Preliminary Results in a Study Regarding the Relationship Between Perlecan Gene Polymorphism and Spinal Muscular Atrophy Type I Disease

Abstract

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by weakness and atrophy of proximal muscles. Despite the fact that the disease transmission suggests an autosomal recessive trait, the wide spectrum of clinical manifestations indicates that other genes may contribute to the SMA phenotype. To identify possible modifier genes, the aim of our study was to investigate the relationship between BamH1 perlecan gene polymorphism and SMA type I, the classical severe form of the disease. We genotyped 40 patients with SMA type I disease and 50 subjects without personal or heredo-colateral neuromuscular problems, using the polymerase chain reaction-restriction fragment length polymorphism method. After statistical analysis of the observed genotypes, a significant difference (p = 0.03) could be observed regarding the incidence of TT genotype and T allele in boys with SMA type I compared with affected girls. However, this result cannot be assessed because of the small and unequal number of subjects. We concluded that there might be no association between perlecan gene polymorphism and SMA type I disease.

Introduction

Spinal muscular atrophy (SMA) is the leading genetic cause of death in infants (Wirth, 2000). Based on the age at onset and the severity of clinical manifestations, several types have been described: SMA type I (Werdnig-Hoffmann disease, the classical severe form, MIM 253300), SMA type II (Dubowitz disease, the intermediate form, MIM 253550), SMA type III (Kugelberg-Welander, the mildest form, MIM 253400), and SMA type IV (adult form, MIM 271150). Also, a type 0 or prenatal form has been reported by some papers to be the very severe form of the disease (Dubowitz, 1999). Exon 7 of the survival motor neuron gene (SMN1) has been found to undergo homozygous deletion in more than 95% of the SMA patients (Lefebvre et al., 1995). However, because of the heterogeneity of clinical manifestations, there is no obvious phenotype-genotype correlation and others genes are supposed to modulate the SMA phenotype.

A special interest was focused on the SMN2 gene which encodes for a small amount of SMN full-length protein, essential for motor neuron survival. The SMN2 gene is reported as a possible SMA modifier gene, based on the correlation relationship established between an increased number of SMN2 genes and a less severe form of the disease (Campbell et al., 1997). More recently (Oprea et al., 2008) it was also demonstrated that plastin 3 (PLS3) is a SMA protective modifier gene, by rescuing the axonogenesis defects observed in the SMA disease. Despite all these findings, the data describing SMA are not yet complete.

Among the axon length and outgrowth defects described in SMA pathology, a lack of nicotinic acetylcholine receptors clustering in the muscle cells of patients with SMA type I was also noticed (Arnold et al., 2004). An essential role in this clustering process is played by perlecan (Peng et al., 1999), the major heparan sulfate proteoglycan of basement membranes.

We proposed in this study to evaluate the relationship between BamH1 perlecan gene polymorphism and SMA type I disease.

Materials and Methods

Subjects

In our study we included 40 patients with SMA type I (diagnosis based on the SMA International Consortium criteria) born in different parts of Romania and who were referred by the “Al.Obregia” Clinical Psychiatry Hospital Bucharest. At the time of enrollment, the patients (23 boys and 17 girls) were between ages 1 and 6 months, except two patients who were 1.6 years old, but who were clinically suspected at 4 and 5 months, respectively. All patients had severe muscle weakness with symmetrical hypotonia (more proximal than distal) and no osteotendinous reflexes. Fasciculations of tongue and tremor of hands were also reported, as well as recurrent respiratory infections. Arthrogryposis was identified in one boy and one girl at the age of 5 months. For six patients, the result of electromyography (EMG) examination was available and it revealed neurogenic atrophy for five of them. The creatine phosphokinase (CPK) serum level was generally normal or slightly elevated.

To confirm the SMA clinical diagnosis, molecular analyses were performed and homozygous deletion of SMN1 gene exon 7 was identified for all the patients, including those with arthrogryposis.

The healthy control group comprised 50 subjects (28 men and 22 women), with age between 18 and 24 years. None of these subjects had personal or heredo-collateral neuromuscular problems. The DraI restriction reaction revealed at least one copy of SMN exon 7 genes.

The subjects were enrolled in the study after informed consent was obtained, according to the Declaration of Helsinki. Taking into account the fact that the majority of patients are at minor age, the consent was given by the parents or their legal representatives.

DNA isolation and perlecan gene genotyping

Blood samples were obtained from the subjects into ethylenediaminetetraacetic acid anticoagulant. A volume of 500 μL was used for DNA extraction, using a commercial kit. For the pertinent polymorphism, the polymerase chain reaction (PCR)-restriction fragment length polymorphism method was employed. The PCR was performed in a 10 μL reaction, using the primer sequences described by Liu et al.: forward 5′-CAT GTC CCA TGC CCC ACG TGT GCT-3′ and reverse 5′-ATT GTA GCT GTG GCA GGC AAA CTC-3′ (Liu et al., 2003a). The reaction temperature and conditions were 94°C for 2 min (1 cycle); 94°C for 30 s, 59°C for 25 s, and 72°C for 35 s (35 cycles); and 72°C for 2 min (1 cycle).

The digestion reaction was performed with 5 U BamH1 restriction enzyme (Fermentas, Vilnius, Lithuania) at 37°C for 3 h. The restriction products were separated in a 8% polyacrilamide gel and visualized after ethidium bromide staining.

Statistical analysis

The values of observed genotypes in case and control groups were tested for the Hardy-Weinberg equilibrium. To compare the observed and expected genotypes, the χ² test was used, and a value of p < 0.05 was considered statistically significant.

Results

The electrophoresis of PCR-restriction fragment length polymorphism products revealed all the three genotypes for BamH1 perlecan gene polymorphism: TT, TG, and GG. The genotype distribution for patients and control subjects fit the Hardy-Weinberg equilibrium, verified by χ² test application (χ² = 0.033 and p = 0.85 for patients, and χ² = 0.53 and p = 0.46 for healthy subjects). The distribution of the genotypes is shown in Table 1.

Table 1.

Genotype Distribution of BamH1 Perlecan Gene Polymorphism

	Genotypes
Groups	Sex	TT	TG	GG
SMA patients	M	18 (78.3%)	4 (17.4%)	1 (4.3%)
	F	7 (41.2%)	9 (53%)	1 (5.8%)
	Total	25 (62.5%)	13 (32.5%)	2 (0.05%)
Control subjects	M	14 (50%)	12 (43%)	2 (7%)
	F	8 (36.3%)	12 (54.5%)	2 (9.2%)
	Total	22 (44%)	24 (48%)	4 (0.08%)

SMA, spinal muscular atrophy.

As can be observed, the TT genotype had a higher frequency in the patient group compared with the control one (62.5% vs. 44%). The odds ratio (OR) calculated using the two-by-two contingency table (Uitenbroek, 1997) had a value of 1.97 (degree of freedom = 1; confidence interval = 95%; 0.8371 < OR < 4.6345), for which the Yates correction was applied, taking into account the small number of subjects with GG genotype. The p-value obtained with this correction (0.1782) indicates that the impact of TT genotype on SMA type I disease is not statistically significant, even though initially an OR = 1.97 suggested an association relationship between the TT genotype and SMA disease. The allele distribution also revealed a higher frequency for T allele in the patient group than in the control group (78.8% vs. 68%), but the differences are not statistically significant: OR for T allele was 1.7439, p = 0.1.

Comparing the genotype distribution (SMA vs. control group) related to sex, we found in the case of male subjects an OR_TT = 3.6 (p-value with Yates correction = 0.07). Regarding the T allele, an OR = 2.66 with a p-value at the statistically significance limit (p = 0.057) was obtained. A significant difference could be observed regarding the incidence of TT genotype in boys with SMA type I (78.2%) compared with affected girls (41.2%): OR = 5.1429; 1.289 < OR < 20.5187; p = 0.03 (Yates correction). Also, a significant value was noticed in case of T allele incidence in the men group with SMA type I: OR = 3.19; 1.0412 < OR < 9.764; p = 0.03.

Discussion

The perlecan gene is mapped on chromosome 1p36.1-p.35 (Dodge et al., 1991; Kallunki et al., 1991) and is composed of 97 exons that code for the five distinct domains of the protein (Nicole et al., 2000). The domain I (NH₂-terminal) contains three heparan sulfate chains and is responsible for the interactions with growth factors and acetylcholinesterase (Iozzo et al., 1994; Dolan et al., 1997). As perlecan has an essential role in the clustering process of nicotinic acetylcholine receptors (Peng et al., 1999), this domain may be more susceptible than other domains to be involved in this process. The most common polymorphism described in this region is BamH1 intron 6 polymorphism, investigated in some populations to evaluate its contribution in different pathologies and complications. A lack of association was reported between BamH1 perlecan gene polymorphism and mitral valve prolapse (Chou et al., 2004), coronary artery disease (Cai et al., 2000), and Alzheimer's disease (Rosenmann et al., 2004). In contrast, the investigated polymorphism may be a factor risk for diabetic nephropathy (Liu et al., 2003b), microalbuminuria (Hansen et al., 1997), severity of atherosclerotic lesions (Radhakrishnamurthy et al., 1998), and lower apoB levels (Dolan et al., 1997). Also, different mutations identified in the perlecan gene were reported to be involved in Schwartz-Jampel syndrome, characterized by myotonic myopathy and chondrodysplasia (Arikawa-Hirasawa et al., 2002; Stum et al., 2006). In the same context of the Schwartz-Jampel syndrome pathology there was further discussion of the roles of perlecan in neuromuscular function (Hirasawa, 2004). Additionally, a very recent paper highlighted the essential role of perlecan for the integrity of somitic muscle and developmental angiogenesis, suggesting the involvement of perlecan in myopathies (Zoeller et al., 2008).

As can be observed, some of the previous research studies sustain our work hypothesis, but we did not obtain conclusive data to establish an association between perlecan gene BamH1 polymorphism and SMA type I disease. A high incidence of TT genotype and T allele was observed in boys with SMA type I, but a study with an increased number, equal for both sexes, is required to assess these results. Our findings do not exclude the involvement of other polymorphisms or mutations in the investigated gene as well as in a relationship with SMA type I disease. Unfortunately, no other reports regarding the involvement of perlecan gene polymorphism in SMA pathology were available.

To our knowledge, this is the first research that focused on the involvement of a perlecan gene polymorphism in SMA disease. The preliminary results of our study indicated that there might be no association between perlecan gene BamH1 intron 6 polymorphism and SMA type I disease. However, additional research and replicate studies in other populations are required to confirm our results.

Footnotes

Acknowledgments

This study was supported in part by the grant CNCSIS-TD 223/2008 funded by the Ministry of Education, Research, and Innovation, Romania. The authors thank Dr. Sanda Magureanu for all her support in performing this study. Also, the authors thank all the subjects who agreed to be included in this research.

Disclosure Statement

The authors declare that they have no competing financial interests.

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