Identification of survival motor neuron (SMN1) single nucleotide variant and SMN gene conversion by PCR-based DNA analysis in Thai spinal muscular atrophy (SMA) patients.
Keywords:
Spinal muscular atrophy (SMA), Survival motor neuron (SMN), Gene conversion, Long range-PCR (LR-PCR), Exonic splicing silencer (ESS)Abstract
Background Spinal muscular atrophy is a genetic disease presenting with proximal muscle weakness and atrophy of varying degrees. It is mostly caused by homozygous deletion of exon 7 in the survival motor neuron 1 (SMN1) gene and its clinical severity is inversely correlated with the copy number of the paralogous SMN2 gene. A small number of patients carry non-deletional SMN1 variants either due to the SMN1-to-SMN2 gene conversion, single nucleotide variants, small deletions, or insertions. High similarity between 2 SMN sequences hinders the accomplishment of the investigating procedure to detect the SMN1-specific single nucleotide variant. A number of laboratory methods were proposed to study the molecular variation in these SMN loci to illustrate the correlation between genotypes and clinical features but still have some limitations and difficulty in application. We therefore proposed another option, based only on a simple PCR and Sanger sequencing, and demonstrated its potential for screening the non-deletional pathogenic variants in SMN1 without requiring next-generation sequencing.
Methods Three long-range PCR (LR-PCR) together with 8 nested PCR followed by sequencing were used for screening the SMN gene conversion and intragenic variants in all coding exons of SMN1 by validating in a group of known reference DNA samples and in another group of 55 patient’s DNA samples which the SMN1 exon 7 homozygous deletion was not detected (totally 204 samples: 149 with SMN1 exon 7 homozygous deletion, 9 with one copy of SMN1 exon 7, and 46 with 2 copies of SMN1 exon 7).
Results Amplification products from both LR-PCR and nested PCR confirmed the practicability of this system to detect the SMN gene conversion and also the coding variant in SMN1. By this method, a likely pathogenic synonymous variant at the first codon of SMN1 exon 2a (NM_000344.4:c.84C>T, p.Ser28=) in heterozygosity was revealed in 2 patients (with one copy of SMN1 exon 7) and was in silico predicted to affect the normal splicing by introducing another exonic splicing silencer site into the transcript. No gene conversion and indel in SMN1 coding exons were detected in any patient’s DNA samples.
Conclusion This LR-PCR/nested PCR-sequencing combination can be another reliable molecular diagnostic tool for SMN1 variant screening.
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