1. Mutation Spectrum of the Survival of Motor Neuron 1 and Functional Analysis of Variants in Chinese Spinal Muscular Atrophy 
Yu-jin Qu, Jin-li Bai, Yan-yan Cao, Hong Wang, Yu-wei Jin, Juan Du, Xiu-shan Ge, Wen-hui Zhang, Yan Li, Sheng-xi He, Fang Song 
The Journal of Molecular Diagnostics 
Volume 18, Issue 5, Pages (September 2016)
DOI: /j.jmoldx
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Pattern diagrams illustrating the partial conversions and partial deletions in SMN1. A: Illustration of the different variations associated with SMN1/SMN2 hybrid genes. B: The types of partial deletions in the SMN1 gene. The exons outlined with dotted lines indicate deletions. del, deletion; E, exon.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Sequence analysis of novel SMN1 variants. Variants c.-7_9del (p.0) and c.835-5T>G were identified both in g.DNA sequencing and in c.DNA. mRNA samples from patients exhibiting p.Tyr109Cys, p.Ile249Tyrfs*16, or p.Tyr272Trpfs*35 variants were not obtained; these variants were identified using g.DNA sequencing and PCR restriction fragment-length polymorphism or allele-specific PCR (data not shown). The sequencing of variant p.Ile249Tyrfs*16 was performed using the reverse primer. c.DNA, cDNA clone sequencing; g.DNA, genomic DNA sequencing.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Level of FL-SMN1 transcript in patients with SMN1 variants. The patients with subtle variants were divided into two groups according to the time of their joining the study. Transcript levels in all cases were detected and compared with those of normal controls and healthy carriers. The normal controls and healthy carrier controls were different between these two groups (normal 1 and normal 2, carrier1 and carrier2). The detected values of the second groups were labeled with yellow background. The FL-SMN1 transcript levels were measured as the number of molecules per nanogram total RNA by absolute real-time PCR as previously described.14,25 The FL-SMN1 transcript levels in the patients with the underlined variants were significantly decreased compared with healthy carriers carrying only one copy of SMN1. ∗Significant difference compared with the carrier controls (P < 0.05). FL, full-length.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Restriction digest of SMN transcripts and ex vivo splicing of the c.835-5T>G variant. A: Transcripts for FL-SMN1, FL-SMN2, and Δ7-SMN2 from the normal controls were separated by 6% polyacrylamide gel electrophoresis after digestion with DdeI. In the patient carrying the c.835-5T>G variant, the FL-SMN1 transcript (1259 bp) was difficult to visualize, but the undigested Δ7-SMN1 fragment (1205 bp, red) was more prominent, which also can be seen in his father. B: Ex vivo splicing of the three variants (c.835-5T>C, c.835-5T>G, and c.835-5T>A). This assay was reported in our previous study.19 Lane 1, wild-type SMN1 control plasmid; lane 2, c.863G>T variant (r.835_*3del, p.Gly279Glufs*5); M, indicates the DNA Marker. Lanes 3 to 5, c.835-5T>C, c.835-5T>G, and c.835-5T>A, respectively; lane 6, splicing-positive control plasmid (c.835-1G>A). Fa, father of this patient who carried this variant (two SMN1 copies); FL, full-length; Mo, mother of this patient who carried only one SMN1 copy; N, normal control; Pa, patient with c.835-5T>G.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
Functional prediction and in vitro expression of the c.-7_9del variant. A: Results of protein prediction using the ORF Finder software ( with the SMN1 mRNA sequence (NM000344). The translation of wild-type SMN1 mRNA initiates from 64 to 948 nt, coding 294 amino acid. B: Results of protein prediction for the variant c.-7_9del. Twelve proteins were predicted by the ORF Finder software using the c.-7_9del SMN1 mRNA sequence. All were much smaller than the wild-type SMN protein (only 38 to 116 amino acid). Among these, only one predicted protein (645 to 932 nt, 95 amino acid) retained a portion of the C-terminal sequence of wild-type SMN1 (661 to 948 nt, red box). The sequence of this predicted protein is MPGPRLGPGKP……EGRCSHSLN. C: In vitro expression of the FLAG-SMN1 protein from the wild-type SMN1 plasmid (WT), the p.(Tyr277Cys) mutant plasmid, and the c.-7_9del plasmid. The Western blot analysis results showed that none of the full-length and truncated FLAG-SMN fusion proteins were detected from the c.-7_9del plasmid. The empty plasmid (pFLAG-CMV-6a) was used for the mock transfection group.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. Figure 6
Schematic representation of SMN1 variants. The 18 variants shown in the upper portion are those identified to date in Chinese patients with SMA. The variants in bold denote novel variants identified in this study; the five mutations shown with underline have also been reported in other populations, and the remaining variants have only been reported in China. The two variants in the black box were the most common variants in the Chinese SMA population. The variants shown below have been reported in other populations and the horizontal arrows indicate the most common variants in the Caucasian populations. SMA, spinal muscular atrophy.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions