Elsevier

Brain and Development

Volume 43, Issue 7, August 2021, Pages 745-758
Brain and Development

Original article
Phenotypes of SMA patients retaining SMN1 with intragenic mutation

https://doi.org/10.1016/j.braindev.2021.03.006Get rights and content

Abstract

Background

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous deletion or intragenic mutation of the SMN1 gene. It is well-known that high copy number of its homologous gene, SMN2, modifies the phenotype of SMN1-deleted patients. However, in the patients with intragenic SMN1 mutation, the relationship between phenotype and SMN2 copy number remains unclear.

Methods

We have analyzed a total of 515 Japanese patients with SMA-like symptoms (delayed developmental milestones, respiratory failures, muscle weakness etc.) from 1996 to 2019. SMN1 and SMN2 copy numbers were determined by quantitative polymerase chain reaction (PCR) method and/or multiplex ligation-dependent probe amplification (MLPA) method. Intragenic SMN1 mutations were identified through DNA and RNA analysis of the fresh blood samples.

Results

A total of 241 patients were diagnosed as having SMA. The majority of SMA patients showed complete loss of SMN1 (n = 228, 95%), but some patients retained SMN1 and carried an intragenic mutation in the retaining SMN1 (n = 13, 5%). Ten different mutations were identified in these 13 patients, consisting of missense, nonsense, frameshift and splicing defect-causing mutations. The ten mutations were c.275G > C (p.Trp92Ser), c.819_820insT (p.Thr274Tyrfs*32), c.830A > G (p.Tyr277Cys), c.5C > T (p.Ala2Val), c.826 T > C (p.Tyr276His), c.79C > T (p.Gln27*), c.188C > A (p.Ser63*), c.422 T > C (p.Leu141Pro), c.835-2A > G (exon 7 skipping) and c.835-3C > A (exon 7 skipping). It should be noted here that some patients with milder phenotype carried only a single SMN2 copy (n = 3), while other patients with severe phenotype carried 3 SMN2 copies (n = 4).

Conclusion

Intragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers.

Introduction

Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by degeneration of anterior horn cells in the human spinal cord and subsequent loss of motor neuron [1]. SMA is clinically divided into five groups; type 0 (the most severe form with onset in the prenatal period; severe respiratory problems after birth), type 1 (Werdnig-Hoffman disease; a severe form with onset before 6 months of age; unable to sit unsupported), type 2 (Dubowitz disease; an intermediate form with onset before 18 months of age; able to sit unaided, but unable to stand or walk), type 3 (Kugelberg-Welander disease; a mild form with onset after 18 months of age; able to stand and walk unaided) and type 4 (the mildest form with age of onset from adolescence to adulthood) [2], [3].

Variations in the survival of motor neuron (SMN) genes, survival motor neuron 1 (SMN1) and survival motor neuron 2 (SMN2), are related to the development of SMA [4]. They reside on chromosome 5q13.2 as highly homologous gene copies [4]. SMN1 is now considered the disease-causing gene for SMA, because SMN1 is completely deleted in more than 95% of SMA patients and is deleteriously mutated (or carry deleterious intragenic mutation) in the remaining patients [4], [5]. SMN2 was considered as a dispensable gene because absence of SMN2 is frequently found in control individuals, but now it is considered to be a modifying factor of the SMA phenotype, because higher copy number of SMN2 may be related to milder SMA phenotypes [6], [7]. This correlation, however, is observed for homozygous SMN1 deleted patients. It is more difficult to conclude a relationship between SMN2 copy number and clinical severity in SMA patients with intragenic mutation [8]. It should also be noted here that all SMA patients, without exception, retain SMN2 [4].

The presence of SMN2 hampers detection of SMN1 mutations, because the two genes are highly homologous, differing in only five nucleotide base pairs in the region, intron 6 – exon 7 – intron 7 – exon 8 [4], [9]. To examine the presence or absence of SMN1 requires the detection of SMN1-specific nucleotides. Several assays using genomic DNA have been established for this purpose, including single-stranded conformation polymorphism (SSCP) analysis [4], restriction enzyme digestion analysis [9], and multiplex ligation-dependent probe amplification (MLPA) [10]. Even so, none of these methods can detect all SMN1 mutations that occur in SMA patients; they cannot detect intragenic SMN1 mutations.

For the detection of intragenic SMN1 mutations, two conditions need to be fulfilled: detection of a mutation in the SMN genes and assignment of the mutation to SMN1. To assign the mutation to SMN1, long-range (LR)- polymerase chain reaction (PCR) analysis of SMN1 in genomic DNA [11], [12] or reverse transcription (RT)-PCR analysis of SMN1 mRNA [13] have been used. Both methods can identify SMN1-specific nucleotides.

We have established accurate SMA diagnostic protocols, including SMN1 deletion detection and intragenic SMN1 mutation identification [8], [13], [14]. We have also established rapid SMA screening systems with filter paper for the detection of SMN1-deletion [15], [16], [17]. Rapid screening and accurate SMA diagnosis has been much more required compared to several years ago, because new effective drugs, such as nusinersen (Spinranza®), onasemnogene abeparvovec-xioi (Zolgensma®), and risdiplam (Evrysdi®) have appeared recently in the world [18], [19], [20].

In this study, we established an algorithm for SMA diagnostic procedures consisting of SMN1 deletion detection and intragenic SMN1 mutation identification, and then elucidated the relationship between clinical phenotypes and SMN2 copy number. We have detected 241 SMA patients in our laboratory, and identified 13 patients with intragenic SMN1 mutations among them. It is well-known that high SMN2 copy number modifies the phenotype of SMA patients with homozygous deletion of SMN1 [5]. However, in the patients with intragenic SMN1 mutation, the relationship between phenotype and SMN2 copy number remains unclear. Here, we compared “patients with homozygous SMN1 deletion” and “patients with intragenic mutation” in the relationship between clinical phenotypes and SMN2 copy number.

Section snippets

Patients

A total of 515 Japanese patients with suspicion of SMA were referred to our laboratory from 1996 to 2019. All patients presented with SMA-like symptoms; delayed developmental milestone, respiratory problem, muscle weakness, etc.

Prior to the genetic analysis, written informed consent was obtained from the patients or their parents. All procedures were reviewed and approved by the Ethics Committee of Kobe University Graduate School of Medicine, and were performed in accordance with the ethical

Results

This section consists of two kinds of data obtained in this study. One is comprehensive analyses of our dataset, which are described in 3.1–3.4. The other is individual analyses of five mutations identified in this study, which are described in 3.5–3.8.

Relationship between clinical phenotype and SMN2 copy number

Our previous data of SMA patient with homozygous SMN1 deletion was in complete agreement with the “inverse correlation between SMN2 copy number and clinical phenotype”, where the more SMN2 copy number, the less clinical severity [8], [26]. However, in the current study on the SMA patients with heterozygous mutations (SMN1 deletion and an intragenic SMN1 mutation), we were unable to find such an inverse correlation between SMN2 copy number and clinical phenotype.

Here, some patients with milder

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are indebted to the SMA patients who participated in this study. This research was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan, Grant No. 20K08197. Our sincere gratitude goes to Chiyo Hayashi for maintenance of the SMA database and for handling SMA patients’ samples. We thank Jeremy Allen, PhD, from Edanz Group (https://en-author-services.edanz.com/ac) for editing a draft of this manuscript.

We are also grateful to all doctors for their helpful

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