- Email: [email protected]
Establishment of a Molecular Diagnostic System for Spinal Muscular Atrophy
The Journal of Molecular Diagnostics, Vol. 13, No. 1, January 2011 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. DOI: 10.1016/j.jmoldx.2010.11.009
Establishment of a Molecular Diagnostic System for Spinal Muscular Atrophy Experience From a Clinical Laboratory in China
Jian Zeng, Yanhong Lin, Aizhen Yan, Longfeng Ke, Zhongyong Zhu, and Fenghua Lan From the Center for Molecular Diagnosis of Genetic Diseases, Fuzhou General Hospital, Xiamen University School of Medicine, Fuzhou City, Fujian Province, China
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of the anterior horn of the spinal cord. The disease gene survival motor neuron 1 (SMN1) is homozygously absent in approximately 95% of patients, and approximately 5% of patients are believed to have subtle mutations. Although methods for molecular diagnosis of SMA have been reported singly, no diagnostic methodological system to tackle different SMA cases has been reported. Thirty-two families affected by SMA enrolled into this study. Our system comprised PCR–restriction fragment length polymorphism and allele-specific PCR for homozygous deletion analysis of SMN1, multiplex ligation– dependent probe amplification analysis for the determination of the copy number of SMN1, and SMN1 subtle mutation analysis at both the transcript and genomic levels. In 23 families, 21 patients had a homozygous deletion of SMN1. The remaining two patients without a deletion had a single SMN1 copy containing the subtle mutations S230L and L228X, respectively. In nine families in whom samples from the index patients were unavailable, parents from eight families showed one SMN1 copy, and one parent in the remaining family showed two SMN1 copies, one being normal and the other carrying the subtle mutation 22_23insA. To our knowledge, our methodological system for the molecular diagnosis of SMA offers the most complete evaluation of family members affected by SMA at this time. ( J Mol Diagn 2011, 13: 41– 47; DOI: 10.1016/j.jmoldx.2010.11.009)
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder caused by degeneration of motor neurons in the anterior horn of the spinal cord. The degeneration process leads to progressive symmetric muscle weakness associated with muscle at-
rophy. The disease has a frequency of approximately 1 in 10,000 live births and a carrier frequency of approximately 1 in 30 to 1 in 50 in the European population.1 In China, a thorough survey of patients with SMA has not been conducted and data for the incidence of SMA in the Chinese population are lacking. However, carrier frequency was calculated as 1 in 42 in the general Chinese population in a population-based study, which is similar to that in the European population.2 On the basis of onset age and severity of clinical course, SMA can be classified into three major types.3 Type I [Mendelian Inheritance in Man (MIM) 253300] is the most severe form, and the involved children usually have symptoms of SMA before the age of 6 months and are never able to sit without support. Type II (MIM 253550) is an intermediate form with an onset before the age of 18 months, and the involved children are unable to stand and walk. Type III (MIM 253400) is a chronic form with an onset after the age of 18 months and can be further divided in type IIIa (onset age, ⬍3 years) and type IIIb (onset age, ⬎3 years). Spinal muscular atrophy with an onset at older than 30 years has been labeled type IV (MIM 271150) SMA. There are also many variants of SMA, but their genetic bases are different.4 The SMA disease-determining gene, termed survival motor neuron 1 [SMN1, Online Mendelian Inheritance in Man (OMIM) #600354], and its closely flanking, nearly identical, copy gene, SMN2 (OMIM #601627), are distinguished by a single base difference in exon 7 in the coding region.5 This difference results in no change in amino acid sequence but disrupts the exonic splicing enhancer in exon 7 of the gene,6 leading to preferential skipping of exon 7.7 Survival motor neuron protein from exon 7 lacking mRNA is unstable in the cell and undetectable using current methods, not to mention the exertion of any functions.8 The full-length SMN protein combines with at least eight other proteins, Gemins 2 through 8 and unrip, to form a macromolecular complex called SMN complex.9 This complex functions in pre-mRNA Accepted for publication July 30, 2010. Address reprint requests to Fenghua Lan, M.D., Center for Molecular Diagnosis of Genetic Diseases, Fuzhou General Hospital, Xiamen University School of Medicine, 156 Xi’erhuanbei Rd., Fuzhou City, Fujian Province 350025, China. E-mail: [email protected].
41
42 Zeng et al JMD January 2011, Vol. 13, No. 1
maturation by recruiting Sm proteins and promoting their transfer on U snRNAs.10 Setola et al11 recently identified an SMN transcript with the retention of SMN intron 3, which encodes an SMN protein selectively expressed in developing motor neurons and mainly localized in an axon (thus, the name axonal SMN). Although functional studies on SMN in model animals have been hampered by the lethality resulting from complete loss of SMN, Briese et al12 showed that deletion of the ortholog of SMN1 in Caenorhabditis elegans results in locomotor dysfunction and reduced life span, and Ebert et al13 successfully established induced human pluripotent stem cells from patients with type I SMA, opening up an alternative avenue to pathological modeling of SMA. The fact that SMN1 is homozygously absent in approximately 95% of patients with SMA because of deletion or conversion to SMN2 justifies the development of an effective PCR–restriction fragment length polymorphism (RFLP) assay for the molecular diagnosis of SMA.14 Confirmation of carrier status in unaffected individuals or support of a diagnosis of SMA in an individual who has typical clinical manifestations of SMA and does not lack both copies of SMN1 is possible by using reliable quantitative methods, such as multiplex ligation– dependent probe amplification (MLPA), for determining SMN1 copy number.15–17 Approximately 5% of patients with SMA are known to have no homozygous SMN1 deletion and are believed to have subtle mutations in one or, in extremely rarely cases, both SMN1 alleles instead.18 The finding of subtle mutations in patients who retain at least one copy of the SMN1 gene (patients with nondeletion SMA) has been considerably improved by the establishment of an SMN1 gene dosage test before subtle mutation analysis. In this article, we describe our experience in the molecular diagnosis of SMA, including homozygous deletion analysis of SMN1, dosage analysis of SMN1 and SMN2, and subtle mutation analysis of SMN1. The concept of a methodological system for molecular diagnosis of SMA is stressed.
Materials and Methods Subjects and DNA Isolation The subjects referred for SMA genetic testing were widely distributed across mainland China, including Guangdong, Fujian, Anhui, Shandong, Hubei, Henan, Hainan, Zhejiang, and Jiangxi Provinces. A total of 103 subjects from 32 families affected by SMA (families 1–32) have been analyzed, including 23 patients with SMA who were clinically suspected to be affected by SMA (9 had type I, 11 had type II, and 3 had type III), 62 parents of patients with SMA, and 18 high-risk fetuses from 15 families affected by SMA. In addition, 200 healthy individuals unaffected by SMA, with no family history of SMA (including five healthy controls with two copies each of the SMN1 and SMN2 gene), also enrolled into this study. Genomic DNA was extracted from peripheral blood using a commercially available kit (SE Blood DNA Kit; Omega, Norcross, GA) and from prenatal samples (amniocytes,
before and/or after culture) using another kit (QIAamp DNA Blood Mini Kit; Qiagen, Duesseldorf, Germany). Informed consent was obtained from all families in our study. The study was approved by the Ethical Committee of Fuzhou General Hospital in China.
PCR-RFLP and Allele-Specific PCR for Homozygous Deletion Analysis of SMN1 The conventional PCR-RFLP used for detecting homozygous deletion of SMN1 exon 7 was performed as originally described by van der Steege et al.14 Primers for allele-specific PCR of SMN exon 7 were described in a previous study.19 The common forward primer RIII was designed as originally described by van der Steege et al. Reverse primers 5=-CCTTCCTTCTTTTTGATTTTGTCAG-3= (for SMN1) and 5=-CCTTCCTTCTTTTTGATTTTGTCAA-3= (for SMN2) were designed to amplify SMN1 or SMN2. A deliberate mismatch (italicized) was introduced 2 bp before the 3= end of reverse primers to increase PCR specificity.19 To monitor PCR efficiency, amplification of SMN exon 4 served as an internal control. Primers for SMN exon 4 were originally described by Parsons et al.20 The optimal ratio of SMN1/2 exon 7 primers to exon 4 primers was 2:1. This optimal ratio was used in all allelespecific PCR assays. The total PCR reaction volume of 25 L contained 50 ng of genomic DNA, 0.5 L of each pair of SMN1/2 exon 7 forward and reverse primers (20 mol/ L), 0.5 L of each pair of SMN exon 4 primers (10 mol/ L), and 2.5 U of rTaq DNA polymerase (Takara, Kyoto, Japan). The following PCR conditions were used: 94°C for 4 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, with a final extension of 72°C for 5 minutes. The results were documented by photographing the 2% gel under UV light.
MLPA Analysis for Determination of SMN1 and SMN2 Copy Number Multiplex ligation– dependent probe amplification was performed in a thermal cycler (Eppendorf, Hamburg, Germany) using a kit (SALSA MLPA Kit P021; MRCHolland, Amsterdam, the Netherlands). All reactions were performed according to the manufacturer’s protocol. Approximately 100 ng of genomic DNA of 23 patients, 62 parents, 18 fetuses, and 50 healthy individuals was used for the MLPA reaction. The MLPA products were run on an analyzer (ABI PRISM 3100-Avant Genetic Analyzer; ABI, Carlsbad, CA) with LIZ 500 as the internal size standard. All samples were analyzed at least twice. Data analysis was performed using manual Excel sheets. The relative peak area (RPA) was obtained by dividing the PA of each probe by the total PA of the control probes: RPA ⫽ PA/Total PA. For each sample, the relative copy number for each probe was obtained by dividing RPA by the median normalized PA of five healthy controls: Relative Copy Number ⫽ RPA/Median Normalized PA. For SMN1/2 exon 7, the absolute copy number of each sample was obtained by multiplying RPA with a
Molecular Diagnostic System for SMA in China 43 JMD January 2011, Vol. 13, No. 1
factor of two. According to the recommendations of the manufacturer, a reduction of 35% to 55% in RPA for a probe’s amplification product is indicative of deletion of one copy of that probe’s target sequence; and an increase between 30% and 55%, a gain in copy number from two to three copies. In this study, only the data of exon 7 of SMN1 and SMN2 were analyzed statistically. Statistical analysis was performed by using a statistical program package (SAS, Cary, NC).
SMN1 Subtle Mutation Analysis at the Transcript Level RNA was isolated from peripheral blood by using a kit (QIAamp RNA Blood Mini Kit, Qiagen, Duesseldorf, Germany). First-strand cDNA synthesis was performed with 2 g of RNA by using oligo(dT) and reverse transcriptase (SuperScript II RT) (200 U/L, Invitrogen, Carlsbad, CA). The single-stranded cDNA was amplified with 10 ng of each primer, SMN forward primer (5=-TGCGCATCCGCGGGTTTGCT-3=) and SMN reverse primer (5=TCATTTAGTGCTGCTCTATGCCA-3=), which was described by Parsons et al.20 The purified RT-PCR products covering the entire coding region were directly sequenced (model 3730 Sequencer; Applied Biosystems, Carlsbad, CA). To determine whether the SMN1 or SMN2 gene contributed to the sequence variation through the RNA approach, purified RT-PCR products of SMN were cloned into a vector (pGEM-T Easy Vector; Promega, Madison, WI), following the supplier’s protocol. The plasmid DNA was purified with a mini plasmid kit (Qiagen) from approximately 6 to 10 white colonies and analyzed by digestion with EcoRI. Recombinant plasmids containing inserts of 956 bp were also sequenced (model 3730 Sequencer, Applied Biosystems).
Mutation Detection at the Genomic Level To confirm the detected mutations, specific regions in genomic DNA were amplified by the following primers: for exon 5, 5=-TGAGTCTGTTTGACTTCAGG-3= (sense primer) and 5=-TATCAAATTGTATGTGAAAGCA-3= (antisense primer); and for exon 1, 5=-GCCGGAAGTCGTCACTCTT-3= (sense primer) and 5=-GGGTGCTGAGAGCGCTAATA-3= (antisense primer). These were described elsewhere by Parsons et al.20 The purified PCR products were directly sequenced (model 3730 Sequencer). Moreover, the restriction enzyme Hin1II was used to cleave the products of exon 5 to check the existence of the mutation C689T (S230L); PCR products of exon 5 from 200 healthy controls were also cleaved by Hin1II to exclude the possibility of a polymorphism. To confirm the mutation in exon 1 (22_23insA), T cloning of the purified PCR products was performed following the supplier’s protocol (previous description) and multiple clones were analyzed.
Figure 1. Pedigree and putative genotypes inferred from families 17 and 24.
Results Thirty-two families were referred to our laboratory for diagnostic SMA testing, and blood samples from 23 patients were available. Of these patients, 21 had a homozygous deletion of SMN1 by conventional PCR-RFLP and an allele-specific PCR. Among the parents of these 21 patients, 36 showed one copy of SMN1 and two (the father in family 2 and the mother in family 5) showed two copies by MLPA analysis. The remaining two patients without a deletion (patients from families 17 and 24) were subsequently subjected to MLPA analysis to determine their copy numbers of the SMN1 gene; these patients were highly suspected to have SMA in terms of clinical manifestations. Both patients possessed one copy of SMN1, making them likely compound heterozygotes (patients with one SMN1 gene lost and one SMN1 gene containing a subtle mutation). The entire SMN coding region was screened for subtle mutations by RT-PCR and T cloning, and two subtle SMN1 mutations (ie, S230L and L228X, identified in patients 17 and 24, respectively) were found. Sequencing of PCR products of SMN exon 5 from the parents showed that both of the mutations were paternal. The SMN genotypes of members from both of the families were shown in Figure 1. In 200 healthy individuals, no S230L mutation was observed. In the nine families in whom the probands’ samples were unavailable because of probands’ death before parental referral to us, carrier testing by MLPA was performed on the 18 parents. Parents from eight families showed one copy of SMN1, confirming carrier status and a diagnosis of SMA of the proband. In the remaining family (family 25), the mother showed one copy and the father showed two copies. Suspecting that this parent was a carrier with one normal SMN1 allele and a subtle mutation on the other allele, mutation analysis was performed at both the transcript and genomic levels. Interestingly, at the genomic level, a frame-shift mutation (ie, 22_23insA) was identified (Figure 2) by direct sequencing of PCR products of exon 1. This mutation was confirmed by sequencing of mutated clones after T cloning of PCR products. However, we were unable to identify any clone containing this mutation at the transcript level. Exhaustive screening of the obtained clones and repeated RT-PCR and T cloning exclusively found clones contain-
44 Zeng et al JMD January 2011, Vol. 13, No. 1
Table 1. Determination of SMN2 Copy Numbers in Patients With SMA by MLPA
Figure 2. Genomic DNA sequence of exon 1 of the survival motor neuron (SMN) gene from the father in family 25. The father had an “A” insertion (indicated by the box) at codon 8 (underlined), resulting in a frame shift on this allele (mutated allele).
ing normal SMN1- or normal SMN2-derived products, suggesting that transcripts from the mutated SMN1 allele were either absent or in extremely low quantity. We also performed prenatal SMA diagnostic testing on 18 amniotic fluid samples of high-risk fetuses from 15 families affected by SMA. In 18 fetuses, five had homozygous deletion of the SMN1 gene, seven had one copy, and six had two copies. The copy number of SMN1 and SMN2 in these 15 fetuses is shown in Figure 3. To look for evidence of correlation of SMN2 copy number and severity of SMA, MLPA was performed on 23 patients to determine SMN2 copy number (Table 1). By Ridit analysis, there was significant correlation between the SMN2 copy number and severity of SMA (P ⬍ 0.05).
Discussion Based on our experience, we propose the following strategy for molecular diagnosis of SMA: (1) Patients are selected for molecular analysis according to clinical criteria. (2) PCR-RFLP and allele-specific PCR are performed to detect a homozygous SMN1 exon 7 deletion. (3) Multiplex ligation– dependent probe amplification is subsequently performed to determine the copy number of SMN1 of a patient if the patient is free from homozygous deletion of SMN1 but the patient’s manifestations fulfill the clinical criteria of SMA. (4) Carrier testing is performed by MLPA to confirm the carrier status of the parents in families in which the index patients with SMA
Figure 3. Histogram showing the results of 18 fetuses by multiplex ligation– dependent probe amplification (MLPA) analysis for SMN1 and SMN2 copy numbers.
Copy No. of SMN2
I (n ⫽ 8)
II (n ⫽ 11)
III (n ⫽ 3)
Total (N⫽22)
1 2 3 4
1 4 3 0
0 0 11 0
0 0 0 3
1 4 14 3
SMA type
The MLPA analysis failed in one patient because of the low quality of DNA. SMA, spinal muscular atrophy; MLPA, multiplex ligation– dependent probe amplification; SMN, survival motor neuron.
have died at the time of reference. (5) Subtle mutation analysis of SMN1 is performed if a patient’s clinical manifestations strongly point to a diagnosis of SMA and if one copy of SMN1 is confirmed by MLPA. (6) Prenatal testing is performed in families in which the genotype of SMN1 in the proband is known or the carrier status of the parents is confirmed. Our strategy represents a comprehensive methodological system for the molecular diagnosis of SMA. It is a tradition of laboratory medicine to search for a single simple method for the laboratory diagnosis for a disease or a kind of disease. In this era of systems medicine and complexity science, such thinking should be abandoned. Instead, the idea of a methodological system in laboratory medicine or a diagnostic system should be established to tackle the complexity of single-gene disorders. The complexity of single-gene disease can be noted in at least three patterns. First, a single-gene disease results from one mutation in one gene but is modulated by many other genes (ie, modifier or epistatic genes) in terms of disease phenotype. Sickle-cell anemia, the first monogenic disease ever described, is typical of this category.21 Second, a monogenic disease can be caused by different mutations in one gene and modulated by many modifier genes. Most single-gene diseases, including SMA, belong to this category. Third, a group of monogenic diseases with common phenotypes is caused by mutations in many different genes. Examples of this category are diseases such as hereditary ataxia and hereditary spastic paraplegia.22,23 Obviously, no single method can tackle the genetic diagnosis of single-gene disorders of the second and third categories. For homozygous deletion analysis of the SMN1 gene, only one single simple method, such as conventional PCR-RFLP, was performed in most laboratories. Actually, every single method has its limits and cannot sufficiently tackle the genetic diagnosis. Moreover, conventional PCR-RFLP requires an enzymatic digestion step and incomplete digestion of PCR products will result in falsenegative results. In our study, the PCR-RFLP method was combined with allele-specific PCR (also as an amplification refractory mutation system) to overcome those disadvantages and both their results can confirm each other. The primers for SMN1 in this method theoretically will only amplify SMN1 exon 7, not SMN2 exon 7, because of a single-nucleotide difference between the exons 7 of SMN1 and SMN2. However, according to the
Molecular Diagnostic System for SMA in China 45 JMD January 2011, Vol. 13, No. 1
report by Xu et al,24 it may not be a method that can be applied in routine genetic testing because the allelespecific reverse primer (for SMN1) designed by Moutou et al25 can easily bind to SMN2, in the presence of higher amounts (20 to 100 ng) of genomic DNA, resulting in substantial amplification of SMN2 in the case of homozygous loss of SMN1. Previous studies have shown that further deliberate mismatches to destabilize the primer/ template complexes render the primers increasingly refractory as the additional mismatch is moved progressively closer to the 3= end.26 Therefore, in our study, we introduce an additional deliberate mismatch 2 bp before the 3= end of reverse primers in amplification refractory mutation system to increase PCR specificity. This amplification refractory mutation system, whose results were in complete agreement with the results of conventional PCR-RFLP and MLPA, has proved to be a simple, quick, and reliable method in a previous study.19 The MLPA technique greatly improves diagnostics in SMA.15–17,27 Multiplex ligation– dependent probe amplification analysis, which is different from PCR-RFLP and allele-specific PCR (which only detect the absence of SMN1), detects the mechanism responsible for SMN1 loss (ie, deletion or conversion to SMN2). Evaluation of the SMN2 copy number is also useful for establishing genotype-phenotype correlation in patients with SMA based on the evidence that the SMN2 copy number relates to severity of the disease.28,29 Although the number of patients studied was few, our data obtained from MLPA, together with the results by statistical analysis, provided further evidence for a disease-modifying role of SMN2, with a greater SMN2 copy number being associated with a milder SMA phenotype, allowing for some prognostic considerations. Another important purpose of MLPA was for the detection of heterozygous SMN1 deletions to support the status of SMA carriers in families affected by SMA and to screen SMA carriers in the general population. Among 50 healthy individuals without a family history of SMA in our study, the percentage of individuals with one copy was 2% (data not shown), which is in accordance with the approximately 2% carrier frequency in the general population.1 Unfortunately, we found unexpected MLPA results in 2.6% (4/153) of the DNA samples. These results included the disappearance of many peaks in one sample and the warning signal of control peaks. It is probably because of contaminants (ie, PCR inhibitors, such as a small amount of ionic impurities, remnant ethanol, and phenol) in these DNA samples. The impurities can have their effect on the PCR reaction or on DNA denaturation by increasing temperature. Actually, MLPA is more sensitive to impurities than ordinary PCR. That is why good results of the same samples, whose MLPA results do not make any sense, can be obtained by conventional PCR-RFLP and allele-specific PCR. In our study of 59 parents whose SMN1 copy number was determined, five had a normal two-copy SMN1 dosage by MLPA. Three (the fathers in families 17, 24, and 25) of these five parents had a mutant non-deleted SMN1 allele identified by subtle mutation analysis. However, the remaining two (the father from family 2 and the mother
from family 5) had a child with homozygous SMN1 deletions. This situation could be caused by two unusual occurrences. First, the occurrence of two (or more) SMN1 genes on a single chromosome is approximately 4% in the normal population.30,31 Given the instability of this genomic region, two (or more) SMN1 genes on a single chromosome may result from gene conversion from SMN2 to SMN1 or alternatively from the remaining duplicated SMN1 genes without conversion to SMN2.31 In reality, such parents would be carriers with a 25% recessive risk of having another affected child with a carrier spouse. Second, SMN1 has a high rate of de novo deletion mutations.32 The high rate of de novo mutations in SMN1 underlies a de novo mutational event in the affected child whose parents have a normal dosage of SMN1. It is important for genetic counseling to distinguish between the normal recessive risk of a two-copy SMN1 carrier and the low risk of recurrence after de novo mutation in noncarrier parents of affected children. For the time being, the case of two or more SMN1 genes on a single chromosome can only be resolved by somatic cell hybridization and fluorescence in situ hybridization and that of de novo deletion mutations by SMN1 gene dosage of extended family members. Subtle mutation analysis of SMN1 is complicated by the presence of SMN2, thus it is not offered routinely in most clinical laboratories. The current approaches used to identify subtle mutations in a few laboratories include single-strand conformation polymorphism, denaturing high-performance liquid chromatography, haplotype studies with Ag1-CA and C212, and sequencing of the complete SMN coding region,18,20,33 which are somewhat labor intensive and time-consuming. Direct sequencing of genomic DNA is inappropriate because of unequally higher amounts of SMN2 copies. Direct sequencing of cDNA is inadequate because of unequal amounts of SMN1/SMN2 and lack of exons 3, 5, and 7 in alternatively spliced transcripts. Furthermore, any mutation found must be assigned to the correct SMN1 copy, which makes subcloning and sequencing of SMN1 absolutely necessary.33 The SSCP analysis or DHPLC of each exon is more time-consuming, in which not all mutations can be identified and sequencing for the exact mutation and assignment to the correct SMN1 copy must be performed. To our knowledge, we were the first to take advantage of the strategy of quantitative analysis, followed by a combination of the RNA approach (sequencing of the complete SMN1 coding region from cloned RT-PCR products) and the genomic DNA approach (sequencing of SMN exon regions from cloned PCR products), which proved effective for SMN1 subtle mutation analysis; this method might offer more reliable results, although it cannot avoid the demerits of being labor intensive and time-consuming. In the present article, we identified three subtle mutations in the SMN1 gene. One is the nonsense mutation L228X, which was described elsewhere.34 Another is the missense mutation S230L, which is described herein for the first time. This novel mutation (S230L) was pinpointed in SMN1 exon 5, together with one SMN2 copy in patient 17 (type I). The region of the SMN protein coded by the
46 Zeng et al JMD January 2011, Vol. 13, No. 1
exon carrying S230L belongs to functionally less important SMN domains (UniprotKB/Swiss-ProtQ16637-SMNHuman), and the amino acid sequence spanning the positions from approximately 194 to 251 corresponds to a domain of low complexity (PridictProtein server for predict_h34345); nevertheless, our finding of a pathogenetic mutation at amino acid residue 230 points to the necessity of reevaluating the function of the relevant region of the SMN protein. The last one is frame-shift mutation 22_23insA. This mutation has previously been identified by Tsai et al.34 Because Tsai et al exclusively used a DNA-based assay to detect the mutation, the presence of transcripts was never checked. This mutation was detected only at the genomic DNA level in the present study. It is well established that transcripts typically undergo nonsense-mediated mRNA decay if they harbor a premature translation termination codon more than 55 nucleotides upstream of the last intron.35 According to this rule, this subtle mutation (22_23insA) detected in exon 1 leads to generation of premature translation termination codon in exon 2a, which is expected to trigger nonsense-mediated mRNA decay and, in turn, reduce the expression of a mutated SMN1 transcript. This phenomenon underscores the importance of combining the RNA approach with the DNA approach to detect subtle mutations. Premature translation termination codon may also be created by the mutation L228X according to the rule; however, why were the mutated transcripts clearly detected in the patient? This question might not be answered until the mechanism of action of the mutations is further studied. In China, there are four SMN1 subtle mutations (ie, S230L, L228X, 22-23insA, and 835-1G⬎A) found in type I Chinese patients. The fact that L228X and 22-23ins A mutations identified more than once by different laboratories (ie, our laboratory and the laboratory of Tsai et al34) have not been reported in the European population so far makes it likely that these subtle mutations might be unique at our location. If these subtle mutations are confirmed to be common subtle mutations in Chinese patients with SMA, rapid assays (eg, allele-specific PCR) for the detection of these mutations can be easily designed to increase the sensitivity of SMA molecular diagnosis. In conclusion, our comprehensive methodological system for the molecular diagnosis of SMA, including SMN1 deletion analysis, SMN1 and SMN2 dosage analysis, and subtle mutation analysis, enables us to resolve almost all of the diagnostic situation of SMA and offers the most complete evaluation of patients with SMA and their family members at this time.
Acknowledgments We thank the families affected by SMA who participated in this study; Dr. Bosheng Yang for family referral; Dr. Lianghu Huang for primer design; Dr. Huijuan Huang for amniotic fluid collection; and Dr. Xiangdong Tu for culture of amniocytes.
References 1. Ogino S, Leonard DG, Rennert H, Wilson RB: Spinal muscular atrophy genetic testing experience at an academic medical center. J Mol Diagn 2002, 4:53–58 2. Zhu S, Xiong F, Chen YJ, Yan TZ, Zeng J, Li L, Zhang YN, Chen WQ, Bao XH, Zhang C, Xu XM: Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population. Eur J Hum Genet 2010. [Epub ahead of press] 3. Munsat TL, Davies KE: International SMA consortium meeting (26 –28 June 1992. Bonn, Germany). Neuromuscul Disord 1992, 2:423– 428 4. Panigrahi I, Kesari A, Phadke SR, Mittal B: Clinical and molecular diagnosis of spinal muscular atrophy. Neurol India 2002, 50:117–122 5. Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M: Identification and characterization of a spinal muscular atrophy-determining gene. Cell 1995, 80:155–165 6. Cartegni L, Krainer AR: Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet 2002, 30:377–384 7. Cartegni L, Hastings ML, Calarco JA, de-Stanchina E, Krainer AR: Determinants of exon 7 splicing in the spinal muscular atrophy genes: SMN1 and SMN2. Am J Hum Genet 2006, 78:63–77 8. Vitte J, Fassier C, Tiziano FD, Dalard C, Soave S, Roblot N, Brahe C, Saugier-Veber P, Bonnefont JP, Melki J: Refined characterization of the expression and stability of the SMN gene products. Am J Pathol 2007, 171:1269 –1280 9. Otter S, Grimmler M, Neuenkirchen N, Chari A, Sickmann A, Fischer U: A comprehensive interaction map of the human survival of motor neuron (SMN) complex. J Biol Chem 2007, 282:5825–5833 10. Pillai RS, Grimmler M, Meister G, Will CL, Lührmann R, Fischer U, Schümperli D: Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component: Lsm11, in histone RNA processing. Genes Dev 2003, 17:2321–2333 11. Setola V, Terao M, Locatelli D, Bassanini S, Gerattini E, Battaglia G: Axonal-SMN (a-SMN), a protein isoform of the survival motor neuron gene, is specifically involved in axonogenesis. Proc Natl Acad Sci U S A 2007, 104:1959 –1964 12. Briese M, Esmaeili B, Fraboulet S, Burt EC, Christodoulou S, Towers PR, Davies KE, Sattelle DB: Deletion of smn-1, the Caenorhabditis elegans ortholog of the spinal muscular atrophy gene, results in locomotor dysfunction and reduced lifespan. Hum Mol Genet 2009, 18:97–104 13. Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN: Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 2009, 457:277–280 14. van der Steege G, Grootscholten PM, van der Vlies P, Draaijers TG, Osinga J, Cobben JM, Scheffer H, Buys CH: PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 1995, 345:985–986 15. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G: Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002, 30:e57 16. Arkblad EL, Darin N, Berg K, Kimber E, Brandberg G, Lindberg C, Holmberg E, Tulinius M, Nordling M: Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy. Neuromuscul Disord 2006, 16:830 – 838 17. Scarciolla O, Stuppia L, De Angelis MV, Murru S, Palka C, Giuliani R, Pace M, Di Muzio A, Torrente I, Morella A, Grammatico P, Giacanelli M, Rosatelli MC, Uncini A, Dallapiccola B: Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification. Neurogenetics 2006, 7:269 –276 18. Wirth B, Herz M, Wetter A, Moskau S, Hahnen E, Rudnik-Schoneborn S, Wienker T, 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 19. Zeng J, Lan FH, Deng XJ, Ke LF, Tu XD, Huang LH, Zheng DZ, Zhu ZY: Evaluation of an in-house protocol for prenatal molecular diagnosis of SMA in Chinese. Clin Chim Acta 2008, 398:78 – 81
Molecular Diagnostic System for SMA in China 47 JMD January 2011, Vol. 13, No. 1
20. Parsons DW, McAndrew PE, Iannaccone ST, Mendell JR, Burghes AH, Prior TW: Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number. Am J Hum Genet 1998, 63:1712–1723 21. Nagel RL, Steinberg MH: Role of epistatic (modifier) genes in the modulation of the phenotypic diversity of sickle cell anemia. Pediatr Pathol Mol Med 2001, 20:123–136 22. Harding AE: Clinical features and classification of inherited ataxias. Adv Neurol 1993, 61:1–14 23. Contino G, Novelli G: Hereditary spastic paraplegia: clinical genomics and pharmacogenetic perspectives. Expert Opin Pharmacother 2006, 7:1849 –1856 24. Xu R, Ogino S, Lip V, Fang H, Wu BL: Comparison of PCR-RFLP with allele-specific PCR in genetic testing for spinal muscular atrophy. Genet Test 2003, 7:277–281 25. Moutou C, Gardes N, Rongières C, Ohl J, Bettahar-Lebugle K, Witterner C, Gerlinger P, Viville S: Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy. Prenat Diagn 2001, 21:498 –503 26. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF: Analysis of any point mutation in DNA: the amplification refractory mutation system (ARMS). Nucleic Acids Res 1989, 17:2503–2516 27. Zapletalová E, Hedvicáková P, Kozák L, Vondrácek P, Gaillyová R, Maríková T, Kalina Z, Jüttnerová V, Fajkus J, Fajkusová L: Analysis of point mutations in the SMN1 gene in SMA patients bearing a single SMN1 copy. Neuromuscul Disord 2007, 17:476 – 481 28. 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 number of
29.
30.
31.
32.
33.
34.
35.
copies of cBCD541 and SMA phenotype. Hum Mol Genet 1996, 5:257–263 Vitali T, Sossi V, Tiziano F, Zappata S, Giuli A, Paravatou-Petsotas M, Neri G, Brahe C: Detection of the survival motor neuron (SMN) genes by FISH: further evidence for a role for SMN2 in the modulation of disease severity in SMA patients. Hum Mol Genet 1999, 8:2525–2532 Scheffer H, Cobben JM, Mensink RGJ, Stulp RP, Van der Steege G, Buys CH: SMA carrier testing: validation of hemizygous SMN exon 7 deletion test for the identification of proximal spinal muscular atrophy carriers and patients with a single allele deletion. Eur J Hum Genet 2000, 8:79 – 86 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 AH, Chapelle A, Prior TW: Hybrids monosomal for human chromosome 5 reveal the presence of a spinal muscular atrophy (SMA) carrier with two SMN1 copies on one chromosome. Hum Genet 2001, 108:109 –115 Wirth B, Schmidt T, Hahnen E, Rudnik-Schöneborn S, Krawczak M, Muller-Myhsok B, Schönling J, Zerres K: De novo rearrangements 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 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 Tsai CH, Jong YJ, Hu CJ, Chen CM, Shih MC, Chang CP, Chang JG: Molecular analysis of SMN: NAIP and P44 genes of SMA patients and their families. J Neurol Sci 2001, 190:35– 40 Wilkinson MF: A new function for nonsense-mediated mRNA-decay factors. Trends Genet 2005, 21:143–148
Establishment of a Molecular Diagnostic System for Spinal Muscular Atrophy Experience From a Clinical Laboratory in China
Jian Zeng, Yanhong Lin, Aizhen Yan, Longfeng Ke, Zhongyong Zhu, and Fenghua Lan From the Center for Molecular Diagnosis of Genetic Diseases, Fuzhou General Hospital, Xiamen University School of Medicine, Fuzhou City, Fujian Province, China
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of the anterior horn of the spinal cord. The disease gene survival motor neuron 1 (SMN1) is homozygously absent in approximately 95% of patients, and approximately 5% of patients are believed to have subtle mutations. Although methods for molecular diagnosis of SMA have been reported singly, no diagnostic methodological system to tackle different SMA cases has been reported. Thirty-two families affected by SMA enrolled into this study. Our system comprised PCR–restriction fragment length polymorphism and allele-specific PCR for homozygous deletion analysis of SMN1, multiplex ligation– dependent probe amplification analysis for the determination of the copy number of SMN1, and SMN1 subtle mutation analysis at both the transcript and genomic levels. In 23 families, 21 patients had a homozygous deletion of SMN1. The remaining two patients without a deletion had a single SMN1 copy containing the subtle mutations S230L and L228X, respectively. In nine families in whom samples from the index patients were unavailable, parents from eight families showed one SMN1 copy, and one parent in the remaining family showed two SMN1 copies, one being normal and the other carrying the subtle mutation 22_23insA. To our knowledge, our methodological system for the molecular diagnosis of SMA offers the most complete evaluation of family members affected by SMA at this time. ( J Mol Diagn 2011, 13: 41– 47; DOI: 10.1016/j.jmoldx.2010.11.009)
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder caused by degeneration of motor neurons in the anterior horn of the spinal cord. The degeneration process leads to progressive symmetric muscle weakness associated with muscle at-
rophy. The disease has a frequency of approximately 1 in 10,000 live births and a carrier frequency of approximately 1 in 30 to 1 in 50 in the European population.1 In China, a thorough survey of patients with SMA has not been conducted and data for the incidence of SMA in the Chinese population are lacking. However, carrier frequency was calculated as 1 in 42 in the general Chinese population in a population-based study, which is similar to that in the European population.2 On the basis of onset age and severity of clinical course, SMA can be classified into three major types.3 Type I [Mendelian Inheritance in Man (MIM) 253300] is the most severe form, and the involved children usually have symptoms of SMA before the age of 6 months and are never able to sit without support. Type II (MIM 253550) is an intermediate form with an onset before the age of 18 months, and the involved children are unable to stand and walk. Type III (MIM 253400) is a chronic form with an onset after the age of 18 months and can be further divided in type IIIa (onset age, ⬍3 years) and type IIIb (onset age, ⬎3 years). Spinal muscular atrophy with an onset at older than 30 years has been labeled type IV (MIM 271150) SMA. There are also many variants of SMA, but their genetic bases are different.4 The SMA disease-determining gene, termed survival motor neuron 1 [SMN1, Online Mendelian Inheritance in Man (OMIM) #600354], and its closely flanking, nearly identical, copy gene, SMN2 (OMIM #601627), are distinguished by a single base difference in exon 7 in the coding region.5 This difference results in no change in amino acid sequence but disrupts the exonic splicing enhancer in exon 7 of the gene,6 leading to preferential skipping of exon 7.7 Survival motor neuron protein from exon 7 lacking mRNA is unstable in the cell and undetectable using current methods, not to mention the exertion of any functions.8 The full-length SMN protein combines with at least eight other proteins, Gemins 2 through 8 and unrip, to form a macromolecular complex called SMN complex.9 This complex functions in pre-mRNA Accepted for publication July 30, 2010. Address reprint requests to Fenghua Lan, M.D., Center for Molecular Diagnosis of Genetic Diseases, Fuzhou General Hospital, Xiamen University School of Medicine, 156 Xi’erhuanbei Rd., Fuzhou City, Fujian Province 350025, China. E-mail: [email protected].
41
42 Zeng et al JMD January 2011, Vol. 13, No. 1
maturation by recruiting Sm proteins and promoting their transfer on U snRNAs.10 Setola et al11 recently identified an SMN transcript with the retention of SMN intron 3, which encodes an SMN protein selectively expressed in developing motor neurons and mainly localized in an axon (thus, the name axonal SMN). Although functional studies on SMN in model animals have been hampered by the lethality resulting from complete loss of SMN, Briese et al12 showed that deletion of the ortholog of SMN1 in Caenorhabditis elegans results in locomotor dysfunction and reduced life span, and Ebert et al13 successfully established induced human pluripotent stem cells from patients with type I SMA, opening up an alternative avenue to pathological modeling of SMA. The fact that SMN1 is homozygously absent in approximately 95% of patients with SMA because of deletion or conversion to SMN2 justifies the development of an effective PCR–restriction fragment length polymorphism (RFLP) assay for the molecular diagnosis of SMA.14 Confirmation of carrier status in unaffected individuals or support of a diagnosis of SMA in an individual who has typical clinical manifestations of SMA and does not lack both copies of SMN1 is possible by using reliable quantitative methods, such as multiplex ligation– dependent probe amplification (MLPA), for determining SMN1 copy number.15–17 Approximately 5% of patients with SMA are known to have no homozygous SMN1 deletion and are believed to have subtle mutations in one or, in extremely rarely cases, both SMN1 alleles instead.18 The finding of subtle mutations in patients who retain at least one copy of the SMN1 gene (patients with nondeletion SMA) has been considerably improved by the establishment of an SMN1 gene dosage test before subtle mutation analysis. In this article, we describe our experience in the molecular diagnosis of SMA, including homozygous deletion analysis of SMN1, dosage analysis of SMN1 and SMN2, and subtle mutation analysis of SMN1. The concept of a methodological system for molecular diagnosis of SMA is stressed.
Materials and Methods Subjects and DNA Isolation The subjects referred for SMA genetic testing were widely distributed across mainland China, including Guangdong, Fujian, Anhui, Shandong, Hubei, Henan, Hainan, Zhejiang, and Jiangxi Provinces. A total of 103 subjects from 32 families affected by SMA (families 1–32) have been analyzed, including 23 patients with SMA who were clinically suspected to be affected by SMA (9 had type I, 11 had type II, and 3 had type III), 62 parents of patients with SMA, and 18 high-risk fetuses from 15 families affected by SMA. In addition, 200 healthy individuals unaffected by SMA, with no family history of SMA (including five healthy controls with two copies each of the SMN1 and SMN2 gene), also enrolled into this study. Genomic DNA was extracted from peripheral blood using a commercially available kit (SE Blood DNA Kit; Omega, Norcross, GA) and from prenatal samples (amniocytes,
before and/or after culture) using another kit (QIAamp DNA Blood Mini Kit; Qiagen, Duesseldorf, Germany). Informed consent was obtained from all families in our study. The study was approved by the Ethical Committee of Fuzhou General Hospital in China.
PCR-RFLP and Allele-Specific PCR for Homozygous Deletion Analysis of SMN1 The conventional PCR-RFLP used for detecting homozygous deletion of SMN1 exon 7 was performed as originally described by van der Steege et al.14 Primers for allele-specific PCR of SMN exon 7 were described in a previous study.19 The common forward primer RIII was designed as originally described by van der Steege et al. Reverse primers 5=-CCTTCCTTCTTTTTGATTTTGTCAG-3= (for SMN1) and 5=-CCTTCCTTCTTTTTGATTTTGTCAA-3= (for SMN2) were designed to amplify SMN1 or SMN2. A deliberate mismatch (italicized) was introduced 2 bp before the 3= end of reverse primers to increase PCR specificity.19 To monitor PCR efficiency, amplification of SMN exon 4 served as an internal control. Primers for SMN exon 4 were originally described by Parsons et al.20 The optimal ratio of SMN1/2 exon 7 primers to exon 4 primers was 2:1. This optimal ratio was used in all allelespecific PCR assays. The total PCR reaction volume of 25 L contained 50 ng of genomic DNA, 0.5 L of each pair of SMN1/2 exon 7 forward and reverse primers (20 mol/ L), 0.5 L of each pair of SMN exon 4 primers (10 mol/ L), and 2.5 U of rTaq DNA polymerase (Takara, Kyoto, Japan). The following PCR conditions were used: 94°C for 4 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, with a final extension of 72°C for 5 minutes. The results were documented by photographing the 2% gel under UV light.
MLPA Analysis for Determination of SMN1 and SMN2 Copy Number Multiplex ligation– dependent probe amplification was performed in a thermal cycler (Eppendorf, Hamburg, Germany) using a kit (SALSA MLPA Kit P021; MRCHolland, Amsterdam, the Netherlands). All reactions were performed according to the manufacturer’s protocol. Approximately 100 ng of genomic DNA of 23 patients, 62 parents, 18 fetuses, and 50 healthy individuals was used for the MLPA reaction. The MLPA products were run on an analyzer (ABI PRISM 3100-Avant Genetic Analyzer; ABI, Carlsbad, CA) with LIZ 500 as the internal size standard. All samples were analyzed at least twice. Data analysis was performed using manual Excel sheets. The relative peak area (RPA) was obtained by dividing the PA of each probe by the total PA of the control probes: RPA ⫽ PA/Total PA. For each sample, the relative copy number for each probe was obtained by dividing RPA by the median normalized PA of five healthy controls: Relative Copy Number ⫽ RPA/Median Normalized PA. For SMN1/2 exon 7, the absolute copy number of each sample was obtained by multiplying RPA with a
Molecular Diagnostic System for SMA in China 43 JMD January 2011, Vol. 13, No. 1
factor of two. According to the recommendations of the manufacturer, a reduction of 35% to 55% in RPA for a probe’s amplification product is indicative of deletion of one copy of that probe’s target sequence; and an increase between 30% and 55%, a gain in copy number from two to three copies. In this study, only the data of exon 7 of SMN1 and SMN2 were analyzed statistically. Statistical analysis was performed by using a statistical program package (SAS, Cary, NC).
SMN1 Subtle Mutation Analysis at the Transcript Level RNA was isolated from peripheral blood by using a kit (QIAamp RNA Blood Mini Kit, Qiagen, Duesseldorf, Germany). First-strand cDNA synthesis was performed with 2 g of RNA by using oligo(dT) and reverse transcriptase (SuperScript II RT) (200 U/L, Invitrogen, Carlsbad, CA). The single-stranded cDNA was amplified with 10 ng of each primer, SMN forward primer (5=-TGCGCATCCGCGGGTTTGCT-3=) and SMN reverse primer (5=TCATTTAGTGCTGCTCTATGCCA-3=), which was described by Parsons et al.20 The purified RT-PCR products covering the entire coding region were directly sequenced (model 3730 Sequencer; Applied Biosystems, Carlsbad, CA). To determine whether the SMN1 or SMN2 gene contributed to the sequence variation through the RNA approach, purified RT-PCR products of SMN were cloned into a vector (pGEM-T Easy Vector; Promega, Madison, WI), following the supplier’s protocol. The plasmid DNA was purified with a mini plasmid kit (Qiagen) from approximately 6 to 10 white colonies and analyzed by digestion with EcoRI. Recombinant plasmids containing inserts of 956 bp were also sequenced (model 3730 Sequencer, Applied Biosystems).
Mutation Detection at the Genomic Level To confirm the detected mutations, specific regions in genomic DNA were amplified by the following primers: for exon 5, 5=-TGAGTCTGTTTGACTTCAGG-3= (sense primer) and 5=-TATCAAATTGTATGTGAAAGCA-3= (antisense primer); and for exon 1, 5=-GCCGGAAGTCGTCACTCTT-3= (sense primer) and 5=-GGGTGCTGAGAGCGCTAATA-3= (antisense primer). These were described elsewhere by Parsons et al.20 The purified PCR products were directly sequenced (model 3730 Sequencer). Moreover, the restriction enzyme Hin1II was used to cleave the products of exon 5 to check the existence of the mutation C689T (S230L); PCR products of exon 5 from 200 healthy controls were also cleaved by Hin1II to exclude the possibility of a polymorphism. To confirm the mutation in exon 1 (22_23insA), T cloning of the purified PCR products was performed following the supplier’s protocol (previous description) and multiple clones were analyzed.
Figure 1. Pedigree and putative genotypes inferred from families 17 and 24.
Results Thirty-two families were referred to our laboratory for diagnostic SMA testing, and blood samples from 23 patients were available. Of these patients, 21 had a homozygous deletion of SMN1 by conventional PCR-RFLP and an allele-specific PCR. Among the parents of these 21 patients, 36 showed one copy of SMN1 and two (the father in family 2 and the mother in family 5) showed two copies by MLPA analysis. The remaining two patients without a deletion (patients from families 17 and 24) were subsequently subjected to MLPA analysis to determine their copy numbers of the SMN1 gene; these patients were highly suspected to have SMA in terms of clinical manifestations. Both patients possessed one copy of SMN1, making them likely compound heterozygotes (patients with one SMN1 gene lost and one SMN1 gene containing a subtle mutation). The entire SMN coding region was screened for subtle mutations by RT-PCR and T cloning, and two subtle SMN1 mutations (ie, S230L and L228X, identified in patients 17 and 24, respectively) were found. Sequencing of PCR products of SMN exon 5 from the parents showed that both of the mutations were paternal. The SMN genotypes of members from both of the families were shown in Figure 1. In 200 healthy individuals, no S230L mutation was observed. In the nine families in whom the probands’ samples were unavailable because of probands’ death before parental referral to us, carrier testing by MLPA was performed on the 18 parents. Parents from eight families showed one copy of SMN1, confirming carrier status and a diagnosis of SMA of the proband. In the remaining family (family 25), the mother showed one copy and the father showed two copies. Suspecting that this parent was a carrier with one normal SMN1 allele and a subtle mutation on the other allele, mutation analysis was performed at both the transcript and genomic levels. Interestingly, at the genomic level, a frame-shift mutation (ie, 22_23insA) was identified (Figure 2) by direct sequencing of PCR products of exon 1. This mutation was confirmed by sequencing of mutated clones after T cloning of PCR products. However, we were unable to identify any clone containing this mutation at the transcript level. Exhaustive screening of the obtained clones and repeated RT-PCR and T cloning exclusively found clones contain-
44 Zeng et al JMD January 2011, Vol. 13, No. 1
Table 1. Determination of SMN2 Copy Numbers in Patients With SMA by MLPA
Figure 2. Genomic DNA sequence of exon 1 of the survival motor neuron (SMN) gene from the father in family 25. The father had an “A” insertion (indicated by the box) at codon 8 (underlined), resulting in a frame shift on this allele (mutated allele).
ing normal SMN1- or normal SMN2-derived products, suggesting that transcripts from the mutated SMN1 allele were either absent or in extremely low quantity. We also performed prenatal SMA diagnostic testing on 18 amniotic fluid samples of high-risk fetuses from 15 families affected by SMA. In 18 fetuses, five had homozygous deletion of the SMN1 gene, seven had one copy, and six had two copies. The copy number of SMN1 and SMN2 in these 15 fetuses is shown in Figure 3. To look for evidence of correlation of SMN2 copy number and severity of SMA, MLPA was performed on 23 patients to determine SMN2 copy number (Table 1). By Ridit analysis, there was significant correlation between the SMN2 copy number and severity of SMA (P ⬍ 0.05).
Discussion Based on our experience, we propose the following strategy for molecular diagnosis of SMA: (1) Patients are selected for molecular analysis according to clinical criteria. (2) PCR-RFLP and allele-specific PCR are performed to detect a homozygous SMN1 exon 7 deletion. (3) Multiplex ligation– dependent probe amplification is subsequently performed to determine the copy number of SMN1 of a patient if the patient is free from homozygous deletion of SMN1 but the patient’s manifestations fulfill the clinical criteria of SMA. (4) Carrier testing is performed by MLPA to confirm the carrier status of the parents in families in which the index patients with SMA
Figure 3. Histogram showing the results of 18 fetuses by multiplex ligation– dependent probe amplification (MLPA) analysis for SMN1 and SMN2 copy numbers.
Copy No. of SMN2
I (n ⫽ 8)
II (n ⫽ 11)
III (n ⫽ 3)
Total (N⫽22)
1 2 3 4
1 4 3 0
0 0 11 0
0 0 0 3
1 4 14 3
SMA type
The MLPA analysis failed in one patient because of the low quality of DNA. SMA, spinal muscular atrophy; MLPA, multiplex ligation– dependent probe amplification; SMN, survival motor neuron.
have died at the time of reference. (5) Subtle mutation analysis of SMN1 is performed if a patient’s clinical manifestations strongly point to a diagnosis of SMA and if one copy of SMN1 is confirmed by MLPA. (6) Prenatal testing is performed in families in which the genotype of SMN1 in the proband is known or the carrier status of the parents is confirmed. Our strategy represents a comprehensive methodological system for the molecular diagnosis of SMA. It is a tradition of laboratory medicine to search for a single simple method for the laboratory diagnosis for a disease or a kind of disease. In this era of systems medicine and complexity science, such thinking should be abandoned. Instead, the idea of a methodological system in laboratory medicine or a diagnostic system should be established to tackle the complexity of single-gene disorders. The complexity of single-gene disease can be noted in at least three patterns. First, a single-gene disease results from one mutation in one gene but is modulated by many other genes (ie, modifier or epistatic genes) in terms of disease phenotype. Sickle-cell anemia, the first monogenic disease ever described, is typical of this category.21 Second, a monogenic disease can be caused by different mutations in one gene and modulated by many modifier genes. Most single-gene diseases, including SMA, belong to this category. Third, a group of monogenic diseases with common phenotypes is caused by mutations in many different genes. Examples of this category are diseases such as hereditary ataxia and hereditary spastic paraplegia.22,23 Obviously, no single method can tackle the genetic diagnosis of single-gene disorders of the second and third categories. For homozygous deletion analysis of the SMN1 gene, only one single simple method, such as conventional PCR-RFLP, was performed in most laboratories. Actually, every single method has its limits and cannot sufficiently tackle the genetic diagnosis. Moreover, conventional PCR-RFLP requires an enzymatic digestion step and incomplete digestion of PCR products will result in falsenegative results. In our study, the PCR-RFLP method was combined with allele-specific PCR (also as an amplification refractory mutation system) to overcome those disadvantages and both their results can confirm each other. The primers for SMN1 in this method theoretically will only amplify SMN1 exon 7, not SMN2 exon 7, because of a single-nucleotide difference between the exons 7 of SMN1 and SMN2. However, according to the
Molecular Diagnostic System for SMA in China 45 JMD January 2011, Vol. 13, No. 1
report by Xu et al,24 it may not be a method that can be applied in routine genetic testing because the allelespecific reverse primer (for SMN1) designed by Moutou et al25 can easily bind to SMN2, in the presence of higher amounts (20 to 100 ng) of genomic DNA, resulting in substantial amplification of SMN2 in the case of homozygous loss of SMN1. Previous studies have shown that further deliberate mismatches to destabilize the primer/ template complexes render the primers increasingly refractory as the additional mismatch is moved progressively closer to the 3= end.26 Therefore, in our study, we introduce an additional deliberate mismatch 2 bp before the 3= end of reverse primers in amplification refractory mutation system to increase PCR specificity. This amplification refractory mutation system, whose results were in complete agreement with the results of conventional PCR-RFLP and MLPA, has proved to be a simple, quick, and reliable method in a previous study.19 The MLPA technique greatly improves diagnostics in SMA.15–17,27 Multiplex ligation– dependent probe amplification analysis, which is different from PCR-RFLP and allele-specific PCR (which only detect the absence of SMN1), detects the mechanism responsible for SMN1 loss (ie, deletion or conversion to SMN2). Evaluation of the SMN2 copy number is also useful for establishing genotype-phenotype correlation in patients with SMA based on the evidence that the SMN2 copy number relates to severity of the disease.28,29 Although the number of patients studied was few, our data obtained from MLPA, together with the results by statistical analysis, provided further evidence for a disease-modifying role of SMN2, with a greater SMN2 copy number being associated with a milder SMA phenotype, allowing for some prognostic considerations. Another important purpose of MLPA was for the detection of heterozygous SMN1 deletions to support the status of SMA carriers in families affected by SMA and to screen SMA carriers in the general population. Among 50 healthy individuals without a family history of SMA in our study, the percentage of individuals with one copy was 2% (data not shown), which is in accordance with the approximately 2% carrier frequency in the general population.1 Unfortunately, we found unexpected MLPA results in 2.6% (4/153) of the DNA samples. These results included the disappearance of many peaks in one sample and the warning signal of control peaks. It is probably because of contaminants (ie, PCR inhibitors, such as a small amount of ionic impurities, remnant ethanol, and phenol) in these DNA samples. The impurities can have their effect on the PCR reaction or on DNA denaturation by increasing temperature. Actually, MLPA is more sensitive to impurities than ordinary PCR. That is why good results of the same samples, whose MLPA results do not make any sense, can be obtained by conventional PCR-RFLP and allele-specific PCR. In our study of 59 parents whose SMN1 copy number was determined, five had a normal two-copy SMN1 dosage by MLPA. Three (the fathers in families 17, 24, and 25) of these five parents had a mutant non-deleted SMN1 allele identified by subtle mutation analysis. However, the remaining two (the father from family 2 and the mother
from family 5) had a child with homozygous SMN1 deletions. This situation could be caused by two unusual occurrences. First, the occurrence of two (or more) SMN1 genes on a single chromosome is approximately 4% in the normal population.30,31 Given the instability of this genomic region, two (or more) SMN1 genes on a single chromosome may result from gene conversion from SMN2 to SMN1 or alternatively from the remaining duplicated SMN1 genes without conversion to SMN2.31 In reality, such parents would be carriers with a 25% recessive risk of having another affected child with a carrier spouse. Second, SMN1 has a high rate of de novo deletion mutations.32 The high rate of de novo mutations in SMN1 underlies a de novo mutational event in the affected child whose parents have a normal dosage of SMN1. It is important for genetic counseling to distinguish between the normal recessive risk of a two-copy SMN1 carrier and the low risk of recurrence after de novo mutation in noncarrier parents of affected children. For the time being, the case of two or more SMN1 genes on a single chromosome can only be resolved by somatic cell hybridization and fluorescence in situ hybridization and that of de novo deletion mutations by SMN1 gene dosage of extended family members. Subtle mutation analysis of SMN1 is complicated by the presence of SMN2, thus it is not offered routinely in most clinical laboratories. The current approaches used to identify subtle mutations in a few laboratories include single-strand conformation polymorphism, denaturing high-performance liquid chromatography, haplotype studies with Ag1-CA and C212, and sequencing of the complete SMN coding region,18,20,33 which are somewhat labor intensive and time-consuming. Direct sequencing of genomic DNA is inappropriate because of unequally higher amounts of SMN2 copies. Direct sequencing of cDNA is inadequate because of unequal amounts of SMN1/SMN2 and lack of exons 3, 5, and 7 in alternatively spliced transcripts. Furthermore, any mutation found must be assigned to the correct SMN1 copy, which makes subcloning and sequencing of SMN1 absolutely necessary.33 The SSCP analysis or DHPLC of each exon is more time-consuming, in which not all mutations can be identified and sequencing for the exact mutation and assignment to the correct SMN1 copy must be performed. To our knowledge, we were the first to take advantage of the strategy of quantitative analysis, followed by a combination of the RNA approach (sequencing of the complete SMN1 coding region from cloned RT-PCR products) and the genomic DNA approach (sequencing of SMN exon regions from cloned PCR products), which proved effective for SMN1 subtle mutation analysis; this method might offer more reliable results, although it cannot avoid the demerits of being labor intensive and time-consuming. In the present article, we identified three subtle mutations in the SMN1 gene. One is the nonsense mutation L228X, which was described elsewhere.34 Another is the missense mutation S230L, which is described herein for the first time. This novel mutation (S230L) was pinpointed in SMN1 exon 5, together with one SMN2 copy in patient 17 (type I). The region of the SMN protein coded by the
46 Zeng et al JMD January 2011, Vol. 13, No. 1
exon carrying S230L belongs to functionally less important SMN domains (UniprotKB/Swiss-ProtQ16637-SMNHuman), and the amino acid sequence spanning the positions from approximately 194 to 251 corresponds to a domain of low complexity (PridictProtein server for predict_h34345); nevertheless, our finding of a pathogenetic mutation at amino acid residue 230 points to the necessity of reevaluating the function of the relevant region of the SMN protein. The last one is frame-shift mutation 22_23insA. This mutation has previously been identified by Tsai et al.34 Because Tsai et al exclusively used a DNA-based assay to detect the mutation, the presence of transcripts was never checked. This mutation was detected only at the genomic DNA level in the present study. It is well established that transcripts typically undergo nonsense-mediated mRNA decay if they harbor a premature translation termination codon more than 55 nucleotides upstream of the last intron.35 According to this rule, this subtle mutation (22_23insA) detected in exon 1 leads to generation of premature translation termination codon in exon 2a, which is expected to trigger nonsense-mediated mRNA decay and, in turn, reduce the expression of a mutated SMN1 transcript. This phenomenon underscores the importance of combining the RNA approach with the DNA approach to detect subtle mutations. Premature translation termination codon may also be created by the mutation L228X according to the rule; however, why were the mutated transcripts clearly detected in the patient? This question might not be answered until the mechanism of action of the mutations is further studied. In China, there are four SMN1 subtle mutations (ie, S230L, L228X, 22-23insA, and 835-1G⬎A) found in type I Chinese patients. The fact that L228X and 22-23ins A mutations identified more than once by different laboratories (ie, our laboratory and the laboratory of Tsai et al34) have not been reported in the European population so far makes it likely that these subtle mutations might be unique at our location. If these subtle mutations are confirmed to be common subtle mutations in Chinese patients with SMA, rapid assays (eg, allele-specific PCR) for the detection of these mutations can be easily designed to increase the sensitivity of SMA molecular diagnosis. In conclusion, our comprehensive methodological system for the molecular diagnosis of SMA, including SMN1 deletion analysis, SMN1 and SMN2 dosage analysis, and subtle mutation analysis, enables us to resolve almost all of the diagnostic situation of SMA and offers the most complete evaluation of patients with SMA and their family members at this time.
Acknowledgments We thank the families affected by SMA who participated in this study; Dr. Bosheng Yang for family referral; Dr. Lianghu Huang for primer design; Dr. Huijuan Huang for amniotic fluid collection; and Dr. Xiangdong Tu for culture of amniocytes.
References 1. Ogino S, Leonard DG, Rennert H, Wilson RB: Spinal muscular atrophy genetic testing experience at an academic medical center. J Mol Diagn 2002, 4:53–58 2. Zhu S, Xiong F, Chen YJ, Yan TZ, Zeng J, Li L, Zhang YN, Chen WQ, Bao XH, Zhang C, Xu XM: Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population. Eur J Hum Genet 2010. [Epub ahead of press] 3. Munsat TL, Davies KE: International SMA consortium meeting (26 –28 June 1992. Bonn, Germany). Neuromuscul Disord 1992, 2:423– 428 4. Panigrahi I, Kesari A, Phadke SR, Mittal B: Clinical and molecular diagnosis of spinal muscular atrophy. Neurol India 2002, 50:117–122 5. Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M: Identification and characterization of a spinal muscular atrophy-determining gene. Cell 1995, 80:155–165 6. Cartegni L, Krainer AR: Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet 2002, 30:377–384 7. Cartegni L, Hastings ML, Calarco JA, de-Stanchina E, Krainer AR: Determinants of exon 7 splicing in the spinal muscular atrophy genes: SMN1 and SMN2. Am J Hum Genet 2006, 78:63–77 8. Vitte J, Fassier C, Tiziano FD, Dalard C, Soave S, Roblot N, Brahe C, Saugier-Veber P, Bonnefont JP, Melki J: Refined characterization of the expression and stability of the SMN gene products. Am J Pathol 2007, 171:1269 –1280 9. Otter S, Grimmler M, Neuenkirchen N, Chari A, Sickmann A, Fischer U: A comprehensive interaction map of the human survival of motor neuron (SMN) complex. J Biol Chem 2007, 282:5825–5833 10. Pillai RS, Grimmler M, Meister G, Will CL, Lührmann R, Fischer U, Schümperli D: Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component: Lsm11, in histone RNA processing. Genes Dev 2003, 17:2321–2333 11. Setola V, Terao M, Locatelli D, Bassanini S, Gerattini E, Battaglia G: Axonal-SMN (a-SMN), a protein isoform of the survival motor neuron gene, is specifically involved in axonogenesis. Proc Natl Acad Sci U S A 2007, 104:1959 –1964 12. Briese M, Esmaeili B, Fraboulet S, Burt EC, Christodoulou S, Towers PR, Davies KE, Sattelle DB: Deletion of smn-1, the Caenorhabditis elegans ortholog of the spinal muscular atrophy gene, results in locomotor dysfunction and reduced lifespan. Hum Mol Genet 2009, 18:97–104 13. Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN: Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 2009, 457:277–280 14. van der Steege G, Grootscholten PM, van der Vlies P, Draaijers TG, Osinga J, Cobben JM, Scheffer H, Buys CH: PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 1995, 345:985–986 15. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G: Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002, 30:e57 16. Arkblad EL, Darin N, Berg K, Kimber E, Brandberg G, Lindberg C, Holmberg E, Tulinius M, Nordling M: Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy. Neuromuscul Disord 2006, 16:830 – 838 17. Scarciolla O, Stuppia L, De Angelis MV, Murru S, Palka C, Giuliani R, Pace M, Di Muzio A, Torrente I, Morella A, Grammatico P, Giacanelli M, Rosatelli MC, Uncini A, Dallapiccola B: Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification. Neurogenetics 2006, 7:269 –276 18. Wirth B, Herz M, Wetter A, Moskau S, Hahnen E, Rudnik-Schoneborn S, Wienker T, 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 19. Zeng J, Lan FH, Deng XJ, Ke LF, Tu XD, Huang LH, Zheng DZ, Zhu ZY: Evaluation of an in-house protocol for prenatal molecular diagnosis of SMA in Chinese. Clin Chim Acta 2008, 398:78 – 81
Molecular Diagnostic System for SMA in China 47 JMD January 2011, Vol. 13, No. 1
20. Parsons DW, McAndrew PE, Iannaccone ST, Mendell JR, Burghes AH, Prior TW: Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number. Am J Hum Genet 1998, 63:1712–1723 21. Nagel RL, Steinberg MH: Role of epistatic (modifier) genes in the modulation of the phenotypic diversity of sickle cell anemia. Pediatr Pathol Mol Med 2001, 20:123–136 22. Harding AE: Clinical features and classification of inherited ataxias. Adv Neurol 1993, 61:1–14 23. Contino G, Novelli G: Hereditary spastic paraplegia: clinical genomics and pharmacogenetic perspectives. Expert Opin Pharmacother 2006, 7:1849 –1856 24. Xu R, Ogino S, Lip V, Fang H, Wu BL: Comparison of PCR-RFLP with allele-specific PCR in genetic testing for spinal muscular atrophy. Genet Test 2003, 7:277–281 25. Moutou C, Gardes N, Rongières C, Ohl J, Bettahar-Lebugle K, Witterner C, Gerlinger P, Viville S: Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy. Prenat Diagn 2001, 21:498 –503 26. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF: Analysis of any point mutation in DNA: the amplification refractory mutation system (ARMS). Nucleic Acids Res 1989, 17:2503–2516 27. Zapletalová E, Hedvicáková P, Kozák L, Vondrácek P, Gaillyová R, Maríková T, Kalina Z, Jüttnerová V, Fajkus J, Fajkusová L: Analysis of point mutations in the SMN1 gene in SMA patients bearing a single SMN1 copy. Neuromuscul Disord 2007, 17:476 – 481 28. 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 number of
29.
30.
31.
32.
33.
34.
35.
copies of cBCD541 and SMA phenotype. Hum Mol Genet 1996, 5:257–263 Vitali T, Sossi V, Tiziano F, Zappata S, Giuli A, Paravatou-Petsotas M, Neri G, Brahe C: Detection of the survival motor neuron (SMN) genes by FISH: further evidence for a role for SMN2 in the modulation of disease severity in SMA patients. Hum Mol Genet 1999, 8:2525–2532 Scheffer H, Cobben JM, Mensink RGJ, Stulp RP, Van der Steege G, Buys CH: SMA carrier testing: validation of hemizygous SMN exon 7 deletion test for the identification of proximal spinal muscular atrophy carriers and patients with a single allele deletion. Eur J Hum Genet 2000, 8:79 – 86 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 AH, Chapelle A, Prior TW: Hybrids monosomal for human chromosome 5 reveal the presence of a spinal muscular atrophy (SMA) carrier with two SMN1 copies on one chromosome. Hum Genet 2001, 108:109 –115 Wirth B, Schmidt T, Hahnen E, Rudnik-Schöneborn S, Krawczak M, Muller-Myhsok B, Schönling J, Zerres K: De novo rearrangements 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 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 Tsai CH, Jong YJ, Hu CJ, Chen CM, Shih MC, Chang CP, Chang JG: Molecular analysis of SMN: NAIP and P44 genes of SMA patients and their families. J Neurol Sci 2001, 190:35– 40 Wilkinson MF: A new function for nonsense-mediated mRNA-decay factors. Trends Genet 2005, 21:143–148