OBJECTIVE: To study the association of copy number of SMN1 and SMN2 with clinical phenotypes in children with spinal muscular atrophy (SMA). METHODS: A total of 45 children with SMA were enrolled. Multiplex ligation-dependent probe amplification was used to measure the gene copy numbers of SMN1 and SMN2. The association of copy number of SMN1 and SMN2 with clinical phenotypes was analyzed. RESULTS: Of the 45 children with SMA, 42 (93%) had a homozygous deletion of SMN1 exons 7 and 8, and 3 (7%) had a deletion of SMN1 exon 7 alone. No association was found between SMA clinical types and the deletion types of SMN1 exons 7 and 8 (P>0.05). There was a significant difference in the distribution of SMN2 gene copy numbers between the children with SMA and the healthy children (P<0.05). The children with SMA usually had two or three copies of SMN2 gene, while the healthy children usually had one or two copies of SMN2 gene. There was a significant difference in the distribution of SMN2 copy numbers among the children with different SMA clinical types (P<0.05). The children with two copies of SMN2 gene had a significantly lower age of onset than those with three or four copies. Most of the children with type I SMA had two or three copies of SMN2 gene. Most of the children with type II SMA had three copies of SMN2 gene. Most of the children with type III SMA had three or four copies of SMN2 gene. Children with a higher copy number of SMN2 gene tended to have an older age of onset and better motor function and clinical outcome, and there was a significant association between SMN2 gene copy number and clinical outcome (P<0.05). CONCLUSIONS The SMN2 gene can reduce the severity of SMA via the dosage compensation effect. SMN2 copy number is associated with the phenotype of SMA, and therefore, it can be used to predict disease severity.
Child ; Humans ; Muscular Atrophy, Spinal ; genetics ; Phenotype ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

BACKGROUNDInfantile proximal spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. Approximately 90% - 95% cases of SMA result from homozygous deletion of survival motor neuron gene 1 (SMN1) and 5% cases are caused by compound heterozygous mutation (a SMN1 deletion on one allele and a subtle mutation on the other allele).

METHODSIn this research, two unrelated patients were clinically diagnosed according to the criteria of proximal SMA. Genetic diagnosis was performed to detect the homozygous deletion of exon 7 of SMN1 by PCR-restriction fragment length polymorphism (RFLP) and genomic sequencing. Multiplex ligation-dependent probe amplification (MLPA) analysis was carried out to measure copy numbers of SMN1, SMN2 and neuronal apoptosis inhibitor protein (NAIP) in the patients. Further sequencing of SMN1 allele-specific PCR (AS-PCR) and SMN1 clones were also performed to analyze the point mutation of SMN1 gene. Additionally, the pedigree analysis of these two families was carried out to identify the transmission of the mutation.

RESULTSThe inconsistent results using PCR-RFLP and genomic sequencing showed homozygous deletion of exon 7 of SMN1 and heterozygous deletion accompanied with a suspicious mutation in SMN1 gene, respectively. MLPA analysis of these two cases exhibited one SMN1 copy deletion. One identical c.863G > T (p.Arg288Met) mutation was found in two cases by sequencing the SMN1 clones, which confirmed that both cases were SMA compound heterozygotes. One case showed partial conversion to form hybrid SMN (SMN2 I7/SMN1 E8) identified by clones sequencing and another case carrying 3 SMN2 implied complete conversion from SMN1 to SMN2.

CONCLUSIONp.Arg288Met is more a disease-causing mutation than a polymorphism variation, and children with this mutation may have more severe phenotypes.

Child, Preschool ; Exons ; genetics ; Female ; Humans ; Infant ; Muscular Atrophy, Spinal ; genetics ; Mutation ; Neuronal Apoptosis-Inhibitory Protein ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; genetics ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

Determination of the copy number of the survival motor neuron (SMN) gene is important for detecting spinal muscular atrophy (SMA) carriers and compound heterozygous patients. Multiplex ligationdependent probe amplification (MLPA) assay is a simple and efficient technique used for detecting variations in the copy numbers of different genes. Race- and ethnicity-based variation in the SMA carrier frequency and the '2+0' genotype of SMN1 are important factors that should be considered when estimating the risk of being an SMA carrier. Since SMN2 plays a disease-modifying role, accurate determination of SMN2 copy numbers in SMA patients can serve as a useful prognostic tool. Therefore, information on the SMN2 genotype distributions in normal populations will be helpful in selecting appropriate reference samples for MLPA analysis. To determine SMA carrier frequencies and SMN genotype distribution, we determined the copy numbers of SMN1 and SMN2 genes using the MLPA assay in 100 unrelated Korean individuals with no family history of SMA. The frequency of SMA carriers in the Korean population appears to be 1 in 50, which indicates that the prevalence of SMA among Koreans is the same as that among individuals in the Western countries. Two of the 100 normal individuals enrolled in this study showed 3 copies of the SMN1 gene. Therefore, 1.0% of the 198 normal alleles in this population was estimated to be 2-copy alleles ('2+0' genotype). SMN2 copy numbers showed a high degree of individual variation. Our results showed that 64% of the individuals had 2 copies of SMN2, but 36% individuals had between 0, 1, or 3 copies of the gene.
Asian Continental Ancestry Group/*genetics ; *Gene Dosage ; Heterozygote ; Humans ; Muscular Atrophy, Spinal/*genetics ; Nucleic Acid Amplification Techniques ; Republic of Korea ; Survival of Motor Neuron 1 Protein/*genetics ; Survival of Motor Neuron 2 Protein/*genetics

OBJECTIVETo introduce the application of denaturing high-performance liquid chromatography (DHPLC) in the diagnosis of childhood type spinal muscular atrophy (SMA).

METHODSExon 7 and flanking area of survival motor neuron (SMN) gene were amplified by PCR in 1 standard sample, 25 normal individuals and 25 patients with SMA. The PCR products were then directly loaded onto the DHPLC system after denaturing and annealing. Different DNA segments were separated by changing the concentration of buffer A relative to that of buffer B.

RESULTSDifferent DNA segments were separable on the DHPLC chromatogram. Three peaks including SMN1/SMN2 heteroduplex peak, SMN2 homoduplex peak and SMN1 homoduplex peak were detected in 23 out of 25 normal individuals. Only SMN1 homoduplex peak was detected in 2 normal individuals and the standard sample, indicating the deletion of SMN2 On the contrary, only the SMN2 homoduplex peak was detected in 22 out of 25 patients with SMA, indicating deletion of SMN1. The three peaks as those of normal individuals were detected in the other 3 patients, indicating no SMN1 or SMN2 deletion.

CONCLUSIONAs a new technology for diagnosing SMA, DHPLC is sensitive, accurate, rapid and convenient.

Chromatography, High Pressure Liquid ; methods ; Exons ; genetics ; Humans ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Polymerase Chain Reaction ; Reproducibility of Results ; SMN Complex Proteins ; genetics ; Sensitivity and Specificity ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein

OBJECTIVESpinal muscular atrophy (SMA) is a common and fatal autosomal recessive disorder. Approximately 94% of SMA patients are caused by homozygous deletion of SMN1 gene. SMA carrier screening is recommended considering the high carrier frequency (1 in 35-50) as well as severity of the disease.

METHODSA prospective population-based cohort study was carried out on 4719 pregnant women from Shanghai region. Copy numbers of SMN1 and SMN2 genes were effectively determined with denaturing high performance liquid chromatography (DHPLC) technique. The method has detected 94% of SMA cases with deletion or conversion of the SMN1 genes.

RESULTSNinety SMA carriers with only one copy of the SMN1 gene were identified among the 4719 pregnant woman. The carrier rate was 1.9%. Respectively, 1.2% and 0.6% of the carriers were caused by SMN1 gene deletion and SMN1 gene conversion.

CONCLUSIONThrough this study, we have determined the frequency of SMA mutation carriers in a population of pregnant women. The result may provide a basis for genetic counseling in order to reduce the rate of SMA affected births.

Adult ; China ; Female ; Gene Deletion ; Genetic Testing ; methods ; Humans ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Pregnancy ; Prenatal Diagnosis ; methods ; Prospective Studies ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics ; Young Adult

OBJECTIVETo detect homozygous deletions of survival motor neuron (SMN) gene with genomic DNA sequencing, and to assess the value of genetic testing for the diagnosis of spinal muscular atrophy (SMA).

METHODSPolymerase chain reaction (PCR) was used for amplifying SMN gene in 100 SMA patients and 110 controls. Four different bases (g.31957, g.32006, g.32154 and g.32269) between SMN1 and SMN2 within the amplified segments were identified with genomic DNA sequencing. Homozygous deletion of SMN1 or SMN2 was determined by the presence or absence of base peaks at such four sites. Multiplex ligation-dependent probe amplification (MLPA) was carried out to confirm the results of genomic DNA sequencing.

RESULTSIn the 100 SMA samples, only SMN2 specific base peaks were detected at the four sites, for which the copy numbers of SMN1 and SMN2 was 0:2 or 0:3, suggesting homozygous deletion of SMN1 gene. By contrast, only SMN1 specific base peaks were detected in 5 samples, for which the ratio of SMN1:SMN2 was 2:0, indicating homozygous deletion of SMN2. At four different sites, SMN1/SMN2 heterozygous peaks were detected in the remaining 105 samples, for which SMN1:SMN2was 2:2, suggesting non-deletion of SMN1 or SMN2. The results of sequencing were consistent with those of MLPA.

CONCLUSIONGenomic DNA sequencing is a rapid, accurate and economic method for the diagnosis of homozygous deletion of SMA.

Base Sequence ; China ; Female ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Muscular Atrophy, Spinal ; genetics ; Sequence Analysis, DNA ; Sequence Deletion ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

OBJECTIVETo investigate the type and frequency of gene conversion from SMN1 to SMN2 in Chinese patients affected with spinal muscular atrophy (SMA), and to explore the relationship between gene conversion and clinical phenotype.

METHODSNon-homozygous deletion of SMN1 gene exon 8 was screened among 417 patients with SMN1 exon 7 homozygous deletions. To analyze and verify the types of gene conversion, genomic DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), and gene subcloning and sequencing were carried out.

RESULTSThirty-one patients (7.4% of all) with non-homozygous deletions of SMN1 exon 8 were detected. Through series of experiments, the fusion genes SMN1/SMN2 in all cases were delineated. Five types of gene conversions were identified, which included SMN2-I7b/SMN1 E8, SMN2-I7a/SMN1 I7b, SMN2-E7/SMN1 I7a, SMN1 I6/SMN2 E7/SMN1 I7a and SMN2-E7/SMN1 I7a/SMN2 I7b. Such conversions were found in the type I-III patients. For 10 patients with type I-III SMA and 3 copies of SMN2 gene produced by conversion, the average survival age was 5 year and 4 months.

CONCLUSIONPartial conversions of SMN1 gene have been found among Chinese SMA patients. The type of conversion and frequency seem to be different from those of other races. Gene conversion to some extent may impact on survival time and rate of SMA patients, especially type I SMA.

Base Sequence ; Child ; Child, Preschool ; Exons ; Female ; Gene Conversion ; Gene Order ; Homozygote ; Humans ; Infant ; Male ; Muscular Atrophy, Spinal ; genetics ; Phenotype ; Sequence Analysis, DNA ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

Background: Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and evaluate a new diagnostic method for SMA. Methods: A total of 1494 children suspected with SMA were enrolled in this study. Traditional strategy, including multiplexed ligation-dependent probe amplification (MLPA) and TA cloning, was used in 1364 suspected SMA children from 2003 to 2014, and the 130 suspected SMA children were tested by a new strategy from 2015 to 2016, who were also verified by MLPA combined with TA cloning. The SMN1 and SMN2 were simultaneously amplified by polymerase chain reaction using the same primers. Mutation Surveyor software was used to detect and quantify the SMN1 variants by calculating allelic proportions in Sanger sequencing. Finally, turnaround time and cost of these two strategies were compared. Results: Among 1364 suspected SMA children, 576 children had SMN1 homozygous deletion and 27 children had SMN1 compound heterozygous mutation. Among the 130 cases, 59 had SMN1 homozygous deletion and 8 had heterozygous deletion: the SMN1-specific peak proportion on exon 7 was 34.6 ± 1.0% and 25.5 ± 0.5%, representing SMN1:SMN2 to be 1:2 and 1:3, respectively. Moreover, five variations, including p.Ser8Lysfs *23 (in two cases), p.Leu228*, p.Pro218Hisfs *26, p.Ser143Phefs*5, and p.Tyr276His, were detected in 6/8 cases with heterozygous deletion, the mutant allele proportion was 31.9%, 23.9%, 37.6%, 32.8%, 24.5%, and 23.6%, which was similar to that of the SMN1-specific site on exon 7, suggesting that those subtle mutations were located in SMN1. All these results were consistent with MLPA and TA cloning. The turnaround times of two strategies were 7.5 h and 266.5 h, respectively. Cost of a new strategy was only 28.5% of the traditional strategy. Conclusion Sanger sequencing combined with Mutation Surveyor analysis has potential application in SMA diagnosis.
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Muscular Atrophy, Spinal ; diagnosis ; genetics ; Mutation ; Sequence Analysis, DNA ; methods ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

OBJECTIVE: To perform carrier screening for spinal muscular atrophy (SMA) among 3049 reproductive-age individuals from Yunnan region and determine the copy number of survival motor neuron (SMN) gene and carrier frequencies. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was used to determine the copy number of exon 7 of SMN1 and SMN2 genes and identify those with a single copy of SMN1 gene. Prenatal diagnosis was performed for couples whom were both found to be SMA carriers. RESULTS: In total 62 SMA carriers were identified among the 3049 subjects, which yielded a carrier frequency of 1 in 49 (2.03%). No statistical difference was found in the carrier frequency between males and females (1.91% vs. 2.30%, P>0.05). Respectively, 1.3% (41/3049) and 0.69% (21/3049) of the carriers were caused by heterozygous deletion and conversion of the SMN1 gene. The average copy number for SMN1 alleles was 1.99. Two couples were found to be both as SMA carriers, for whom the birth of an affected fetus was avoided by prenatal diagnosis. CONCLUSION No difference was found in the carrier frequency of SMA-related mutations between the two genders in Yunnan region, which was in keeping to an autosomal recessive inheritance pattern. Determination of the carrier frequency for SMA and SMN gene variants may provide a basis for genetic counseling and prenatal diagnosis for the disease.
China ; Female ; Genetic Carrier Screening ; Genetic Counseling ; Genetic Variation ; Heterozygote ; Humans ; Male ; Muscular Atrophy, Spinal ; genetics ; Pregnancy ; Prenatal Diagnosis ; Survival of Motor Neuron 1 Protein ; genetics ; Survival of Motor Neuron 2 Protein ; genetics

OBJECTIVETo detect the correlation between the clinical phenotype of spinal muscular atrophy (SMA) and survival motor neuron gene (SMN2) copy number.

METHODSThe SMN2 gene copy numbers of 57 different types of SMA were detected by real-time fluorescence quantitative PCR method with TaqMan technique.

RESULTSAverage SMN2 copy number was 1.017 +/- 0.090, 2.019+/- 0.080, 3.104+/- 0.170 in predicting one, two, three copy numbers, respectively, and CV was 8.9%, 3.9%, 5.4%, respectively. Average SMN2 copy number was 1.926+/- 0.460, 2.508+/- 0.460, 2.876+/- 0.270, in type I, II and III SMA, respectively. The SMN2 gene copy number in type II and III SMA were higher than that of type I SMA (P < 0.01). The SMN2 gene copy number in type III SMA was higher than that of type II SMA (P < 0.01). 85.72% of type I SMA patients usually had 2 SMN2 copies; 40% and 60% of type II SMA patients had 2 and 3 SMN2 copies, respectively; 82% of type III SMA patients had 3 SMN2 copies.

CONCLUSIONThere is significant correlation between the change of SMA clinical phenotype and SMN2 cope number. The distributions of the SMN2 gene copy number are various in different types of SMA patients. All types of SMA patients have at least one copy SMN2. The SMN2 gene copy numbers in type II, III SMA are higher than that of type I. All of these findings suggest that the severity of SMA patients depend on the change of the SMN2 copy numbers.

Gene Dosage ; Genetic Predisposition to Disease ; genetics ; Humans ; Muscular Atrophy, Spinal ; genetics ; pathology ; Phenotype ; Polymerase Chain Reaction ; SMN Complex Proteins ; genetics ; Survival of Motor Neuron 2 Protein