Deletion analysis of SMN1 and NAIP genes in southern Chinese children with spinal muscular atrophy

Deletion analysis of SMN1 and NAIP genes in southern Chinese children with spinal muscular atrophy 1 2 3 first-author 2 3 4 2 3 2 3 2 3 4 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhangxianning@zju.edu.cn †E-mail:zhangxianning@zju.edu.cn zhangxianning@zju.edu.cn Department of Bioscience, Bengbu Medical College, Bengbu 233000, China Department of Bioscience, Bengbu Medical College, Bengbu 233000, China Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, Hangzhou 310058, China Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, Hangzhou 310058, China National Education Base for Basic Medical Sciences, Zhejiang University, Hangzhou 310058, China National Education Base for Basic Medical Sciences, Zhejiang University, Hangzhou 310058, China Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of lower motor neurons and occasionally bulbar motor neurons leading to progressive limb and trunk paralysis as well as muscular atrophy. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1, SMA2, and SMA3. The survival of motor neuron ( SMN ) gene has been identified as an SMA determining gene, whereas the neuronal apoptosis inhibitory protein ( NAIP ) gene is considered to be a modifying factor of the severity of SMA. The main objective of this study was to analyze the deletion of SMN1 and NAIP genes in southern Chinese children with SMA. Here, polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was performed to detect the deletion of both exon 7 and exon 8 of SMN1 and exon 5 of NAIP in 62 southern Chinese children with strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients and 76% (13/17) of SMA2 patients showed homozygous deletions for exon 7 and exon 8, and all the 13 SMA3 patients showed single deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients. Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and none of SMA2 and SMA3 patients was found to have NAIP deletion. The findings of homozygous deletions of exon 7 and/or exon 8 of SMN1 gene confirmed the diagnosis of SMA, and suggested that the deletion of SMN1 exon 7 is a major cause of SMA in southern Chinese children, and that the NAIP gene may be a modifying factor for disease severity of SMA1. The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis of SMA. Spinal muscular atrophy (SMA) Survival motor neuron ( SMN ) gene Neuronal apoptosis inhibitory protein ( NAIP ) gene Mutation INTRODUCTION 1 Spinal muscular atrophy (SMA) is a clinically and genetically heterogeneous group of neuromuscular disorders characterized by progressive muscle weakness because of degeneration and loss of the anterior horn cells of the spinal cord and the brain stem nuclei. SMA is the second most common lethal autosomal recessive disorder after cystic fibrosis (CF) in Caucasian populations with an overall incidence of 1 in 6000 live births and a carrier frequency of approximately 1 in 50 (Frugier et al., 2002 2002 ; Ogino and Wilson, 2004 2004 ; Darras and Kang, 2007 2007 ). The onset of weakness ranges from birth to adolescence or young adulthood. Diagnostic criteria vary by age of onset. Three types of SMA are recognized depending on the age of onset, the maximum muscular activity achieved, and survivorship: SMA1 (OMIM: 253300), SMA2 (OMIM: 253550) and SMA3 (OMIM: 253400). Among all the candidate genes, the survival of motor neuron ( SMN ) is believed to be the primary SMA disease-causing gene (Lefebvre et al., 1995 1995 ; Battaglia et al., 1997 1997 ; Hsieh-Li et al., 2000 2000 ; Andreassi et al., 2004 2004 ; Azzouz et al., 2004 2004 ; Schmutz et al., 2004 2004 ; Trülzsch et al., 2004 2004 ; Burnett and Sumner, 2008 2008 ; Girardet et al., 2008 2008 ). In humans, SMN is contained in a 500-kb sequence on chromosome 5q12.2-q13.3 which consists of 9 exons, and is present in two copies: a telomeric one ( SMN1 , or SMNt ) and a centromeric one ( SMN2 , or SMNc ). SMN1 gene has a highly homologous copy with SMN2 . This copy is present in 90%~95% of normal controls and hampers detection of deletions and mutations within the SMN1 gene (Frugier et al., 2002 2002 ; Ogino and Wilson, 2004 2004 ). The coding sequence of SMN2 exon 7 differs from that of SMN1 by a single nucleotide (840C < T), which alters a restriction enzyme site and allows one to easily distinguish SMN1 from SMN2 using a polymerase chain reaction (PCR)-based assay. The finding of homozygous deletions of exon 7 and/or exon 8 of SMN1 patients with consistent clinical features is generally considered to be diagnostic of SMA (Lefebvre et al., 1995 1995 ; Chang et al., 1995 1995 ; Chen K.L. et al., 1999 1999 ; Chen W.J. et al., 2007 2007 ; Tsai et al., 2001 2001 ; Su et al., 2005 2005 ; Watihayati et al., 2007 2007 ). The neuronal apoptosis inhibitory protein ( NAIP ) gene located on 5q12.2-q13.3 has been hypothesized to be an SMA modifying gene because of its deletion in approximately two-thirds SMA1 chromosomes and its homology with baculoviral apoptosis inhibitory proteins (Roy et al., 1995 1995 ; Gotz et al., 2000 2000 ). In this study, we try to confirm the clinical diagnosis of southern Chinese SMA patients and to correlate the frequency of deletions within SMN and NAIP genes with SMA. SUBJECTS AND METHODS 1 Study population 2 A total of 62 unrelated southern Chinese children (aged 1 month to 11 years) with strongly suspected SMA from the Affiliated Children’s Hospital, School of Medicine, Zhejiang University, China, between the period of January 2000 and September 2007, and 100 healthy controls ( > 24 years of age) were investigated. Thirty-two children had a diagnosis of SMA1, 17 had SMA2, and the remaining 13 were diagnosed with SMA3. All of the patients fulfilled the diagnostic criteria on the basis of clinical, electrophysiological, and/or histological examination. The clinical manifestations included reduced muscle tension, decreased or absent tendon reflexes, different degrees of muscle atrophy. Levels of serum creatine phosphokinase (CPK) were found to be characteristically normal. Electromyography (EMG) revealed spontaneous discharge activity in resting muscles, increased amplitude, and prolonged duration of motor unit potentials during voluntary efforts. The muscle biopsies of some patients showed different degrees of atrophy in muscle fibers. The family history is in accordance with autosomal recessive inheritance (Table 1 1 ). Eleven patients had been studied previously (Yu et al., 2001 2001 ). M: male; F: female; Pos.: positive; Neg.: negative; E: exon; EMG: electromyography. EMG shows neurogenic abnormalities; Muscle biopsies of skeletal muscle show different changes of denervation with small groups of atrophic muscle fibers associated with markedly hypertrophied fibers. Most patients (46 cases) refused to take this procedure 3 SMA type 3 Age 10 Patient number 2 Gender 2 Family history 2 EMG * * 2 Muscle biopsy (+) ** ** 2 SMN1 deletion 2 NAIP -E 5 deletion 2 Deletion of both SMN1 -E 7 & E 8 and NAIP -E 5 M F Pos. Neg. E 7 & E 8 E 7 only SMA1 1~18 months 17 15 12 20 32 7 32 (100%) 0 (0%) 11 (34%) 11 (34%) SMA2 2~3 years 11 6 4 13 17 4 13 (76%) 4 (24%) 0 (0%) 0 (0%) SMA3 3.5~11 years 8 5 3 10 13 5 0 (0%) 13 (100%) 0 (0%) 0 (0%) 12 Total 36 26 19 43 62 16 45 (73%) 17 (27%) 11 (18%) 11 (18%) This study was approved by the ethics committee for the protection of human subjects of Zhejiang University School of Medicine, and informed consent was obtained from all individuals. Extraction of genomic DNA and PCR 2 Peripheral blood samples were collected and genomic DNA was extracted using the standard protocol. The primers used for the amplification were as follows (van der Steege et al., 1995 1995 ; Roy et al., 1995 1995 ): exon 7 of SMN : 5′-AGACTATCAACTTAATTTCTGATCA-3′ (forward), 5′-CCTTCCTTCTTTTTGATTTTGTTT-3′ (reverse); exon 8 of SMN : 5′-GTAATAACCAAATGCAATGTGAA-3′ (forward), 5′-CTACAACACCCTTCTCACAG-3′ (reverse); exon 5 of NAIP : 5′-CTCTCAGCCTGCTCTTCAGAT-3′ (forward), 5′-AAAGCCTCTGACGAGAGGATC-3′ (reverse); exon 13 of NAIP : 5′-ATGCTTGGATCTCTAGAATGG-3′ (forward), 5′-CCAGCTCCTAGAGAAAGAAGGA-3′ (reverse). PCR was performed on an ABI-2720 Thermal Cycler (Applied Biosystems, Foster City, California, USA) with a heated lid. Each reaction was carried out in a 50-μl volume containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl 2 , 0.2 μmol/L each primer, 200 μmol/L each dNTPs, approximately 200 ng genomic DNA, and 1 U TaKaRa Ex Taq polymerase (TaKaRa, Shiga, Japan) (He et al., 2004 2004 ). For SMN gene, amplification consisted of an initial denaturation step at 94 °C for 3 min, then 35 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, and a final extension step at 72 °C for 7 min. One normal sample as a negative control and one sample from a patient known to have exon 7 and exon 8 deletion (courtesy by Dr. Ke-lian Chen at University of Pennsylvania Medical Center) as a positive control were included in each PCR run. PCR amplification produced 190-bp and 187-bp fragments, respectively. For NAIP gene, a multiplex PCR was carried out, the annealing temperature was 60 °C, and the 435-bp (exon 5) and the 241-bp (exon 13) bands appeared (exon 13 taken as positive control). Restriction enzyme digestion for SMN and gel electrophoresis 2 SMN PCR products (4 μl) were subsequently digested with restriction enzyme Dra I or Dde I (1 U) (New England Biolabs, Beverly, Massachusetts, USA), and the resultant bands were visualized in 4% (w/v) agarose gel stained with ethidium bromide under UV light. For Dra I digestion of exon 7, SMN2 was cut into 170-bp and 20-bp fragments, and SMN1 remained as a 190-bp fragment. For exon 8, after Dde I digestion, SMN2 had 119-bp and 68-bp fragments, while SMN1 had a 187-bp band. RESULTS 1 Thirty-two SMA1 children with age ranged from 1 month to 18 months were unable to sit or hold up their heads and were floppy toddler. They were found to have homozygous deletions for SMN1 gene (exon 7 and exon 8), while 11 of 32 (34%) had a deletion in the NAIP gene (exon 5). Seventeen SMA2 patients, from 2 to 3 years old, were unable to walk. The majority of them showed wasting and areflexia of the lower limbs. Thirteen (76%) had deletions in both exon 7 and exon 8 of SMN1 , while 4 (24%) had deletions only in exon 7 of the SMN1 gene. All the 13 SMA3 patients aged from 3.5 to 11 years had slowly progressive weakness of the lower limbs. These patients revealed SMN1 gene deletion of exon 7 only. None of SMA2 and SMA3 patients lacked NAIP exon 5. In 100 normal controls, only 2 individuals deleted exon 7 of SMN1 . The results are summarized in Table 1 1 . DISCUSSION 1 According to the survey of Chung et al.( 2003 2003 ), SMA was the second most frequent inherited neuromuscular disease in southern Chinese children. The prevalence of SMA in Hong Kong, a major city in southern China with a population of around 6.7 million, is 18.7×10 −6 (about 1 in 53 000 children). The carrier rate for deletional SMA amongst the general population in Hong Kong is 1.6%, which is close to the 2% quoted in western countries (Chan et al., 2004 2004 ). The clinical manifestation of SMA is similar to many other neuromuscular diseases. It is difficult to identify SMA only from patients’ clinical symptoms and physical signs. So far, SMA is diagnosed primarily through a blood DNA testing including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single-strand conformation polymorphism (SSCP), denatured high-performance liquid chromatography (DHPLC), real-time quantitative PCR, and multiplex ligation-dependent probe amplification (MLPA). MLPA detects the presence or absence of the SMN1 gene that is present in normal individuals, in conjunction with a suggestive history and physical examination (Huang et al., 2007 2007 ). PCR-RFLP method was constructed by van der Steege et al.( 1995 1995 ) in 1995, which can diagnose SMA quickly and has been applied by clinicians for many years (van der Steege et al., 1995 1995 ; Watihayati et al., 2007 2007 ). For exon 7, there is no known difference in restriction site. Therefore, a specific oligonucleotide prime directly adjacent to the variant site was introduced, which allows a mismatch such that a restriction site for Dra I is created in the PCR product of exon 7 of the SMN2 , i.e., Dra I will cleave SMN2 exon 7 specifically. And SMN1 / 2 exon 8 homozygous absence can be distinguished by a restriction enzyme ( Dde I) digestion and gel electrophoresis. SMN2 is cut with Dde I while the SMN1 is not. Here we concentrated on the alterations in these 2 exons and NAIP exon 5 in 62 southern Chinese children in Zhejiang Province, who were strongly suspected to have SMA. It was observed that the majority of patients with more SMN1 gene deletions resulted in a severe phenotype. Our results of exon 7 deletion were consistent with the studies from Taiwan, Hong Kong, and Fujian Province, China (Chang et al., 1995 1995 ; 1997 1997 ; Tsai et al., 2001 2001 ; Wong and Chan, 2001 2001 ; Su et al., 2005 2005 ; Chen W.J. et al., 2007 2007 ). Thus, deletion of the SMN1 exon 7 is a major cause of SMA in southern China. Several investigations suggested the possibility of NAIP involvement in the development of SMA (Roy et al., 1995 1995 ; Gotz et al., 2000 2000 ; Watihayati et al., 2007 2007 ). All the patients enrolled in this study showed the deletion of the SMN1 gene, so we eliminated the clinical bias and looked at only the frequency of NAIP deletion among these SMA patients lacking SMN1 . After SMN1 deletion was confirmed in these patients, NAIP deletion was analyzed. In our study, 34% SMA1 patients were found to lack NAIP exon 5. Therefore, the NAIP gene deletion seems to affect disease severity. In fact, 24 SMA1 patients have died later, and we had no further details of 8 patients because of missing follow-up. The PCR-RFLP test used in this study is fast, sensitive, and inexpensive, and forgoes the need for invasive diagnostic procedure like a muscle biopsy from the patients (mostly children). Also, it is particularly applicable for prenatal diagnosis and preimplantation genetic diagnosis (PGD). The limitation of this method is that smaller rearrangements or point mutations of SMN gene can also result in a large series of SMA patients (Wirth, 2000 2000 ; Tsai et al., 2001 2001 ; Su et al., 2005 2005 ). Further analyses revealed that SMN2 copy number has been well established as a modifying factor of clinical severity. The absence of SMN gene in SMA1 is associated with gene dosage effect, whereas no gene dosage effect was detected in SMA2 or SMA3 (Lefebvre et al., 1995 1995 ; Campbell et al., 1997 1997 ; Frugier et al., 2002 2002 ; Yamashita et al., 2004 2004 ). These observations raised the hypothesis of a gene deletion event in SMA1 and a gene conversion event in SMA2 or SMA3, which would result in an increased number of SMN2 copies (Lefebvre et al., 1995 1995 ; Campbell et al., 1997 1997 ; Talbot et al., 1997 1997 ; Frugier et al., 2002 2002 ; Yamashita et al., 2004 2004 ). Therefore, the copy numbers of SMN1 and SMN2 genes should be determined by the point mutation and gene dosage analysis. In addition, PCR-based assay for determining the presence or absence of SMN1 is not quantitative, and therefore, cannot identify SMA carriers. The genomic complexity of the SMN region and its high degree of variability hamper the ability to directly screen the SMA carriers. 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Gene diagnosis of spinal muscular atrophy in Children We are very grateful to the SMA patients and their families who collaborated with us in this study, and to Prof. Ronald Scott of Division of Genetics, School of Medicine, University of Washington, USA, for his critical help. 10.1631/jzus.B0820125 Project supported by the National Natural Science Foundation of China (No. J0710043), and the Natural Science Foundation of Zhejiang Province (No. 2007C33049), China 2008-04-14 2008-12-05 2008-09-23 2022-01-05 Copyright © 2009, Journal of Zhejiang University Science 2009 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 10