Elsevier

Gene

Volume 518, Issue 2, 15 April 2013, Pages 325-329
Gene

Methods Paper
Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy

https://doi.org/10.1016/j.gene.2012.12.109Get rights and content

Abstract

Spinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disorder, which is caused by mutations of the survival motor neuron 1 (SMN1) gene. Additionally, the phenotype is modified by several genes nearby SMN1 in the 5q13 region. In this study, we analyzed mutations in SMN1 and quantified the modifying genes, including SMN2, NAIP, GTF2H2, and H4F5 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), multiplex ligation-dependent probe amplification (MLPA), TA cloning, allele-specific long-range PCR, and Sanger sequencing in 157 SMA patients. Most SMA patients (94.90%) possessed a homozygous SMN1 deletion, while 10 patients demonstrated only the absence of exon 7, but the presence of exon 8. Two missense mutations (c.689 C > T and c.844 C > T) were identified in 2 patients who both carried a single copy of SMN1. We found inverse correlations between SMN2, the NAIP copy number, and the clinical severity of the disease. Furthermore, 7 severe type I patients possessed large-scale deletions, including SMN1, NAIP, and GTF2H2. We conclude that SMN1 gene conversion, SMN1 subtle mutations, SMN2 copy number, and the extent of deletion in the 5q13 region should all be considered in the genotype–phenotype analysis of SMA.

Highlights

► SMN1 and modifying genes were analyzed simultaneously in 157 Chinese SMA patients. ► Two missense mutations were identified in 2 non SMN1-deleted patients. ► Severe type I patients with large-scale deletions were analyzed in detail. ► Variants of SMN1 and modifying genes should all be considered in the analysis of SMA.

Introduction

Spinal muscular atrophy (SMA), first described in 2 infant brothers by Guido Werdnig in 1891, is a common autosomal recessive neurodegenerative disorder characterized by symmetric and progressive myasthenia and amyotrophia owing to the dysfunction and loss of motor neurons in the anterior horn of the spinal cord. The prevalence of SMA is 1 in 6000 to 1 in 10,000 live births, with a carrier frequency of 1 in 40 in the general population (Iannaccone, 1998, Kolb and Kissel, 2011) these figures are 14.34 in 100,000 and 1 in 42, respectively, in mainland China (Sheng-Yuan et al., 2010). Childhood onset SMA can be classified into 3 types on the basis of age at onset and severity of clinical course (Munsat and Davies, 1992). In the most severe form, patients (type I) suffer from muscular weakness before 6 months and cannot sit without aid, and approximately 60–70% of affected individuals die from respiratory failure within the first 2 years (Meldrum et al., 2007, Munsat and Davies, 1992, Prior et al., 2011). Type II SMA is an intermediate form with onset before the age of 18 months, and patients of this type are able to sit, but unable to stand or walk without aid. Type III SMA (Kugelberg–Welander disease) is the mildest form, with onset during the juvenile period; these patients can walk and have a normal life span (Kolb and Kissel, 2011, Munsat and Davies, 1992).

The SMA-causative gene, the survival of motor neuron gene (SMN), was first identified by Lefebvre in 1995 (Lefebvre et al., 1995). In human beings, SMN is present in 2 highly homologous copies, SMN1 and SMN2, both of which are located in an inverted repeat area on chromosome 5q13. The functional difference between these 2 genes is a C-T variation in exon 7 (Burghes and Beattie, 2009, Lefebvre et al., 1995). About 95% of SMA cases are attributed to the homozygous loss of SMN1, while the remaining 5% have intragenic subtle mutations, such as missense mutations, small deletions, and splice site mutations (Alías et al., 2009, Lefebvre et al., 1995). Compared with the full-length SMN protein encoded by SMN1 gene, 85% of protein from SMN2 transcripts is truncated and unstable, which fails to compensate for the loss of SMN1 in SMA patients (Kolb and Kissel, 2011). However, the severity and duration of SMA largely depends on the copy number variations of SMN2 (Feldkötter et al., 2002). In a previous study, we analyzed the SMN2 copy number of 51 SMA patients by real-time PCR and found that there was a negative correlation between SMN2 copy number and severity of the disease (Chen et al., 2005). Moreover, within the 5q13 region, NAIP, GTF2H2, and H4F5 are regarded as disease-modifying genes, correlating with disease severity and duration of survival. (Bürglen et al., 1997, Ma et al., 1999, Roy et al., 1995, Scharf et al., 1998).

Because of the lack of specific manifestations, a definite diagnosis of SMA largely relies on genetic testing. In standard genetic testing, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) is only capable of detecting homozygous deletion of SMN1; hence, a series of quantitative techniques, including denaturing high-performance liquid chromatography (DHPLC), real-time PCR, and multiplex ligation-dependent probe amplification (MLPA), have been developed to detect copy number variants related to SMA phenotype (Arkblad et al., 2006, Chen et al., 2007, Feldkötter et al., 2002). In particular, MLPA, which possesses the ability to analyze up to 50 DNA sequences in a single reaction, has become a widely used technique for copy number variation analysis (Schouten et al., 2002). Here, we adopted PCR-RFLP, sequencing, and MLPA to analyze the correlation between SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes variants and the SMA phenotype in 157 Chinese patients.

Section snippets

Subjects and DNA isolation

Our study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University and all subjects offered written consent.

From September 10, 1998, to April 30, 2012, 157 SMA patients from 143 unrelated families of Chinese origin were recruited, including the 87 patients who had been analyzed by PCR-RFLP and DHPLC in our previous study (Chen et al., 2007). These included 80 men and 77 women. The average age at onset is 5.795 years (SD = 7.833, range from 3 days to 30 

SMN1 gene mutation analysis

Using PCR-RFLP and MLPA, we found that 94.9% (149/157) of patients harbored homozygous deletions of SMN1; among these, 10 patients demonstrated absence of exon 7, but presence of exon 8. The remaining 5.1% (8/157) patients possessed 1 copy (5 patients) or 2 copies (3 patients) of SMN1. The 5 patients with a single copy of SMN1 were likely compound heterozygotes, with an SMN1 deletion on 1 allele and a subtle mutation on the other allele; 2 missense mutations were identified: c.689 C > T (p.S230L)

Discussion

At present, no specific clinical features or examinations enable the diagnosis of SMA; hence, genetic testing plays an important role in identifying and classifying this condition. SMA is attributed to SMN1 mutations, and the absence of SMN1 includes homozygous deletion and conversion to SMN2 (Burghes, 1997). Here, we show that of 157 Chinese patients, 94.90% demonstrated homozygous loss of SMN1, which is consistent with that reported previously (Lefebvre et al., 1995). We also detected 10

Funding

This work was supported by grant 30900481 from the National Natural Science Foundation of China, grant 2012J06016 from the Natural Science Foundation of Fujian Province of China, grant JA12129 from the Program for New Century Excellent Talents in Fujian Province University, grant 2009-CXB-25 from the Fujian Medical Innovating Program, Program for Innovative Research Team in Science and Technology in Fujian Province University (Min Jiao Ke [2012] 3), a program for clinical medical key discipline

Acknowledgments

The authors sincerely thank the SMA families for their help and willingness to participate in this study.

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    Author contributions: Study concept and design (Drs. W-J Chen and Wang); acquisition of data (Drs. He, Zhang, Q-F Lin, Y-F Chen, X-Z Lin, M-T Lin, Murong, Wang and W-J Chen); analysis and interpretation of data (Drs. He, Zhang, Q-F Lin, Y-F Chen, X-Z Lin, Wang and W-J Chen); drafting of the article (Drs. He, Zhang and W-J Chen); critical revision of the article for important intellectual content (Drs. W-J Chen and Wang); obtaining of funding (Drs. W-J Chen and Wang); administrative, technical, or material support (Drs. He, Zhang, Q-F Lin, Y-F Chen, X-Z Lin, Wang and W-J Chen); study supervision (Drs. W-J Chen and Wang).

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    These authors contributed equally to this work.

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