PMC7701169_01
Results of the mini-gene splicing assay. (A) Schematic diagram of mini-gene construction and the c.154 + 9A > G variant related abnormal splicing. The asterisk indicates the location of the c.154 + 9A > G variant. (B) Sequencing results of the target fragment of NONO-wt (upper panel) and NONO-mut (lower panel) mini-gene. (C) Gel electrophoresis of RT-PCR products: the band of the mutant was bigger than the wild-type. (D) Mini-gene product sequencing results: (a) The wild-type mini-gene (NONO-wt) formed normal mRNA composed of exons 4 and 5; (b) The mutant mini-gene (NONO-mut) caused a splicing abnormality, resulting in the retention of the 4 bp in intron 4.
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F2
The newly detected variant was predicted by HSF to insert 4 bp of intron 4 into the mature mRNA (Figure 2A). RT-PCR analysis using wild-type and mutant minigene constructs revealed that, compared with the wild-type construct, the PCR products generated by the mutant c.154 + 9A > G construct were slightly larger (Figure 2C). The amplicons confirmed by Sanger sequencing showed that the cDNA fragment obtained from the c.154 + 9A > G plasmid contained an additional 4 bp in intron 4 to exon 4 (c.154_155ins GTGT) (Figure 2D).
The echocardiography of the fetus identified aorta hypoplasia (A,B) hypoplastic left ventricle and Mitral valve dysplasia (C,D). (A) Two-dimensional echocardiography showed the thin, small ascending aorta (arrow). (B) Color Doppler echocardiography showed the narrowed aortic arch (arrows) and retrograde flow in the aortic arch. (C) Two-dimensional echocardiography showed that the thickened mitral leaflets with enhanced echo. The ratio of the right heart to the left heart was increased. (D) Color Doppler showed a limited mitral valve (arrows) opening, reduced forward flow, and reduced left ventricular filling. DA, descending aorta; LA, left atrium; LV, left ventricle; PA, pulmonary artery; RA, right atrium; RV, right ventricle; SVC, superior vena cava; T, trachea.
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F3
Detailed fetal echocardiographic examination revealed hypoplastic left ventricle, Mitral valve dysplasia, and aorta hypoplasia (Figure 3).
Sanger sequencing shows that the mutation is heterozygous in the mother.
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F4
Subsequent Sanger sequencing confirmed the heterozygous mutation in the mother (Figure 4).