SMN1,1,42 and the variation in exon 7 of SMN2 is sufficient to greatly diminish the amount of full-length transcript and consequently the amount of full-length protein produced by the gene.43,44 This base change affects an exonic splice enhancer site,45 resulting in the diminished ability to correctly splice out intron 7. Absence of SMN2 does not cause spinal muscular atrophy, however SMN2 copy number has been correlated with modification of the SMA phenotype in a transgenic mouse model46–48 and in humans, where increased copy number of SMN2 is associated with a more mild presentation of the disease.27
Several studies have shown that SMN2 gene copy number can modify the SMA phenotype.17,23,26,28,31,47,48 We have performed a large-scale study comparing the SMN2 copy number between 52 type I and 90 type III patients. Our results conclusively demonstrate a greater number of copies of the SMN2 gene in type III versus type I patients.
Chimeric SMN alleles (exon 7SMN2, exon 8SMN1) were found by DiDonato et al.29 to be more common in patients with mild SMA, suggesting that these alleles might be more mild than normal alleles.27–29,49 Other studies found all SMA types represented in the population of patients with chimeric SMN genes.22,23,25,26,30,50 An analysis of SMN2 copy number as it correlates to severity in SMA patients with chimeric SMN genes has not previously been reported. We hypothesized that SMN2 copy number might account for the variation in phenotype observed within the population of chimeric SMA patients and tested this by comparing SMN2 copy number between type I and type III patients with chimeric SMN genes.
PATIENTS AND METHODS
Patient clinical classification
We do not receive extensive clinical information about the majority of patients sent for diagnostic testing and almost never receive follow-up data. For these reasons, we were not able to reliably place all of our patients into SMA types I, II, and III. Lacking motor milestone information and dates of the death of these patients, we instead categorized 52 patient samples sent for molecular diagnosis between the ages of zero and 6 months as type I. Our group of 90 type III patients were taken from DNA samples of individuals classified as type III before enrollment in a gabapentin therapeutic drug trial. We realize that by analyzing the most severe and the most mild patients, we have chosen our data from the extreme ends of the diagnostic spectrum. This method was the only way for us to ensure the segregation of these populations, and we believe that these extremes legitimately represent acute and chronic SMA cases, without providing information about intermediate presentation.
Allele-specific PCR panel
A multiplex panel of allele-specific primers (Table 1) was used to preferentially amplify the mutant over the normal allele. For each primer pair, the nonallele-specific primer was fluorescently labeled. Part of the APC gene was used as an internal control and was amplified in every sample regardless of the presence of mutations to confirm that sufficient template was present in each reaction. Every gel had the following controls: blank, normal, and each of the seven mutations being tested. Amplification was performed on a Perkin Elmer 9600 thermocycler (Applied Biosystems, Foster City, CA) starting with a 5-minute denaturation at 94°C; followed by 30 cycles of 1 minute at 95°C, 2 minutes at 55°C, 3 minutes at 72°C; and concluding with a 7-minute extension at 72°C. Product was electrophoresed through a 5% LongRanger (FMC Bioproducts, Rockland, ME) polyacrylamide gel on an ABI Prism 377 DNA Sequencer (Applied Biosystems) at 3 kV/h for 1.5 hours. GeneScan TAMRA 500 (Applied Biosystems) was used as a size standard. Intensity of bands were analyzed using GeneScan software (Applied Biosystems). Although the A2G primer pair produces a 194-bp band, a smaller more visible product is used for diagnosis.
Genomic DNA isolation and identification of SMN1 copy number
DNA was extracted from peripheral venous blood from patients by a simple salting out technique.51 DNA was analyzed for SMA carrier status by an SMN dosage assay.17 Paternity was confirmed in the cases of parents of affected patients found to have two copies of the SMN1 gene.
SMN2 dosage assay
Carrier analysis for the SMN1 and SMN2 genes was based on the protocol of McAndrew et al.;17 however, the same primers were fluorescently labeled with 6-FAM. When testing for SMN2 copy number in patients deleted for SMN1, controls with zero copies of SMN1 and with 1, 2, 3, or 4 copies of SMN2 were run on each gel. Amplification was carried out in a Perkin Elmer 9600 thermocycler (Applied Biosystems) using the following conditions: 5 minutes denaturation at 94°C; followed by 21 cycles of 1 minute at 95°C, 2 minutes at 55°C, 3 minutes at 72°C; and concluding with a 7-minute extension at 72°C. Product was electrophoresed through a 5% LongRanger polyacrylamide gel (FMC Bioproducts) on an ABI Prism 377 DNA Sequencer (Applied Biosystems) at 3 kV/h for 1.5 hours. Dosage ratios were analyzed using GeneScan software (Applied Biosystems).
RESULTS AND DISCUSSION
Since 1995, our laboratory has offered diagnostic SMN1 deletion analysis to detect the homozygous absence of the gene.52 This deletion test was most often performed on DNA extracted from whole blood. Muscle biopsies, newborn blood spots, paraffin-embedded autopsy material, and fixed tissue on glass slides have been used for postmortem diagnosis. Postmortem molecular testing is often important for the confirmation of clinical diagnosis, thus allowing for accurate genetic counseling.
It has been the experience of this laboratory that the clinical symptoms of SMA are not sufficiently specific to make a reliable diagnosis. Patients that are sent for the molecular test generally have similar symptoms, including hypotonia, proximal muscle weakness, and loss of ambulation. These symptoms are not specific to 5q-spinal muscular atrophy. Of the 610 patients tested, 232 (38.0%) were deleted for exons 7 and 8 and 29 (4.8%) were deleted only for exon 7. The remaining 349 (57.2%) were not deleted. Our data indicate that the clinical diagnosis of SMA is not straightforward, and oftentimes the test is being ordered to exclude the disease. Muscle biopsies are sometimes ordered for hypotonic infants before the molecular test. Our data would suggest that such an invasive procedure should be reserved for infants with negative SMN1 deletion results.
Diagnostic testing for intragenic mutations
Although many researchers have identified intragenic mutations in the SMN1 gene in compound heterozygotic patients,53 testing for these mutations is not performed on a routine diagnostic basis. The difficulties in screening for intragenic mutations include the fact that there is no highly prevalent mutation found in these patients, and that mutations are found throughout the gene. Adding to the complexity of finding intragenic mutations is the presence of SMN2, which makes for technical difficulties in identification of mutant sequence in SMN1 through the background of normal sequence from the copy gene. Also important is that newly identified mutations must be shown to be present on the SMN1 gene and not on SMN2. Several mutations were found more often than others, and these mutations have not been found in the SMN2 gene.31,32 This finding led us to develop a rapid and inexpensive assay that detects the more common intragenic mutations. The test entails the amplification of patient DNA using a panel of allele-specific primers that preferentially amplify the mutant allele. Seven mutations that have been observed more than once are detected (Fig. 1), based on mismatches in the 3′ end of the primer that are homologous to the mutant sequence (Table 1). Amplification of the APC gene is used as an internal control to confirm that sufficient template is present and amplification conditions are appropriate. The 800ins11 primer pair also acts as an internal control. This primer set is not allele-specific. Rather, it detects the mutation based on an 11- bp size differential; a 153-bp band if a normal allele is present, and a 164-bp band indicates a mutation. The 153-bp product acts as an amplification control, because this primer pair should amplify at least one normal SMN2 allele. If any of the seven mutations are identified, SMN1 copy number is determined to confirm the compound heterozygosity of the sample.
This panel was used to screen 366 patients shown not to homozygously lack the SMN1 gene based on results of the diagnostic deletion test.52 Ninety-five percent to 98% of SMA patients homozygously lack the SMN1 gene. Approximately 43% of the patients screened were deleted. Therefore, we estimated that 44% of the patients tested by our laboratory actually had SMA. Based on this estimation, we expected to find between one and three intragenic mutations. One mutation was identified (T274I). The patient with this mutation was subsequently found to possess only a single copy of the SMN1 gene and was, therefore, determined to be a compound heterozygote. This panel detects only the seven most common intragenic mutations, representing approximately 60% of all intragenic mutations reported in the literature.53 It is possible that less common mutations were present in patients for which mutations were not identified by this assay. This multiplex panel was able to detect every patient previously shown to have one of these common mutations. For this reason, we believe that it is an effective and appropriate diagnostic assay to increase the sensitivity of SMA testing. Because of the low intragenic mutation rate, this application might be reserved for patients with a very high probability of having proximal spinal muscular atrophy.
A carrier test for SMA was developed in our laboratory in 1997,17 which compares the ratios of the coamplified products of the SMN1 and CFTR genes. This competitive radiolabeled PCR reaction is optimized such that the copy numbers of the SMN1 and SMN2 genes can be determined in comparison to the two copies of the CFTR gene. Currently, 399 individuals have been tested for carrier status. Parents were most often tested as shown in Table 2. Unlike most autosomal recessive disorders, one cannot assume that parents of affected children are obligate carriers. De novo mutations40 and the presence of chromosomes with two copies of SMN141 allow for the possibility of affected individuals born to parents with normal SMN1 copy number. Ninety-five percent of the parents tested were shown to be carriers (Table 2). The remaining individuals present a diagnostic challenge. Because we cannot distinguish the de novo events (which confer a very low recurrence risk) from two-copy SMN1 chromosomes (which confer the normal 25% recurrence risk) based solely on the SMN1 dosage test, linkage studies, dosage testing of extended family members, or monosomal hybrid studies would be helpful in providing accurate genetic counseling to such parents.
The second most common population requesting carrier testing are the unrelated spouses of carriers. Three percent (3 of 100) of unrelated spouses were positive for carrier status, indicating a carrier frequency of 3%, which is in agreement with the previously described frequency of 1 of 50.16 Eighty-eight aunts and uncles of probands were tested of which 41 (47%) were carriers. Additionally, we found that 73% (27 of 37) of sibs, 21% (3 of 14) of first cousins, and 40% (8 of 20) of grandparents were carriers. None of these values are different from what would be expected from a recessive model with a 2% de novo mutation rate (Table 2).
Because more than 57% of the patients tested are not deleted for the critical exon 7, we only performed carrier tests on families known to have an SMN1 deletion. There are two important reasons for our policy. It is possible that one would perform dosage testing on a point mutation, which may result in false negatives. Another reason is that the clinical diagnosis is not straightforward. One may, therefore, perform dosage testing on the carrier of a phenotypically similar disorder, thereby falsely reducing the recurrence risk.
SMN2 copy number
Previous studies state that 5% of unaffected people homozygously lack SMN2.1 In this study, 14 of 97 normal controls with at least two copies of SMN1 had zero copies of SMN2, indicating a much higher percentage (14.4%). The difference between studies may be attributed to variation between populations. The majority of normal individuals have one or two copies of SMN2 (Fig. 2). It was also found that 4% of this sample of the normal population had three copies of the SMN1 gene.
To determine whether mild SMA patients have more SMN2 copies than severe patients, we have performed a large-scale comparison of SMN2 copy number in SMA patients with disparate ages of onset, including 52 type I cases that were sent for molecular diagnosis before they were 6 months old and 90 patients who were at least 18 years of age and diagnosed as having type III SMA. The results are shown in Table 3 and clearly demonstrate that there are more copies of SMN2 in mild SMA cases compared with severe cases based on a chi-squared test (P < 0.0001). Interestingly, 100% of type III patients had at least three copies of SMN2 and 20 of 90 had four copies. This finding is in contrast to only 3.8% of type I patients with three copies, whereas none had more than three copies. No type III patients were identified with one or two copies of SMN2. These data indicate that the presence of three or more copies of SMN2 is clearly correlated with a milder phenotype. Based on this information, it can be concluded that the presence of one or two copies of SMN2 predicts a severe phenotype, and three or more copies of SMN2 is a good prognostic indicator that a patient will at least sit unaided and live more than 2 years.
This study sought to test whether the variation in severity between patients with chimeric SMN genes was correlated with SMN2 copy number by analyzing the number of SMN2 genes in one type I and 10 type III patients who lacked exon 7 and retained exon 8 of SMN1, while possessing exon 7 of SMN2. We observed that every chimeric type III patient had at least three copies of SMN2, whereas the only type I chimeric patient tested had two copies. There were two additional chimeric patients who were 8 and 8.5 months at molecular diagnosis (possible type I) that had two copies of SMN2. If chimeric alleles were milder than normal SMN2 alleles, we might have expected to find some type III patients with two copies of SMN2. SMN2 copy number modifies the phenotype in deleted patients17,23,26,28,31,47,48 as well as in compound heterozygotes.32 These data are suggestive that the variation in phenotype between patients with chimeric SMN genes is also correlated with SMN2 copy number and not with the fact that chimeric alleles are inherently mild. Our population does not contain a large proportion of type I chimeric patients. It would be interesting to analyze SMN2 copy number in a population with a larger number of such patients.
The experience from 6 years of spinal muscular atrophy testing leads us to the following conclusions. There is a high frequency of patients with some symptoms consistent with SMA that are negative for the standard SMA deletion test. This fact emphasizes the importance of the molecular test in the diagnosis of spinal muscular atrophy. To further increase the sensitivity of molecular testing, a cost-effective and rapid PCR-based intragenic mutation panel has been described. There is great interest in SMA carrier testing, particularly by parents of affected patients, which has uncovered the interesting finding that 5% of parents possess two intact copies of SMN1 and require linkage analysis or monosomal hybrid studies for accurate risk assessment. With such a high rate of SMA-like patients testing negative for the common deletion, we recommend that carrier testing be reserved for at-risk relatives of patients that are positive for the SMN1 deletion. Our data indicate that, if a molecular diagnosis of SMA is not made in a family, then carrier testing will be performed on the wrong gene in more than half of the cases. Finally, SMN2 copy number has been shown to clearly correlate with the clinical type of SMA. Our comparison of severe type I and mild type III patients is the largest study to date and demonstrates that SMN2 can provide prognostic information.
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, Melki J . Identification and characterization of a spinal muscular atrophy-determining gene. Cell 1995; 80: 155–165.
Brzustowicz LM, Lehner T, Castilla LH, Penchaszadeh GK, Wilhelmsen KC, Daniels R, Davies KE, Leppert M, Ziter F, Wood D, Dubowitz V, Zerres K, Hausmanowa-Petrusewicz I, Ott J, Munsat TL, Gilliam TC . Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3. Nature 1990; 344: 540–541.
Gilliam TC, Brzustowicz LM, Castilla LH, Lehner T, Penchaszadeh GK, Daniels RJ, Byth BC, Knowles J, Hislop JE, Shapira Y, Dubowitz V, Munsat TL, Ott J, Davies KE . Genetic homogeneity between acute and chronic forms of spinal muscular atrophy. Nature 1990; 345: 823–825.
Melki J, Abdelhak S, Sheth P, Bachelot MF, Burlet P, Marcadet A, Aicardi J, Barois A, Carriere JP, Fardeau M, Fontan D, Ponsot G, Billette T, Angelini C, Barbosa C, Ferriere G, Lanzi G, Ottolini A, Babron MC, Cohen D, Hanauer A, Clerget-Darpoux F, Lathrop M, Munnich A, Frezal J . Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature 1990; 344: 767–768.
Daniels RJ, Thomas NH, MacKinnon RN, Lehner T, Ott J, Flint TJ, Dubowitz V, Ignatius J, Donner M, Zerres K, Rietschel M, Cookson WOC, Brzustowicz LM, Gilliam TC, Davies KE . Linkage analysis of spinal muscular atrophy. Genomics 1992; 123: 335–339.
Simard LR, Vanasse M, Rochette C, Morgan K, Lemieux B, Melancon SB, Labuda D . Linkage study of chronic childhood-onset spinal muscular atrophy (SMA): confirmation of close linkage to D5S39 in French Canadian families. Genomics 1992; 14: 188–190.
Francis MJ, Morrison KE, Campbell L, Grewal PK, Christodoulou Z, Daniels RJ, Monaco AP, Frischauf A-M, McPherson J, Wasmuth J, Davies KE . A contig of non-chimaeric YAC's containing the spinal muscular atrophy gene in 5q13. Hum Mol Genet 1993; 2: 1161–1167.
MacKenzie A, Roy N, Besner A, Mettler G, Jacob P, Korneluk R, Surh L . Genetic linkage analysis of Canadian spinal muscular atrophy kindreds using flanking microsatellite 5q13.3 polymorphisms. Hum Genet 1993; 90: 501–504.
Soares VM, Brzustowicz LM, Kleyn PW, Knowles JA, Palmer DA, Asokan S, Penchaszadeh GK, Munsat TL, Gilliam TC . Refinement of the spinal muscular atrophy locus to the interval between D5S435 and MAP1B. Genomics 1993; 15: 365–371.
Brahe C, Velona I, van der Steege G, Zappata S, van de Veen AY, Osinga J, Tops CMG, Fodde R, Khan PM, Buys CHCM, Neri G . Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis. Hum Genet 1994; 93: 494–501.
Burghes AHM, Ingraham SE, Kote-Jarai Z, Rosenfeld S, Herta N, Nadkarni N, DiDonato CJ, Carpten J, Hurko O, Florence J, Moxley RT, Cobben JM, Mendell JR . Linkage mapping of the spinal muscular atrophy gene. Hum Genet 1994; 93: 305–312.
Wirth B, El-Agwany A, Baasner A, Burghes AHM, Koch A, Dadze A, Piechaczek-Wappenschmidt B, Rudnik-Schoneborn S, Zerres K, Schonling J . Mapping of the spinal muscular atrophy (SMA) gene to a 750 kb interval flanked by two microsatellites. Eur J Hum Genet 1995; 3: 56–60.
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, Munnich A . De novoand inherited deletions of the 5q13 region in spinal muscular atrophies. Science 1994; 264: 1474–1477.
Roberts DF, Chaves J, Court SDM . The genetic component in child mortality. Arch Dis Child 1970; 45: 33–38.
Pearn J . The gene frequency of acute Werdnig-Hoffman disease (SMA Type 1). A total population survey in North-East England. J Med Genet 1973; 10: 260–265.
Pearn JH . Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet 1978; 15: 409–413.
McAndrew PE, Parsons DW, Simard LR, Rochette C, Ray PN, Mendell JR, Prior TW, Burghes AHM . Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am J Hum Genet 1997; 60: 1411–1422.
Bussaglia E, Clermont O, Tizzano E, Lefebfvre S, Burglen L, Cruaud C, Urtizberea JA, Colomer J, Munnich A, Baiget M, Melki J . A frame-shift deletion in the survival motor neuron gene in Spanish spinal muscular atrophy patients. Nat Genet 1995; 11: 335–337.
Cobben JM, van der Steege G, Grootscholten P, de Visser M, Scheffer H, Buys CHCM . Deletions of the survival motor neuron gene in unaffected siblings of patients with spinal muscular atrophy. Am J Hum Genet 1995; 57: 805–808.
Hahnen E, Forkert R, Merke C, Rudnik-Schoneborn S, Schonling J, Zerres K, Wirth B . 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 1995; 4: 1927–1933.
Rodrigues NR, Campbell L, Owen N, Rodeck CH, Davies KE . Prenatal diagnosis of spinal muscular atrophy by gene deletion analysis. Lancet 1995; 345: 1049.
Devriendt K, Lammens M, Scholen E, Van Hole C, Dom R, Devlieger H, Cassiman J-J, Fryns J-P, Matthijs G . Clinical and molecular genetic features of congenital spinal muscular atrophy. Ann Neurol 1996; 40: 731–738.
Hahnen E, Schonling J, Rudnik-Schoneborn S, Zerres K, Wirth B . Hybrid survival motor neuron genes in patients with autosomal recessive spinal muscular atrophy: new insights into molecular mechanisms responsible for the disease. Am J Hum Genet 1996; 59: 1057–1065.
Matthijs G, Schollen E, Leguis E, Devriendt K, Goemans N, Kayserili H, Apak MY, Cassiman J-J . Unusual molecular findings in autosomal recessive spinal muscular atrophy. J Med Genet 1996; 33: 469–474.
van der Steege G, Grootscholten PM, van der Vlies P, Draaijers TG, Osinga J, Cobben JM, Scheffer H, Den Dunnen JT, van Ommen GJ, Brahe C, Buys CHCM . Apparent gene conversions involving the SMN gene in the region of the spinal muscular atrophy locus on chromosome 5. Am J Hum Genet 1996; 59: 834–838.
Velasco E, Valero C, Valero A, Moreno F, Hernandez-Chico C . Molecular analysis of the SMN and NAIP genes in Spanish spinal muscular atrophy (SMA) families and correlation between copies ofCBCD541 and SMA phenotype. Hum Mol Genet 1996; 5: 257–263.
Burghes AHM . When is a deletion not a deletion? When it is converted. Am J Hum Genet 1997; 61: 9–15.
Campbell L, Potter A, Ignatius J, Dubowitz V, Davies K . Genomic variation and gene conversion in spinal muscular atrophy: implications for disease process and clinical phenotype. Am J Hum Genet 1997; 61: 40–50.
DiDonato CJ, Ingraham SE, Mendell JR, Prior TW, Lenard S, Moxley R, Florence J, Burghes AHM . Deletions and conversion in spinal muscular atrophy patients: is there a relationship to severity?. Ann Neurol 1997; 41: 230–237.
Talbot K, Rodrigues NR, Ignatius J, Muntoni F, Davies KE . Gene conversion at the SMN locus in autosomal recessive spinal muscular atrophy does not predict a mild phenotype. Neuromuscul Disord 1997; 7: 198–201.
Wirth B, Herz M, Wetter A, Moskau S, Hahnen E, Rudnik-Schoneborn S, Zerres K . Quantitative analysis of survival motor neuron copies: identification of subtle SMN1 mutations in patients with spinal muscular atrophy, genotype-phenotype correlation, and implications for genetic counseling. Am J Hum Genet 1999; 64: 1340–1356.
Parsons DW, McAndrew PE, Iannaccone ST, Mendell JR, Burghes AHM, Prior TW . Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of SMA phenotype by cenSMN copy number. Am J Hum Genet 1998; 63: 1712–1723.
Munsat TL, Davies KE . Meeting report: International SMA consortium meeting. Neuromuscul Disord 1992; 2: 423–428.
Zerres K, Schoneborn SR . Natural history in proximal spinal muscular atrophy: clinical analysis of 445 patients and suggestions for a modification of existing classifications. Arch Neurol 1995; 52: 518–523.
Werdnig G . Zwei fruhinfantile hereditare Falle von progressiven Mukelatrophie unter dem Bilder der Dystrophie, aber auf neurotische Grundlage. Arch Psychiat 1891; 22: 437–481.
Hoffmann J . Ueber Familiare progressive spinale muskelatrophie. Arch Psych Berlin 1892; 24: 644–646.
Kugelberg E, Welander L . Familial neurogenic (spinal?) muscular atrophy simulating ordinary proximal dystrophy. Arch Psych Scand 1954; 29: 42–43.
Kugelberg E, Welander L . Heredo-familial juvenile muscular atrophy simulating muscular dystrophy. Arch Neurol Psychiatry 1956; 75: 500–509.
Russman BS, Buncher CR, White M, Samaha FJ, Iannaccone ST, the DCN/SMA group. Function changes in spinal muscular atrophy II and III. Neurology 1996; 47: 973–976.
Wirth B, Schmidt T, Hahnen E, Rudnik-Schoneborn S, Krawczak M, Muller-Myhsok B, Schonling J, Zerres K . De novorearrangements found in 2% of index patients with spinal muscular atrophy: mutational mechanisms, parental origin, mutation rate, and implications for genetic counseling. Am J Hum Genet 1997; 61: 1102–1111.
Mailman MD, Hemingway T, Darsey RL, Glasure CE, Huang Y, Chadwick RB, Heinz JW, Papp AC, Snyder PJ, Sedra MS, Schafer RW, Abuelo DN, Reich EW, Theil KS, Burghes AHM, de la Chapelle A, Prior TW . Hybrids monosomal for human chromosome 5 reveal the presence of spinal muscular atrophy (SMA) carrier with two SMN1 copies on one chromosome. Hum Genet 2001; 108: 109–115.
Burglen L, Lefebvre S, Clermont O, Burlet P, Viollet L, Cruaud C, Munnich A, Melki J . Structure and organization of the human survival motor neuron (SMN) gene. Genomics 1996; 32: 479–482.
Lorson CL, Hahnen E, Androphy EJ, Wirth B . A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci U S A 1999; 96: 6307–6311.
Monani UR, Lorson CL, Parsons DW, Prior TW, Androphy EJ, Burghes AHM, McPherson JD . A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum Mol Genet 1999; 8: 1177–1183.
Lorson CL, Androphy EJ . An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN. Hum Mol Genet 2000; 9: 259–265.
Schrank B, Gotz R, Gunnersen JM, Ure JM, Toyka KV, Smith AG, Sendtner M . Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos. Proc Natl Acad Sci U S A 1997; 94: 9920–9925.
Hsieh-Li HM, Chang JG, Yong YJ, Wu MH, Wang NM, Tsai CH, Li H . A mouse model for spinal muscular atrophy. Nat Genet 2000; 24: 66–70.
Monani UR, Sendtner M, Coovert DD, Parsons DW, Andreassi C, Le TT, Jablonka S, Schrank B, Rossol W, Prior TW, Morris GE, Burghes AHM . The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(−/−) mice and results in a mouse with spinal muscular atrophy. Hum Mol Genet 2000; 9: 333–20.
Wirth B, Hahnen E, Morgan K, DiDonato CJ, Dadze A, Rudnik-Schoneborn S, Simard LR, Zerres K, Burghes AHM . Allelic association and deletions in autosomal recessive proximal spinal muscular atrophy: association of marker genotype with disease severity and candidate cDNAs. Hum Mol Genet 1995; 4: 1273–1284.
Nishio H, Horikawa H, Yakura H, Sugie K, Nakamuro T, Koterazawa K, Ishikawa Y, Lee MJ, Wada H, Takeshima Y, Matsuo M, Sumino K . Hybrid survival motor neuron genes in Japanese patients with spinal muscular atrophy. Acta Neurol Scand 1999; 99: 374–380.
Miller SA, Dykes DD, Polesky HF . A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215.
van der Steege G, Grootscholten PM, van der Vlies P, Draaijers TG, Osinga J, Cobben JM, Scheffer H, Buys CHCM . PCR-based DNA. test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 1995; 345: 985–986.
Wirth B . An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mutat 2000; 15: 228–237.
Laken SJ, Petersen GME, Gruber SB, Oddoux C, Ostrer H, Giardiello FM, Hamilton SR, Hampel H, Markowitz A, Klimstra D, Jhanwar S, Winawer S, Offit K, Luce MC, Kinzler KW, Vogelstein B . Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC. Nat Genet 1997; 17: 79–83.
This study was supported by Families of SMA, NIH grant NS38650, MDA of America; the Madison, Preston, and Matthew Funds, Families of SMA (BW), and Deutsche Forschungsgemeinschaft (BW).
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Mailman, M., Heinz, J., Papp, A. et al. Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2. Genet Med 4, 20–26 (2002). https://doi.org/10.1097/00125817-200201000-00004
- spinal muscular atrophy
- polymerase chain reaction
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