Ping Fang and Liang Li contributed equally to this work.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XMX conceived and coordinated the study. L Li designed the experiments, analyzed the data and prepared the manuscript. PF, ZYZ and L Lin performed the experiments, assisted with data analysis and manuscript preparation. WJZ and JH recruited the healthy participants. JZ, WQW, TZY, JSX and YZ recruited the SMA patients and collected the samples. All authors read and approved the final manuscript.
Abbreviations
MLPA
Multiple ligation-dependent probe amplification
NAIP
Neuronal apoptosis inhibitory protein
SMA
Spinal muscular atrophy
SMN
Survival motor neuron
Background
Spinal muscular atrophy (SMA) is one of the most common autosomal recessive diseases and is characterized by degeneration of spinal cord motor neurons, atrophy of skeletal muscles, and generalized weakness [
1]. The incidence of SMA is approximately 1/6,000 to 1/10,000 live births, and the carrier frequency is about 1/42 in the Chinese population [
2]. SMA is divided into four clinical types according to age of onset and achieved motor function: (1) severe type I; (2) intermediate type II; (3) mild type III; and (4) adult-onset type IV.
SMA is caused by the dysfunction of the survival motor neuron (
SMN) gene on chromosome 5q13.2. The two versions of
SMN,
SMN1 and
SMN2, differ by only five nucleotides.
SMN1 produces a full-length transcript that encodes functional SMN protein. About 94% of SMA patients have a homozygous deletion of
SMN1 exon 7.
SMN1 can be absent because of deletion or SMN1-to-SMN2 conversion [
3]. A single nucleotide transition in
SMN2 exon 7 (c.840C > T) relative to
SMN1 causes most of the
SMN2 pre-mRNA to lack exon 7 and encode nonfunctional SMNΔ7 protein [
4]. However, about 10% of
SMN2 pre-mRNA is normal and can be translated into full-length SMN protein.
SMN2 partially functionally compensates an
SMN1 homozygous deletion [
5]. Thus, the
SMN2 copy number influences SMA severity. In addition to the
SMN genes, the neuronal apoptosis inhibitory protein (
NAIP) gene, also located at chromosome 5q13.2, is an SMA disease-related gene. The copy number of
NAIP is reported to be correlated with SMA severity. SMA patients with fewer copies of
NAIP have more severe phenotypes than patients with more copies of
NAIP [
6,
7]. Moreover, both
SMN2 and
NAIP copy numbers were associated with the onset age, risk of death and survival probability of SMA patients [
8]. Therefore, elucidating the gene copy numbers and structures of
SMN1,
SMN2 and
NAIP is important for analyzing the molecular mechanism of SMA and for SMA clinical diagnosis.
Anzeige
The
SMN1,
SMN2 and
NAIP genes are all located at 5q13.2, an unstable chromosomal region that is prone to deletion, duplication, and gene conversion. Copy numbers of
SMN1 and
SMN2 vary in humans. In Chinese populations, up to 4
SMN1 or
SMN2 genes are reported in both healthy people and SMA patients [
2,
9]. In addition,
SMN gene deletions and rearrangements are found in different populations [
6,
7,
10]. Detecting copies and gene structures of SMA-related genes is difficult because of the high homology between
SMN1 and
SMN2. However, the multiple ligation-dependent probe amplification (MLPA) assay has been established for detecting deletions and duplications of SMA-related genes [
11,
12].
To determine the copy numbers and structures of SMA-related genes in a Chinese population, we analyzed
SMN1,
SMN2 and
NAIP in 42 Chinese SMA patients and 212 Chinese healthy individuals using MLPA assays.
Methods
Population and patient samples
A total of 212 peripheral blood samples were obtained from unrelated healthy adults (108 men and 104 women) at the Third Affiliated Hospital of Southern Medical University. In addition, we enrolled 42 unrelated SMA patients from four hospitals (18 patients from the Fuzhou General Hospital of Nanjing Military Command, 18 from the Shenzhen Maternity and Child Healthcare Hospital, 4 from the Liuzhou Municipal Maternity and Child Healthcare Hospital, and 2 from the Prenatal Diagnosis Center of Dongguan Maternal and Child Health Hospital). All SMA patients were confirmed by MLPA assay to have a homozygous deletion of
SMN1 gene exon 7. All healthy individuals and SMA patients were of Han ethnicity.
Ethical approval
Ethical approval for this study has been obtained from the Ethical Committee of the Southern Medical University as well as from partners including the Ethics Committee of the Third Affiliated Hospital of the Southern Medical University, the Ethics Committee of Fuzhou General Hospital of Nanjing Military Command, the Ethics Committee of Shenzhen Maternal and Child Health Care Hospital, the Ethics Committee of Liuzhou Maternal and Child Health Care Hospital and the Ethics Committee of Reproductive Medical Center of Dongguan Maternal and Child Health Care Hospital. Written informed consent was obtained from all participants or guardians prior to the study.
Anzeige
MLPA technique
Human DNA was extracted from leukocytes in peripheral blood using a standard phenol/chloroform method.
SMN1,
SMN2 and
NAIP copy numbers were determined by an MLPA technique using SALSA P021-A2 SMA kits (MRC-Holland, Amsterdam, Netherlands) that amplify 37 regions throughout the genome for products between 140 and 463 bp. Of the amplified regions, 15 were target sequences at the SMA locus on 5q13.2 and 22 were reference sequences. Ten probes were complementary to
SMN1,
SMN2 and
NAIP sequences. Because of the sequence similarity of
SMN1 and
SMN2, probes SMN1-1
*, SMN1-5
*, SMN1-6
* and SMN1-8
* were complementary to common regions in exons 1, 4, 6, 8 of either
SMN1 or
SMN2. Probes SMN1-7
# and SMN1-8
# were specific for
SMN1 in exons 7 and 8. Probes SMN2-7
# and SMN2-8
# were specific for
SMN2 in exons 7 and 8. Probes NAIP-1
# and NAIP-08
# were specific for
NAIP in exons 13 and 5 (Figure
1). MLPA assays are a multiplex PCR strategy with four steps: DNA denaturation and probe hybridization, ligation of two probes, PCR of ligated probes, and separation of amplified fragments and data analysis. All experimental procedures were carried out according to kit instructions. MLPA products were separated and quantified by capillary electrophoresis using an ABI 3130XL Genetic Analyzer with LIZ 500 as the internal size standard. Data were analyzed with the GeneMapper software v3.2 package (Applied Biosystems, Foster city, CA). Intrasample normalization was by dividing the peak area of each probe’s amplification product by the total area of reference probes only. Intersample normalization was by dividing the intranormalized probe ratio for a sample by the average intranormalized probe ratio of two reference samples. Reference samples had two copies of
SMN1,
SMN2 and
NAIP genes. Intervals for estimating the copy number of each probe were defined according to the predefined standards of Alías et al. [
13].
Figure 1
Schematic representation of SMA-related genes in MLPA assays. Gray,
RAD17,
SERF1B and
GTF2H2, near the disease-determining gene. Arrows, relative position and 5′-to-3′ direction of SMA-related genes. Blank boxes, exons (E). Important MLPA probes are above related genes. *, probes complementary to common regions in exons 1, 4, 6, 8 of either
SMN1 or
SMN2; #, probes specific for
SMN or
NAIP genes.
×
Statistical analysis
The distributions of samples with different
SMN2 and
NAIP copy numbers from SMA patients and healthy individuals were compared using the Mann–Whitney
U test. A p-value of less than 0.05 was considered statistically significant. All statistical analyses used the SPSS software package (version 13.0, SPSS Inc., Chicago, IL).
Results
Gene copy number and structure in SMA patients
All SMA patients had a homozygous deletion of
SMN1 exon 7. We also identified a homozygous deletion of
SMN1 exon 8 in 37 of 42 patients (88.1%); the other 5 SMA patients (11.9%) had a single copy of
SMN1 exon 8. The proportions of SMA patients with various numbers of normal
SMN2 copies were 1 copy in 2 patients (4.8%), 2 copies in 14 (33.3%), 3 copies in 24 (57.1%) and 4 copies in 2 (4.8%). The proportions of SMA patients with various numbers of normal
NAIP were 0 copies in 4 patients (9.5%), 1 copy in 26 (61.9%) and 2 copies in 12 (28.6%). In addition, 10 patients (23.8%) had an
NAIP gene lacking exon 5 (pattern
a in Figure
2), and 1 patient (2.4%) had an
SMN1 gene lacking exon 7 (pattern
b in Figure
2).
SMN2 genes lacking partial sequences were also found in SMA patients. Three patients (7.1%) had an
SMN2 lacking exon 1 to exon 7 (pattern
d in Figure
2), and two (4.8%) had an
SMN2 lacking exon 7 (pattern
c in Figure
2). We also found hybrid
SMN genes in SMA patients. A hybrid
SMN gene in which exon 8 of
SMN2 was converted to exon 8 of
SMN1 (pattern
e in Figure
2) was found in 3 patients (7.1%), and a hybrid
SMN gene in which exon 7 of
SMN1 was converted to exon 7 of
SMN2 (pattern
f in Figure
2) was found in 1 (2.4%). MLPA probe copy numbers of 42 SMA patients are in Table
1 and inferred copy numbers are in Table
2. Abnormal gene structures are in Figure
2.
Figure 2
Gene structures inferred from MLPA results. (a)NAIP gene lacking exon 5;
(b)SMN1 gene lacking exon 7;
(c)SMN2 gene lacking exon 7;
(d)SMN2 gene lacking exon 1 to exon 7;
(e) Hybrid
SMN gene in which exon 8 of
SMN2 was converted to exon 8 of
SMN1;
(f) Hybrid
SMN gene in which exon 7 of
SMN1 was converted to exon 7 of
SMN2;
(g)NAIP gene lacking exon13;
(h) Hybrid
SMN gene in which exon 8 of
SMN1 was converted to exon 8 of
SMN2.
Table 1
The MLPA probe copy numbers of SMA-related genes in 42 Chinese SMA patients
MLPA probe copy number
Total
SMN1/
SMN2
SMN1
SMN2
NAIP
Group
N = 42
Exon1
Exon4
Exon6
Exon8
Exon7
Exon8
Exon7
Exon8
Exon13
Exon5
1
3(7.1%)
2
2
2
2
0
0
2
2
1
1
2
3(7.1%)
2
2
2
2
0
0
2
2
1
0
3
1(2.4%)
2
2
2
2
0
1
2
1
1
0
4
1(2.4%)
2
2
2
2
0
0
2
2
2
1
5
1(2.4%)
3
3
3-4
3
0
0
3
3
2
1
6
1(2.4%)
3
3
3-4
3
0
0
3
3
2
2
7
4(9.5%)
3
3
3
3-4
0
0
3
3
2
2
8
2(4.8%)
4
4
4
4
0
0
4
4
2
2
9
1(2.4%)
3
3-4
3
3
0
1
2
2
2
1
10
2(4.8%)
3
3-4
3-4
3
0
0
2
3
2
2
11
9(21.3%)
3
3-4
3
3-4
0
0
3
3
2
1
12
6(14.2%)
3
3
3
3
0
0
3
3
1
1
13
1(2.4%)
3
3
3
3
0
1
3
2
1
1
14
1(2.4%)
2
2
2
2
0
1
2
1
1
1
15
1(2.4%)
2-3
2-3
3
4
0
0
3
4
2
2
16
2(4.8%)
2-3
3
3
3-4
0
0
3
4
2
1
17
2(4.8%)
1-2
2
2
2
0
0
2
2-3
2
2
18
1(2.4%)
2-3
2-3
3
3
0
1
3
2
2
1
Table 2
Copy numbers of SMA-related genes in 42 Chinese SMA patients determined by MLPA
Total
Gene copy number
Pattern
Group
N = 42
Normal
SMN1gene
Normal
SMN2gene
Normal
NAIPgene
Hybrid
SMNgene
Gene lacking partial sequences
A
1
3(7.1%)
0
2
1
0
0
B
2
3(7.1%)
0
2
0
0
1
a
C
3
1(2.4%)
0
1
0
1
e
1
a
D
4
1(2.4%)
0
2
1
0
1
a
E
5
1(2.4%)
0
3
1
0
1
a
F
6/7
5(11.9%)
0
3
2
0
0
G
8
2(4.8%)
0
4
2
0
0
H
9
1(2.4%)
0
2
1
0
1
a and 1
b
I
10
2(4.8%)
0
2
2
0
1
c
J
11
9(21.3%)
0
3
1
0
1
a
K
12
6(14.2%)
0
3
1
0
0
L
13
1(2.4%)
0
2
1
1
e
0
M
14
1(2.4%)
0
1
1
1
e
0
N
15
1(2.4%)
0
3
2
0
1
d
O
16
2(4.8%)
0
3
1
0
1
a and 1
d
P
17
2(4.8%)
0
2
2
0
0
Q
18
1(2.4%)
0
2
1
1
f
1
a
a:
NAIP gene lacking exon 5,
b:
SMN1 gene lacking exon7,
c:
SMN2 gene lacking exon7,
d:
SMN2 gene lacking exon 1 to exon 7,
e:Hybrid
SMN gene in which exon 8 of
SMN2 gene was converted to exon 8 of
SMN1,
f:Hybrid
SMN gene in which exon 7 of
SMN1 gene was converted to exon 7 of
SMN2.
×
Gene copy number and structure analysis of unrelated healthy individuals
Using MLPA assays, 212 healthy Chinese participants were analyzed. We found that 117 (55.2%) had normal gene copy numbers for
SMN1/
SMN2/NAIP (2/2/2) and 54 (25.5%) had different gene copies numbers for
SMN1/
SMN2 NAIP (2/1/2). The proportions of the 212 individuals with different copy numbers for the normal
SMN1 gene were 1 copy in 4 individuals (1.9%), 2 copies in 203 (95.7%) and 3 copies in 5 (2.4%). The proportions of the 212 with different gene copy numbers for normal
SMN2 were 0 copies in 10 (4.7%), 1 copy in 70 (33.0%), 2 copies in 130 (61.4%) and 3 copies in 2 (0.9%). The proportions of the 212 with different gene copies for normal
NAIP were 1 copy in 15 (7.1%) and 2 copies in 197 (92.9%). Genes lacking partial sequences were also found in healthy participants. Five (2.4%) had an
NAIP gene lacking exon 5 (pattern
a in Figure
2) and 27 (12.7%) had an
NAIP gene lacking exon 13 (pattern
g in Figure
2). One person appeared to have an
SMN1 gene lacking exon 7 (pattern
b in Figure
2) and an
SMN2 gene lacking exon 7 (pattern
c in Figure
2). Three types of hybrid
SMN genes were found in 6 of the healthy participants. Four (1.9%) had a hybrid
SMN gene in which exon 8 of
SMN2 was converted to exon 8 of
SMN1 (pattern
e in Figure
2). Two (0.9%) had a hybrid
SMN gene in which exon 8 of
SMN1 was converted to exon 8 of
SMN2 (pattern
h in Figure
2). MLPA results for 212 healthy individuals are in Table
3 and inferred gene copy numbers are in Table
4. Abnormal gene structures are in Figure
2.
Table 3
The MLPA probe copy numbers of SMA-related genes in 212 Chinese healthy individuals
MLPA probe copy number
Total
SMN1/
SMN2
SMN1
SMN2
NAIP
Group
N = 212
Exon1
Exon4
Exon6
Exon8
Exon7
Exon8
Exon7
Exon8
Exon13
Exon5
1
117(55.2%)
4-5
4-5
4
4-5
2
2
2
2
2
2
2
1(0.5%)
4
4
4
4
2
3
2
1
2
2
3
1(0.5%)
4
4-5
4
4
2
1
2
3
2
2
4
54(25.4%)
3-4
3-4
3
3-4
2
2
1
1
2
2
5
1(0.5%)
3
3
3
3
2
3
1-2
0
2
2
6
7(3.3%)
3-4
3
3-4
3-4
2
2
1
1
2
3
7
1(0.5%)
3-4
3-4
3-4
3
2
2
1
1
2
4
8
4(1.9%)
2
2
2
2
2
2
0
0
1
2
9
5(2.3%)
2
2-3
2-3
2-3
2
2
0
0
1
3
10
1(0.5%)
3
3
3-4
3-4
2
2
1
1
1
1
11
5(2.3%)
4
4-5
4
4
2
2
2
2
2
3
12
1(0.5%)
4
4-5
4
4-5
2
1
2
3
2
3
13
2(0.9%)
4
4-5
4
4-5
2
3
2
1
2
3
14
2(0.9%)
4
4
4
4
3
3
1
1
2
2
15
3(1.4%)
4
4-5
4
4-5
2
2
2
2
2
1
16
2(0.9%)
4-5
5
5
4-5
2
2
3
3
2
2
17
1(0.5%)
4
4-5
4
5
1
2
1
2
2
2
18
1(0.5%)
4-5
5
5
4-5
3
3
2
2
2
3
19
1(0.5%)
4-5
5
4-5
5
3
3
2
2
2
1
20
1(0.5%)
3
3-4
3-4
3-4
1
1
2
2
2
1
21
1(0.5%)
4
4
4
4
3
3
1
1
2
3
Table 4
Copy numbers of SMA-related genes in 212 Chinese healthy individuals determined by MLPA
Total
Gene copy number
Pattern
Group
N = 212
Normal
SMN1gene
Normal
SMN2gene
Normal
NAIPgene
Hybrid
SMNgene
Gene lacking partial sequences
A
1
117(55.2%)
2
2
2
0
0
B
2
1(0.5%)
2
1
2
1
e
0
C
3
1(0.5%)
1
2
2
1
h
0
D
4
54(25.5%)
2
1
2
0
0
E
5
1(0.5%)
2
0
2
1
e
0
F
6/7
8(3.8%)
2
1
2
0
1
g
G
8/9
9(4.2%)
2
0
1
0
1
g
H
10
1(0.5%)
2
1
1
0
0
I
11
5(2.3%)
2
2
2
0
1
g
J
12
1(0.5%)
1
2
2
1
h
1
g
K
13
2(0.9%)
2
1
2
1
e
1
g
L
14
2(0.9%)
3
1
2
0
0
M
15
3(1.4%)
2
2
1
0
1
a
N
16
2(0.9%)
2
3
2
0
0
O
17
1(0.5%)
1
1
2
0
1
b and 1
c
P
18
1(0.5%)
3
2
2
0
1
g
Q
19
1(0.5%)
3
2
1
0
1
a
R
20
1(0.5%)
1
2
1
0
1
a
S
21
1(0.5%)
3
1
2
0
1
g
a:
NAIP gene lacking exon 5,
b:
SMN1 gene lacking exon 7,
c:
SMN2 gene lacking exon 7,
e:Hybrid
SMN gene in which exon 8 of
SMN2 was converted to exon 8 of
SMN1,
g:
NAIP gene lacking exon13,
h:Hybrid
SMN gene in which exon 8 of
SMN1 was converted to exon 8 of
SMN2.
Differences in distribution of participants by SMN2 and NAIP gene copy numbers
We examined the distribution of different copy numbers for the normal
SMN2 and
NAIP genes in SMA patients and normal individuals. Distributions by copy number for normal
SMN2 were significantly different in SMA patients compared to participants without SMA (
P < 0.001) (Table
5). Distributions by copy number for normal
NAIP were significantly different for SMA patients compared to healthy participants (
P < 0.001) (Table
6).
Table 5
Comparison of the distribution ofSMN2copy number in SMA patients and healthy individuals
Number of normal
SMN2
SMA patients (N = 42)
Healthy individuals (N = 212)
P
0
0 (0%)
10 (4.7%)
1
2 (4.8%)
70 (33.1%)
2
14 (33.3%)
130 (61.3%)
<0.001
3
24 (57.1%)
2 (0.9%)
4
2 (4.8%)
0 (0%)
Table 6
Comparison of the distribution ofNAIPcopy number in SMA patients and healthy individuals
Number of normal
NAIP
SMA patients (N = 42)
Healthy individuals (N = 212)
P
0
4 (9.5%)
0 (0%)
1
26 (61.9%)
15 (7.1%)
<0.001
2
12 (28.6%)
197 (92.9%)
Discussion
In this study, the copy numbers for
SMN1,
SMN2 and
NAIP were determined in Chinese SMA patients and healthy controls using MLPA assays and plausible gene structures were inferred. We found that 37 (88.1%) people with SMA had deletions in both exon 7 and exon 8 of
SMN1 and the other 5 SMA patients (11.9%) had a single copy of
SMN1 exon 8. These results were similar to several other studies in different populations [
14,
15]. Chen et al. reported that a distribution for
SMN2 copy numbers in 94 Chinese SMA patients of 5 copies in 1 patient (1.1%), 4 copies in 24 (25.5%), 3 copies in 47 (50%) and 2 copies in 22 (23.4%). No patients with 0 or 1 copy of
SMN2 were found in their population [
16]. In addition, Qu et al. reported that a distribution for
SMN2 copy numbers in 232 Chinese SMA patients of 4
SMN2 copies in 13 patients (5.6%), 3 copies in 153(65.9%), 2 copies in 66(28.5%) and no patients having only 0 or 1 copy of
SMN2 [
8]. In our results, the proportion of SMA patients with a single normal
SMN2 copy was 4.8% and no SMA patients completely lacked copies of
SMN2. Although two SMA patients had a single normal copy of
SMN2, they also had a hybrid
SMN gene. These findings indicated that the proportion of SMA patients with 0 or 1 copy of
SMN2 was low in Chinese SMA patients. Similar to the previous report, 2 patients (4.8%) had 4
SMN2 copies, and no patients had 5 or 6
SMN2 copies. The results revealed that the proportion of SMA patients with more than 4
SMN2 copies was low in Chinese population. Most of Chinese SMA patients had 2 or 3
SMN2 copies. The distribution of normal copy numbers of
NAIP in 42 SMA patients were: homozygous deletion (9.5%), 1 copy (61.9%), and 2 copies (28.6%). This result is similar to data reported by another two studies of Chinese SMA patients [
8,
17]. Using conversions between
SMN1 and
SMN2 in exons 7 and 8, six possible hybrid
SMN genes were determined [
10]. The hybrid
SMN genes of patterns
e and
f have been reported in Chinese SMA patients [
10,
16]. In addition, four types of genes lacking partial sequences were found in SMA patients in this study. Ten SMA patients had an
NAIP gene lacking exon 5, and this type (pattern
a in Figure
2) of deletion was also found in several studies [
7,
18,
19]. Three patients had an
SMN2 lacking exon 1 to exon 7 (pattern
d in Figure
2), and two had an
SMN2 lacking exon 7 (pattern
c in Figure
2). We also inferred that one patient had an
SMN1 lacking exon 7 (pattern
b in Figure
2). For all we know, this study is the first report of three types of
SMN (pattern
b,
c,
d) lacking partial sequence.
The SMA-related genes of 212 Chinese healthy individuals were analyzed using MLPA assays. Zhu et al. reported that 1% of healthy Chinese individuals have four
SMN1 copies [
2]. However, no individuals with 4 copies of
SMN1 were found in our study. In addition, we identified 4 participants with a single normal
SMN1 copy, for a carrier frequency of 1.9% in our study. The reported frequency of SMA carriers is 2.4% in the general Chinese population [
2,
16], therefore, our carrier frequency was lower. In contrast to the SMA patients, no homozygous deletions of
NAIP were found in healthy Chinese participants. In addition to the hybrid
SMN gene
e in SMA patients, the hybrid
SMN gene in which exon 8 of
SMN1 was converted to exon 8 of
SMN2 (pattern
h in Figure
2) was found in healthy participants; this type of the hybrid
SMN gene has been reported previously [
10,
20]. Four types of genes lacking partial sequences (pattern
a,
b,
c,
g in Figure
2) were found in healthy individuals of our study. The proportion of healthy individuals (2.4%) with an
NAIP gene lacking exon 5 was lower than for SMA patients (23.8%). This result might indicate that deletion of exon 5 in
NAIP is involved in the molecular basis of SMA. Of the healthy participants, 27 (12.7%) had an
NAIP gene with an exon 13 deletion, and this type of
NAIP gene was not found in SMA patients. One SMA carrier had a ratio of 1:2 for exon 7 and 8 of
SMN1 and a ratio of 1:2 for exon 7 and 8 of
SMN2. We hypothesized that she had an
SMN1 lacking exon 7 (pattern
b in Figure
2) and an
SMN2 lacking exon 7 (pattern
c in Figure
2).
The distributions of different copy numbers for normal
SMN2 were compared between Chinese SMA patients and healthy individuals and distributions were significantly different (
P < 0.001) between SMA patients and people without SMA (Table
5). Similarly, in a study of 108 SMA patients and 22 healthy controls, Crawford et al. found that the
SMN2 copy number was significantly lower in control subjects [
21]. Thus, SMA patients appeared to be prone to having more
SMN2 copies than the controls. Because of the lack of SMN protein, fewer copies of
SMN2 decrease the likelihood of survival to birth. This might explain the difference in the distribution of people with different
SMN2 copy numbers. We also analyzed the distribution of copy numbers of normal
NAIP in SMA patients and people without SMA and found significant differences (
P < 0.001) (Table
6). Compared with healthy people, SMA patients had fewer
NAIP copies.
NAIP modifies the clinical phenotypes of SMA patients. Patients with fewer copies of
NAIP might have more severe clinical symptoms of SMA [
6,
7]. This might be why that distribution of
NAIP copies was significantly different between people with and without SMA. The
NAIP gene locus is near the
SMN1 gene locus, and both are located at 5q13.2. We determined that some SMA patients had a large deletion in the
SMN1 and
NAIP loci. This could partially explain why SMA patients had fewer
NAIP copies than people without SMA.
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Currently,
SMN1 is recognized as an SMA causing gene. In contrast, the
SMN2 and
NAIP have been characterized as a modifying factor of the clinical severity of SMA. So, the clinical diagnosis and prenatal diagnosis of SMA are mainly based on the identification of
SMN1 copy numbers and the situation of the
SMN1 gene exon7 [
22-
24]. The copy numbers of
SMN2 and
NAIP were used to evaluate the clinical phenotype of SMA patients [
25,
26]. According to our results, the differences between the SMA patients and healthy controls were not only the distribution of SMA-related genes, the gene structures were also different. Moreover, the proportions of abnormal gene structures were high in both SMA patients (36%) and healthy controls (17%). Our data revealed that only confirming the gene copy numbers may not be sufficient to the clinical diagnosis, prenatal diagnosis, phenotype evaluation and carrier screening of SMA. The abnormal gene structures should be taken into account in the clinical diagnosis of SMA. So the analyses of SMA-related gene structures were also important to the molecular diagnostics of SMA patients and carriers.
At present, most of previous studies about Chinese SMA disease only focused on SMA patients or healthy population. There were few articles about comparison and analysis of SMA-related genes and gene structures in the Chinese people with and without SMA. In this study, we compared copy numbers of
SMN1,
SMN2 and
NAIP between Chinese SMA patients and healthy controls and inferred gene structures. We summarized the subdivision of SMA-related gene patterns (Tables
2 and
4) based on the MLPA technique. The genetic characteristics would be helpful for the further identification of the clinical subdivision of SMA patients. There are limitations of our study. All gene copy numbers and gene structures were inferred from MLPA results, so we didn’t know the detailed haplotype of each participant. Therefore, further investigations are needed.
Conclusions
In conclusion, we analyzed and compared gene copy numbers and gene structures in Chinese SMA patients and healthy individuals. For the first time three types of
SMN lacking partial sequence were found. The distributions of different copy numbers for normal
SMN2 and
NAIP were significantly different between SMA patients and healthy controls. The gene structures of
SMN and
NAIP were also different between the SMA patients and healthy controls. Further studies are required to address the molecular mechanisms and clinical diagnoses of SMA.
Acknowledgements
This work was partially supported by the National Natural Science Fund of China (NSFC, No. 81101328), Pearl River S&T Nova Program of Guangzhou (No. 2013 J2200050), the Natural Science Fund of Guangdong (No. S2012040007745) and the research fund for the distinguished young teachers of Southern Medical University to Liang Li.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XMX conceived and coordinated the study. L Li designed the experiments, analyzed the data and prepared the manuscript. PF, ZYZ and L Lin performed the experiments, assisted with data analysis and manuscript preparation. WJZ and JH recruited the healthy participants. JZ, WQW, TZY, JSX and YZ recruited the SMA patients and collected the samples. All authors read and approved the final manuscript.
