Deletion analysis of SMN and NAIP genes in Tunisian patients with spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder involving degeneration of anterior horn cells of spinal cord, resulting in progressive muscle weakness and atrophy. AIMS: The purpose of our study was to determine the frequency of SMN and NAIP deletions in Tunisian SMA patients.
MATERIALS AND METHODS: Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was used to detect the deletion of exon 7 and exon 8 of SMN1 gene, as well as multiplex PCR for exon 5 and 13 of NAIP gene.
RESULTS: Fifteen (45.4%) out of 33 SMA patients were homozygously deleted for exons 7 and/or 8 of SMN1. Homozygous deletion of NAIP gene was observed in 20% (3 / 15) of patients.
CONCLUSIONS: The molecular diagnosis system based on PCR-RFLP analysis can conveniently be applied in the clinical testing, genetic counseling, prenatal diagnosis, and pre-implantation genetic diagnosis of SMA.

Introduction

Spinal muscular atrophy (SMA) is an autosomal recessive (AR) neuromuscular disorder characterized by symmetrical muscle weakness and atrophy caused by degeneration of the anterior horn cells in the spinal cord.[1]

The International SMA Consortium classification defines several types of SMA depending on the age of onset and clinical severity (Type I - IV).[2]

The majority of SMA cases are caused by homozygous deletion or mutation in the SMN1 gene. 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: Atelomeric one (SMN1) and a centromeric one (SMN2). 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.[34] 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.[56] The neuronal apoptosis inhibitory protein (NAIP) gene located on 5q12.2-q13.3 has been hypothesized to be an SMA modifying gene.[78]

The purpose of our study was to determine the percentage of SMN and NAIPgenes deletions in Tunisian SMA patients.

Materials and Methods

Patient's selection

We selected 33 patients from 14 different families originating from southern Tunisia that fulfilled the diagnostic criteria of SMA according to the International SMA Consortium. The diagnoses were confirmed by clinical symptoms, electroneuromyographic (ENMG) evaluation, and some time by muscle biopsy.

Clinically, all patients could be classified as SMA type III according to the classification of Pearn et al.[9] and Serratrice.[10]

After written and informed consent had been obtained, blood was sampled and DNA was extracted using a standard protocol.

Detection of SMN and NAIP gene deletions by PCR- RFLP method

SMN exon 7 and exon 8 deletions were detected by PCR amplification and restriction-enzyme digestion as described elsewhere.[11]

The 33 patients included in the study were studied for SMN1 and NAIP gene deletions. Eight samples from non-SMA patients were also run as controls.

Polymerase chain reaction (PCR) amplification of SMN exons 7 and 8 was carried out based on the method described by Wirth et al.[12] With slight modifications. The exon 7 PCR products (20 μL) were digested with 1.5 u HinfI (Fermentas life sciences) for 2 hours at 37°C and run on a 4% agarose gel at 150 V in 1X tris-borate-EDTA (TBE) (Amresco Inc., Solon, Ohio, USA) during 30 minutes. PCR products of SMN exon 8 (20 μl) were subsequently digested with restriction enzyme Dde I (1.5 units).

For NAIP, exon 5 and 13 (exon 13 taken as positive control), a multiplex reaction was carried out for 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min using primers 1864 and 1863 for exon 5 and primers 1258 and 1343 for exon 13.[713]

Results

The clinical and paraclinical features of 15 patients from 14 families with SMN and/or NAIP deletion are summarized in Table 1.

Table 1

Molecular analysis using PCR-RFLP assay revealed 15 patients (n = 15/33, 45.4%) with homozygous deletions in SMN1 or/and NAIP genes [Table 1].

Deletion analysis of SMN and NAIP genes in adult SMA patients using PCR/RFLP are shown in [Figures 1a, b and c].

Figure 1

(a) Detection of deletions in the SMN gene exon 7. Column 1: SMN gene exon 7; Column 2: SMN gene exon 7 cleaved with HinfI from an unaffected subject; Column 3: SMN gene exon7 cleaved with HinfI from SMA patients; Column M: 20 pb DNA Ladder (b) DdeI digestion of SMA patients for SMN exon 8. Column 1: SMN exon 8; Column 2: SMN exon 8 control; Column 3: SMN gene exon8 cleaved with DdeI from SMA patients; Column M: 100 pb DNA Ladder (c) The PCR analysis of the NAIP gene. Column 1: normal control; Column 2: normal pattern; Column 3: homozygous deletion of exon 5

A PCR product of exon 7 of SMN gene (135 bp) was obtained at the end of PCR. SMN/exon 7/HinfI digestion result in 4fragments corresponding to the SMN1 (101 bp and 34 bp) and SMN2 (78 bp, 34 bp and 23 bp). All patients (n = 15/15, 100%) have a deletion on exon 7 (missing of 78 pb and 23 pb bands) [Figure 1a]. For exon 8, the nucleotide, which differs between SMN1 and SMN2 genes, is G>A. The nucleotide A in the SMN2 gene (located at position 1155 of the cDNA) creates a Dde I site, which will digest the amplified DNA into two products of 122 bp and 78 bp.[1114] The exon 8 of SMN1 gene does not contain any Dde I site, hence it will not be cut and will remain as 200 bp.Exon 8 deletions were indicated by the absence of a 200 bp band [Figure 1b]. Twelvepatients (n = 12/15, 80%) showed a homozygous deletion of exon 7 and exon 8, while 3 (n = 3/15, 20%) lacked the SMN exon 7, but retained the exon 8.

In NAIP gene analysis, only control band presentation indicates deletions of exon 5 (435 pb), which is specific for the functional gene. The 241 bp (exon 13) bands appeared (exon 13 taken as positive control) [Figure 1c]. Exon 5 of NAIP gene was homozygously deleted in 20% (n = 3/15). No patient had a deletion in NAIP gene without a deletion in the SMN1 gene. No homozygous deletion of SMN and NAIP exon 5 genes was detected in the 8 control individuals.

Discussion

Spinal muscular atrophies are hereditary disorders characterized by degeneration of spinal cord motor neurons. The majority of SMA cases showed AR inheritance and are caused by homozygous deletion or mutation of the SMN1 gene on 5q (OMIM 253300, 253550, 253400, and 271150). Non-5q SMA is rare, clinically diverse, and genetically heterogeneous.[1516]

Among all the candidate genes, SMN1 is believed to be the primary SMA disease-causing gene. The finding of homozygous deletions of exons 7 and/or 8 of SMA patients with consistent clinical features is generally considered to be diagnostic of SMA.[17]

In the present study, we demonstrate that the percentage of homozygosity for the deletion of SMN1 exon 7 for all Tunisian patients was 45.4%, similar to those reported in Iran (100% in type I, 66% in type II, and 50% in type III).[18]

A higher frequency was observed in other populations, especially among SMA patients from China,[19] Netherlands,[20] Finland, UK,[21] and Tunis.[22]

The majority of SMA patients are characterized by homozygous deletions in exon 7 and 8 of the SMN1 gene.[141921] Van der Steege et al.[23] Identified a gene conversion event that changed the sequence of the SMN1 gene into that of an SMN2 gene in some SMA patients, in which the SMN exon 7 had been deleted, but exon 8 was retained. The deletion of exon 8 alone is very rare. Deletions involving both exons 7 and 8 (80%) were much more frequent than deletions of only exon 7 (20%) or only exon 8 (0%).

In addition, there was no correlation between deletions in the SMN gene and phenotype severity in accordance with previous reports in the literature.[24]

Deletions in exon 5 of NAIP (20% in our study), which is specific for the functional NAIP gene, showed wide frequency variation (0 to 67%) in different population studies.[1921] NAIP deletion alone, without SMN1 is being deleted, was not seen in any of our patients. This is contrary to the results of Japanese patients where two unaffected mothers of patients with NAIP and SMN1 deletions showed deletions of only NAIP gene.[25]

The role of NAIP gene in the pathogenesis of SMA remains controversial. However, several studies suggest that NAIP gene can be a predictive marker of SMA prognosis and acts a cofactor for the differentiation and survival of neuronal cells including the motor neurons. Further studies will be necessary to clarify its exact functional role.

Conclusion

With the advent of molecular biology techniques, SMN gene deletion study represents nowadays a useful and reliable tool to confirm the diagnosis of SMA suspected clinically. Demonstrate of homozygous deletions of exons 7 and/or 8 of SMN gene confirm the diagnosis of SMA, even though the clinical features are incomplete or atypical. Deletions of NAIP gene were mainly seen in severely affected patients, hence is useful to predict prognosis.