Background
Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders, with an incidence of 1 in 10,000 births [
1]. The disease is characterized by the degeneration of the anterior horn cells of the spinal cord, resulting in symmetrical limb muscle atrophy and weakness. SMA is classified into three clinical subtypes: Type I SMA (Werdnig-Hoffmann disease, MIM253300), Type II SMA (MIM253550) and Type III (Kugelberg-Welander disease, MIM253400) [
2]. The phenotypes of these three subtypes of SMA are based on previous reports [
3].
Previously, a clinical diagnosis of SMA is confirmed by muscle biopsy and electromyography (EMG). These procedures are time-consuming, affected by cross-talk and post-processing artefacts and yield non-conclusive results in young infants [
4,
5]. Thus, the diagnosis of SMA is usually dependent on the doctor’s experience. When a diagnosis is made, these infants often suffer from irreversible loss of neurological function. Early diagnosis of SMA can improve outcomes, and the efficacy of early diagnosis of SMA is also motivated by progress in therapeutic development [
6].
Due to the shortcomings of the traditional diagnosis of SMA, molecular diagnosis for SMA has gradually been developed in recent years. For the molecular genetics of SMA, all three clinical subtypes of SMA are associated with mutations in the survival of motor neuron (
SMN) gene, which is mapped to chromosome 5q13. Normally, this region contains one telomeric
SMN1 gene (Genbank: NG_008691.1) and one centromeric paralogue
SMN2 gene (Genbank: NG_008728.1), which differs in exon 7 at cDNA residue 840 (C for
SMN1 and T for
SMN2) [
7,
8]. Patients with a homozygous deletion of
SMN1 and a high
SMN2 copy number have a phenotype [
8‐
10] due to the small fraction of normal transcripts, which indicates that infants with a homozygous mutation on c.840 of C > T will have symptoms of SMA. Moreover, the phenotype is correlated with the exon loss of several genes in the 5q13 region, such as apoptosis inhibitory protein (
NAIP) (Genbank: NG_008724.1) and general transcription factor IIH, polypeptide 2 gene (
GTF2H2) (Genbank: NT_187651.1) [
11]. The absence of exon 4 and 5 of
NAIP may result from an unequal crossing over, leading to severe SMA [
12]. The
GTF2H2 gene and/or exon deletion might be related to the severity of the disease, but its clinical significance is still unclear [
13].
The “gold standard” to detect a single nucleotide difference is DNA sequencing, and the best method to test for the exon loss of several genes is multiplex ligation-dependent probe amplification (MLPA) [
11]. Only trace DNA can be extracted from a dry blood spot (DBS), which limits the application of DNA sequencing and MLPA during newborn screening, when is the best time to make an early diagnosis of SMA and treat in time, based on DBS being the only specimen could be obtained during newborn screening. Moreover, both of these methods are time-consuming. For restriction fragment length polymorphism (RFLP), which was established by Van’s group [
14], it is also time-consuming, taking nearly 20 h to get the result, because the procedure contains PCR and enzyme digestion; on the other hand, it is hard to judge the results in daily work only by 22 base pairs differences through electrophoresis. Multiple real-time PCR has been used to detect single nucleotide differences and exon loss in adults and newborn screening [
15,
16], which means multiple real-time PCR diagnosing SMA is very likely to be successful. Moreover, the technique is high sensitivity, specificity and rapid, so we chose it to establish a new method for SMA diagnosis. We screened and chose the best multiple pairs of primers and probes to establish the new real-time PCR method, which was fast (<3 h), cheap (<US$2), accurate, to analyse exons deletion of
SMN, NAIP and
GTF2H2 genes.
In our study, the clinical and molecular characteristics of a pediatric population with SMA from Southwest China were presented. Real-time PCR was developed to measure the gene mutation or deletion of key genes for SMA and to further analyse genotype-phenotype correlation; subsequently, 2000 cases of DBS were randomly selected for trace DNA, and real-time PCR was performed.
Discussion
Here, we established a novel real-time PCR method for detecting mutations in the
SMN,
NAIP and
GTF2H2 genes. The accuracy of this real-time PCR method for detecting the presence of a
SMN point mutation or a homozygous deletion in
NAIP,
GTF2H2 exons was at least 98.8 % compared with DNA Sanger sequencing and MLPA. Real-time PCR is a fast and an ideal method to replace DNA Sanger sequencing, multiplex PCR, and MLPA for the diagnosis of SMA and requires small amounts of DNA [
19]. Real-time PCR is also suitable for application in newborn screening for SMA, similar to Somech’s report of T cell receptor excision circles in combined T and B cell immunodeficiency [
20]. However, real-time PCR was not able to distinguish between different sub-types of SMA patients. This may be due to the correlation between the genotypes and phenotypes of SMA with hundreds of genes that are not located on chromosome 5q [
21,
22], and thus, whole genome sequencing should be further explored.
The incidence of SMA was 4.65 % (75/1613) in children with limb movement disorders in the southwest part of China. Interestingly, most of our SMA patients were classified as Type I and II (Table
1). Type I SMA children were more susceptible to congenital heart diseases and respiratory failure and were more prone to decreased fetal movement compared with Type II SMA children, consistent with previous reports [
23‐
25]. Neither the proportion of muscular atrophy abnormalities nor EMG were found in different subtypes of SMA patients.
In our SMA children, 94.67 % (71/75) displayed a homozygous
SMN1 exon 7 deletion, which was similar to the findings obtained in a previous report [
26]. However, the odds ratios of
NAIP and
GTF2H2 homozygous deletions were only 12 % and 4 %, respectively, among SMA patients, which was much lower than the report in east China [
11], indicating the different inheritance characteristics for SMA in southwest China. In our study, there was no heterozygous mutations or deletions of
SMN,
NAIP and
GTF2H2 found in SMA patients. Indeed, the multiplex real-time PCR detected a
SMN heterozygous exon 7 deletion, as shown in Fig.
1a. In addition, real-time PCR detected heterozygous deletions of the
NAIP or
GTF2H2 gene when a normal sample with no deletion of the genes was used as an internal control and an equal amount of DNA from patient was amplified (data not shown). Not only patients with DMD/BMD or ME and normal children, as shown in Table
4, but also 20 pairs of the parents of SMA patients (data not shown), who had no symptom, were found to have heterozygous mutations of
SMN1,
NAIP and
GTF2H2. Some of them had no clinical symptom and some of them had clinical symptoms not related to mutations of
SMN1,
NAIP and
GTF2H2. There was no clinical significance for heterozygous mutations of
SMN1,
NAIP and
GTF2H2.
Among the Type I SMA patients, the development of SMA was much faster in patients with a homozygous deletion in NAIP or GTF2H2 compared to patients without this type of deletion. For the 11 Type I SMA patients, 3 patients demonstrated decreased fetal movement during pregnancy. Among these 3 patients, the patient with a homozygous deletion in exon 4 of NAIP had a poor prognosis (the child died within the first 2 months). The amount of samples was too small to obtain a conclusion that decreased fetal movement may signal a bad prognosis for SMA patients. Thus, more cases of SMA children should be further investigated.
The relationship between the prognosis of patients and SMN2 copy number in our study was different from others [
11,
27]. There were no significant differences between the possibility of developing to different sub-types of SMA and
SMN2 copy numbers in our study according to Table
5, whereas an inverse relationship between
SMN2 copy number and possibility of developing to Type I SMA in He J’s research. Maybe different genetic background result in this different prognosis comparing with He J’s research. The onset age of most of our type I SMA patients (38/41, 92.7 %) was <2 months and 72.4 % (21/29) of type II SMA patients carried more than 3 copies of
SMN2, whereas the onset age of Qu’s type I patients was 31.1 % (33/106) and copies of
SMN2 of Qu’s type II patients was 96 %. As Qu’s research, the clinical and genetic characters of our patients were more severe, leading to worse survival rate.
Only one DBS-positive sample, whose ID number was NS-13050012, was analysed in our study. The doctor diagnosed that the baby suffered from Type I SMA using DNA sequencing and MLPA. The child was nursed as a Type I SMA patient and was still alive until September 2014, with no obvious symptoms except non-sitting. If we performed the newborn screening for SMA for newborn babies, children with SMA will benefit from being diagnosed as Type I SMA at an early age and can begin an early specific nursing program. Thus, their prognosis will improved, similar to the child in our study.
There are four main methods to detect SMA: Sanger DNA sequencing, MLPA, RFLP and multiple routine PCR. No one was used in newborn screening. Sanger DNA sequencing and MLPA are both time consuming (>2 weeks), expensive (>US$21) and high amount of DNA required (amount of DNA is trace in newborn screening); RFLP is also time consuming (20 h) and not able to detect exons deletion of NAIP and GTF2H2; multiplex routine PCR is not able to detect exon 7 deletion of SMN1 and it is more subjective because of being judged by electrophoresis gel image; even the method of RFLP with multiplex routine PCR is also time consuming and subjective to judge results. Our new approach is fast (<3 h), cheap (<US$2), objective and able to analyse exons deletion of SMN, NAIP and GTF2H2 genes simultaneously with trace DNA.
According to Prior’s and Stabley’s studies [
28,
29], there are several limitations of real-time PCR. At the same time, digital PCR (dPCR) is able to detect SMN mutation. Moreover, for the coefficients of variation, dPCR is even better than real-time PCR.
For limitations of real-time PCR, (1) we recommended eathylene diamine tetraacetic acid (EDTA) as anticoagulant for periphery blood sample, avoiding heparin for its inhibiting Taq polymerase activity. Our method detected more than 200 EDTA peripheral blood and 2000 DBS samples, and the amplification efficiency was excellent, so the compounds in specimen did not shown inhibition; (2) it is possible that DNA sequence variants located under the primer binding sites may be a problem to influence the results. To avoid the question, we need to add another pair of primers to cover outside of binding sites of the primers we used to amplify samples in future; (3) for data analysis, repeating reactions twice and evaluating the capability of equipment could ensure the quality of results to a certain extent.
The recent new method of dPCR is suitable and promising for detecting SMN mutation. However, the ability of dPCR to simultaneously detect NAIP and GTF2H2 mutation and for DBS is not available and need further evaluation. Moreover, dPCR is more expensive (>US$15) in China and specific equipment is required. In a word, the Real-time PCR for SMN, NAIP and GTF2H2 is cheaper and feasibility for clinical usage at present in China.
Acknowledgements
We thank all patients and guardians involved in the study.