Identification of two novel SMN1 point mutations associated with a very severe SMA-I phenotype

https://doi.org/10.1016/j.ejmg.2020.104006Get rights and content

Highlights

  • We report two Chinese patients presented with SMA type I.

  • The two SMA patients are both compound heterozygous, with one deleted SMN1 allele and a small mutation in the other.

  • We identified a novel missense mutation (c.280G > T, p. (Val94Phe)) and a novel splicing mutation c.*3+3A > T in SMN1 gene.

  • Our results provide important information for genetic counseling and prenatal diagnosis in these families.

Abstract

Spinal muscular atrophy (SMA) is a common autosomal recessive genetic disorder characterized by degeneration of motor neurons and weakness and muscle atrophy. Approximately 95% of SMA patients are caused by homozygous deletions of the SMN1 gene, whereas the remaining 5% of patients harbor compound heterozygous mutations such as an SMN1 deletion allele and an intragenic mutation (insertions, deletions, or point mutations) in the other SMN1 allele. Although analysis for the SMN1/SMN2 copy number is relatively easy, molecular genetic testing for patients with subtle mutations is still compromised due to the presence of a highly homologous SMN2 gene. Herein, we analyzed the SMN1/SMN2 copy number by multiplex ligation-dependent probe amplification (MLPA) and subtle mutations by long-range PCR (LR-PCR) for two “nondeletion” SMA patients. We identified a missense mutation (c.280G > T, p. (Val94Phe)) and a splicing mutation c.*3+3A > T in SMN1 gene not previously described in the scientific literature. Giving the severe phenotype of the two patients, we speculated that these two point mutations could significantly affect the function of SMN proteins. Our results provide important information for genetic counseling and prenatal diagnosis in these families and enrich the SMN1 mutation database.

Introduction

SMA is an autosomal recessive disease characterized by progressive loss of lower motor neurons, resulting in weakness and muscle atrophy (Ogino and Wilson, 2004). SMA is caused by deletion of the survival motor neuron (SMN) gene located on chromosome 5 (5q13). The incidence of the disease has been estimated at 1 in 6000–10,000 live births, with a carrier frequency of 1 in 40–60 (Ogino et al., 2004; Cusin et al., 2003).

SMN is present in two homologous copies, a telomeric SMN1 and a centromeric SMN2; the difference between these two genes is only five base pairs: one nucleotide in intron 6, one in exon 7, two in intron 7, and one in exon 8 (Wirth, 2000; Tsai et al., 2001). Both SMN genes encode the SMN protein, which has a role in pre-messenger RNA (mRNA) splicing in the anterior horn cells in the spinal cord. Although transcription of SMN1 produces full-length mRNA, the transition of cytosine-to-thymine (C-T) results in only 15% full-length SMN2 mRNA (85% of the mRNA is lacking exon 7) (Lorson et al., 1999). Approximately 95% of patients have homozygous deletions of SMN1 owing to deletion or conversion of SMN1 to SMN2. The remaining 3–5% of SMA patients retains at least one copy of SMN1 carrying insertions, deletions, or point mutations (Markowitz et al., 2012; Alias et al., 2009). Real-time polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification are used to analyze SMN1 copy number. The analysis of SMN1-derived transcripts by reverse-transcription PCR (RT-PCR) followed by clone sequencing and long-range PCR (LR-PCR) specifically amplifying SMN1 sequences can be considered to screen for intragenic SMN1 mutations in SMA patients with only one SMN1 copy (Sun et al., 2005; Yu-Jin et al., 2012; Clermont et al., 2004; Cusco et al., 2004). Generally, analysis of copy number was firstly performed for SMA patients by RT-PCR or MLPA. For SMA patients with a SMN1 copy, intragenic mutation screening was performed by LR-PCR.

In this study, we identified two novel point mutations in two chinese patients presenting with SMA symptoms who have only one copy of SMN1 using LR-PCR.

Section snippets

Ethical compliance

The study and procedures were approved by the Research Ethics Committee of Zhengzhou University. All subjects gave signed informed consent forms, and the consents of the probands 1 and 2 (<18 years) was obtained from their patents.

Samples and DNA extraction

Genomic DNA was extracted from EDTA peripheral blood samples using Lab-Aid® 824 DNA Extraction Kit according to the manufacturer's protocol (ZEESAN, Xiamen, China).

Analyses of the SMN1 copy number and intragenic mutation screening by LR-PCR

To detect the copy numbers of SMN1 and SMN2, MLPA analysis was performed using the SALSA MLPA KIT P060

Results

The proband 1 was an 11-month-old male presented to our hospital with hypotonia, muscle weakness, respiratory insufficiency, and neurogenic damage by electromyogram (EMG), and was clinically diagnosed with SMA type I. He cries low after birth, cough without power at 3 months age and have feeding problems (choking cough choking milk). He failed to acquire head control and sitting position at 6 months. A heterozygous deletions variation of exon7 and 8 in SMN1 was detected by MLPA, which inherited

Discussion

SMA is an autosomal recessive neuromuscular disorder characterized by degeneration of anterior horn cells in the spinal cord (Ogino and Wilson, 2004). On the basis of age at the onset and degree of motor disability, SMA is divided into types I (severe, Werdnig–Hoffmann, OMIM 253300), II (intermediate), III (mild) and IV (adult-onset) (Kolb and Kissel, 2011). It has been reported that mildly affected patients with SMA are partially protected by an increased SMN2 copy number (Wirth et al., 2006)

Declaration of competing interest

The authors report no relevant conflicts of interests related to the manuscript.

Acknowledgments

The authors wish to thank the subjects and family for participating in the study.

This work was supported by National Natural Science Foundation of China of China [grant number 81701125].

References (19)

There are more references available in the full text version of this article.
View full text