Abstract
Background
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a mutation in SMN1. SMA is classified into three subtypes (types 1, 2, 3) based on achieved motor milestones. Although NAIP and SMN2 are widely accepted as SMA-modifying factors, gender-related modifying factors or gender effects on the clinical phenotype are still controversial.
Methods
A total of 122 Japanese patients with SMA, of which SMN1 was homozygously deleted, were analyzed from the perspective of the achieved motor milestone, NAIP status and SMN2 copy number.
Results
A predominance of male patients was observed in SMA type 3 (the walker group) without NAIP-deletion or with high SMN2 copy number (3 or 4 copies).
Conclusion
We suggest the presence of gender-related modifiers on disease severity in SMA patients. The modifiers may contribute only in the presence of NAIP and a high copy number of SMN2.
Keywords: Spinal muscular atrophy, gender, clinical phenotype, SMN2, NAIP
INTRODUCTION
Spinal muscular atrophy (SMA) is a common neuromuscular disorder with autosomal recessive inheritance. SMA is clinically classified into three subtypes: type 1 (severe form, onset of symptoms at age of <6 months, unable to sit without aid); type 2 (intermediate form, onset of symptoms at age of 6–18 months, able to sit alone but unable to stand or walk without aid); and type 3 (mild form, onset of symptoms at age of >18 months, able to stand and walk without aid) (11). The responsible gene of the disease is the survival motor neuron 1 gene (SMN1), which was identified in the SMA locus in chromosome 5q (7). SMN1 is homozygously deleted in 95% of SMA patients and deleteriously mutated in the remaining patients (7).
The neuronal apoptosis inhibitory protein gene (NAIP) was also identified as an SMA-related gene within the SMA locus (14). NAIP deletion is found most frequently in SMA type 1 (~50% of the patients with SMA type 1), and seldom in other subtypes (14). On the other hand, the survival motor neuron 2 gene (SMN2) in the SMA locus, a highly homologous gene of SMN1, encodes the same SMN protein as SMN1 does, and multiple copies of SMN2 may compensate to some degree for the lack of SMN1 (17). Thus, NAIP and SMN2 are now widely accepted as SMA-modifying genetic factors.
Gender effect on the clinical phenotype of SMA has been controversial until now. Some researchers reported that the symptoms in female siblings were milder than those of the male siblings in the affected SMA families (4,13). In addition, predominance of female patients with SMA type 3 was reported (3). However, other researchers observed a predominance of male patients in milder forms of SMA (5,6,18). In this study, to clarify the gender effects on the disease severity or clinical phenotype of SMA, we analyzed the SMA patients referred to our laboratory.
PATIENTS AND METHODS
Patients
A total of 122 Japanese patients (60 females and 62 males) were enrolled in this study. They were referred to Kobe University from 1996 to 2015, and fulfilled the diagnostic criteria defined by the International SMA Consortium (11). Informed consent was obtained from these patients and/or their parents. This study, including the genetic analysis, was approved by the Ethics Committee of Kobe University Graduate School of Medicine, Japan.
SMN and NAIP deletion test
To detect SMN and NAIP deletion, PCR restriction fragment length polymorphism analysis was performed according to the method of van der Steege et al. (16). NAIP exon 5 was detected using the PCR method of Roy et al. (14).
Copy number analysis of the SMN genes
The copy number of the SMN genes was determined by real-time PCR (15) or the multiplex-dependent probe amplification (MLPA) assay (1).
Statistics
Correlation of SMN2 copy number with clinical subtype was determined using chi-squared tests and logistic regression analysis. A P-value of less than 0.05 was considered to indicate a significant difference. To avoid α-error inflation in the separate analyses of NAIP-deleted and non-NAIP-deleted patients, and of low- and high-SMN2-copy-number patients, a P-value of less than 0.025 was considered to indicate a significant difference. The software used for statistical analysis was Statistical Program for Social Science (SPSS) version 16 (IBM Corporation, Palo Alto, CA, USA).
RESULTS
To clarify the effect of gender on the disease severity of SMA, we analyzed the relations between gender and clinical subtypes in 122 Japanese SMA patients. A significant predominance of male patients (or a rarity of female patients) was found in SMA type 3 (p<0.01) (Table I). Then, the patients were divided into two groups based on the “obtaining the ability to walk without aid in the life”, the non-walkers (SMA types 1 and 2) and the walkers (SMA type 3). A 2×2 contingency table ([male and female] × [non-walker and walker]) demonstrated a significantly higher frequency of male patients (or a significantly lower frequency of female patients) in the walkers (p<0.01) (Table I).
Table I.
Gender and motor function
| (A) Gender and clinical subtype | |||
|---|---|---|---|
|
| |||
| Type 1 | Type 2 | Type 3 | |
| M (n=62) | 28 | 15 | 19 |
| F (n=60) | 33 | 22 | 5 |
χ2 = 9.87, df = 2, P < 0.01
| (B) Gender and walking ability | ||
|---|---|---|
|
| ||
| Non-walker | Walker | |
| M (n=62) | 43 | 19 |
| F (n=60) | 55 | 5 |
χ2 = 9.61, df = 1, P < 0.01
Then, we examined the gender effect in the patients with or without NAIP deletion, and we found a significant difference between males and females only in the patients without NAIP deletion. Only in the patients without NAIP deletion, the 2×2 contingency table demonstrated a significantly higher frequency of male patients (or a significantly lower frequency of female patients) in the walkers (p<0.05) (Table II).
Table II.
Gender, NAIP and walking ability
| (A) Patients with NAIP deletion | ||
|---|---|---|
|
| ||
| Non-walker | Walker | |
| M (n=17) | 15 | 2 |
| F (n=25) | 25 | 0 |
| (B) Patients without NAIP deletion | ||
|---|---|---|
|
| ||
| Non-walker | Walker | |
| M (n=45) | 28 | 17 |
| F (n=35) | 30 | 5 |
χ2=5.45, df=1, P<0.05
We also examined the gender effect in the patients with low SMN2 copy number (1 or 2) and high SMN2 copy number (3 or 4). Only in the patients with high SMN2 copy number, the 2×2 contingency table demonstrated a significantly higher frequency of male patients (or a significantly lower frequency of female patients) in the walkers (p<0.01) (Table III).
Table III.
Gender, SMN2 and walking ability
| (A) Patients with 1–2 SMN2 copies | ||
|---|---|---|
|
| ||
| Non-walker | Walker | |
| M (n=25) | 24 | 1 |
| F (n=29) | 29 | 0 |
| (B) Patients with 3–4 SMN2 copies | ||
|---|---|---|
|
| ||
| Non-walker | Walker | |
| M (n=37) | 19 | 18 |
| F (n=31) | 26 | 5 |
χ2=5.45, df=1, P<0.05
To clarify which of the studied parameters, gender (male or female), NAIP (presence or absence), and SMN2 (copy number) affects the ability to walk without aid in the life, we performed a multiple logistic regression analysis with “the ability to walk without aid in the life” as a dependent variable. Here, we used the forward stepwise selection method to evaluate the relative contribution of gender, NAIP, and SMN2 to the outcome. The best model we obtained in this stepwise selection process included gender and SMN2, but not NAIP (Table IV). According to our calculation, the adjusted odds ratios of <male to female> and <high copy number of SMN2 to low copy number of SMN2> for “the ability to walk without aid” were 3.902 and 37.382, respectively.
Table IV.
Logistic regression analysis
| Variables | B | Standard Error | Wald | df | P Value | Exp (B) | Confidence Intervals |
|---|---|---|---|---|---|---|---|
| Gender | 1.362 | 0.633 | 4.623 | 1 | <0.05 | 3.902* | 1.128–13.501 |
| SMN2 | 3.621 | 1.022 | 12.547 | 1 | <0.001 | 37.382* | 5.040–277.243 |
“B” designates coefficient values. “Exp(B)” designates odds ratios.
designates odds ratio of male to female.
DISCUSSION
Our study revealed that a relatively larger number of male patients were observed in SMA type 3, though there were no significant gender differences with regard to the number of patients in SMA type 1 or 2.
This finding suggested two possible gender-related modifiers: (1) One is the presence of a male-related modifier which lead to increasing the number of male patients with SMA type 3, and (2) the other is the presence of a female-related modifier which lead to decreasing the number of female patients with SMA type 3. The male-related modifier might delay the onset of SMA, while the female-related modifier might prevent the development of SMA. Oprea et al. reported that asymptomatic SMN1-deleted females exhibited significantly higher expression of plastin 3 (PLS3, T-plastin or T-fimbrin; MIM 300131, Xq23) in their lymphoblasts than did their SMA-affected counterparts (13), supporting the hypothesis of female-related modifiers. PLS3 is an actin-binding protein that is expressed in normal cells of solid tissues and transformed fibroblasts (8). PLS3 has a calcium-binding domain (9), and calcium binding may be essential for plastin 3 function in SMN-deficient motoneurons (10).
As for the relationships between gender and NAIP, or between gender and SMN2, we found a significantly higher frequency of male patients in the walker group without NAIP-deletion or with a high SMN2 copy number (3 or 4 copies). It means that gender-related modifiers may have some effects on the clinical phenotype of SMA in the presence of NAIP and a high copy number of SMN2. According to the report of Oprea et al., non-symptomatic individuals with SMN1 deletion carried a high SMN2 copy number (3 or 4 copies) (13).
Then, a question arises why the gender-related modifiers have effects only in the presence of NAIP and a high copy number of SMN2. The absence of NAIP may mark the extent of the deletion involving SMN1 in SMA chromosome (2). On the other hand, the presence of NAIP implies no deletion of SMN1; but it suggests that an SMN1-to-SMN2 gene conversion event has occurred at least on one chromosome, which may decrease the SMN1 copy numbers and increase the SMN2 copy numbers (12). Thus, the question is paraphrased into “why the gender-related modifiers have effects only in the patients with a high copy number of SMN2.” Our answer to this question is that in SMA patients, negative effect of a low SMN2 copy number on the clinical phenotype may be much larger than positive effect of the gender-related modifiers on the clinical phenotype. To say in other words, positive effect of the gender-related modifiers may be masked by negative effect of a low SMN2 copy number.
In this study, we suggested the presence of gender-related modifiers on disease severity in SMA patients, and the modifiers work only in the presence of NAIP and a high copy number of SMN2. Indeed, we could not fully neglect the possibility that our study may be subject to selection bias or sampling bias, i.e. the patients studied may not be representative of the Japanese SMA population. If more Japanese patients with SMA could have been recruited for our study, we could draw more solid conclusions about gender-related modifiers.
ACKNOWLEDGMENTS
This research was supported in part by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, Grant No. 16ek0109086h0002 (title “Practical study for multicenter cooperative and investigator initiated clinical trial using valproic acid in childhood onset spinal muscular atrophy”).
Footnotes
DECLARATION OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this paper.
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