Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population

Abstract

Screening for carriers of spinal muscular atrophy (SMA) is necessary for effective clinical/prenatal diagnosis and genetic counseling. However, a population-based study of SMA prevalence in mainland China has not yet been conducted. In this study, the copy number of survival motor neuron (SMN) genes was determined in 1712 newborn cord blood samples collected from southern China and from 25 core families, which included 26 SMA patients and 44 parents, to identify SMA carriers. The results presented 13 groups with different SMN1/SMN2 ratios among 1712 newborn individuals, which corresponded to 1535 subjects with two copies of SMN1, 119 with three copies of SMN1, 17 with four copies of SMN1, and 41 with a heterozygous deletion of SMN1 exon 7. Simultaneously, two '2+0' genotypes and two point mutations were found among the 44 obligate carriers in the core families, including a novel SMN1 splice-site mutation that was identified in the junction between intron 6 and exon 7 (c. 835-1G>A). These results indicated that the carrier frequency is 1/42 in the general Chinese population and that duplicated SMN1 alleles and de novo deletion mutations are present in a small number of SMA carriers. In addition, we developed and validated a new alternative screening method using a reverse dot blot assay for rapid genotyping of deletional SMA. Our research elucidated the genetic load and SMN gene variants that are present in the Chinese population, and could serve as the basis for a nationwide program of genetic counseling and clinical/prenatal diagnosis to prevent SMA in China.

Figure 1

Diagnostic flow chart for targeted detection of SMN1 and SMN2 gene dosages linked to SMA in a population-based study. The sample numbers detected at each step are indicated in brackets. ^*Some samples with abnormal peaks screened by DHPLC were sequenced to distinguish point mutations or SNPs.

Figure 2

Representative DHPLC profiles of single/multiplex PCR analysis for different SMN1/SMN2 ratios. ^*Female individuals with SMN1/SMN2=1:1;^**male individuals with various SMN1/SMN2 ratios.

Figure 3

Representative results for RDB detection of different SMN1/SMN2 ratios. (a) Typing of a normal individual with SMN1/SMN2=2:N; (b) typing of an SMA carrier with SMN1/SMN2=1:N; (c) typing of a normal individual with SMN1 only; (d) typing of SMA patients with SMN1/SMN2=0:N.

Figure 4

SMN analysis for a family carrying the novel SMN1 splice-site mutation c.835–1G>A. (a) Pedigree and SMN copy numbers for the patient's family. (b) RT-PCR amplification of SMN gene exons 5–8 to determine SMN1/ SMN2 expression in the RNA sample obtained from the proband's father. The transcript product for Δexon7 was 294 bp and the normal transcript product including SMN1 exon 7 was 348 bp in length. M: DNA molecular weight marker; lanes 1 and 2: normal individuals; lanes 3 and 4: the father carried the SMN1 splice-site mutation at the junction between intron 6 and exon 7 (c.835–1G>A). (c) RDB analysis of the SMN gene in the proband and his father. The DNA fragment hybridized with the probe for SMN1 exon 7 contained the site with the splice-site mutation c.835G>A, which provided a negative result by the RDB assay for the proband (panel b). However, the father was an SMA carrier with one normal SMN1 gene, which provided a positive result by the RDB assay (panel a). (d) Quantitative DHPLC analysis of the SMN1/SMN2 gene dosage in the proband and his father. (Panel a) The father exhibited a gene ratio of SMN1/SMN2=2:2; (panel b) the proband exhibited a gene ratio of SMN1/SMN2=1:3. (panel e) DNA sequencing of the SMN1 gene carried by the proband and his father. (Panel a) Sequence of genomic DNA containing SMN gene intron 6 and exon 7 from the father; (panel b) sequence of the genomic DNA containing SMN gene intron 6 and exon 7 from the proband. The downward arrow indicates a G → A mutation at the acceptor splice site of intron 6.