Functional analysis of novel MMP21 gene compound heterozygous mutations in a prenatal case with heterotaxy

Heterotaxy is a class of congenital disorders resulting from failure to establish normal left-right asymmetry during embryonic development, which causes abnormal positioning and morphology of the thoraco-abdominal organs. The pathogenesis of heterotaxy is multifactorial and involves both genetic and environmental factors. With the application of whole exome sequencing (WES), pathogenic biallelic variants in the matrix metalloproteinase 21 (MMP21) gene have been increasingly identified in patients with heterotaxy and congenital heart defects.

In this study, two novel compound heterozygous MMP21 variants, specifically, a frame shift variant c.414del; p.(Arg139Glufs*38) and an intron variant c.980-16 T > A, were identified in a fetus diagnosed with heterotaxy through WES. In vitro assays were performed to evaluate the effects of the two variants.

Western blotting revealed that c.414del variant resulting in premature translation termination and the production of a truncated protein, which was found to completely lack the hemopexin-like repeats domain and to almost entirely lose its catalytic domain. In silico analysis and minigene assay suggested that c.980-16 T > A variant lead to aberrant splicing pattern including exon 5 skipping and 14-bp intron retention. To our knowledge, both of the two variants were reported for the first time, and the c.980-16 T > A is the first intron variant founded to affect splicing in the MMP21 gene.

Functional analysis confirmed the pathogenicity of the two variants. Our findings expand the mutational spectrum of MMP21 and provide support for the critical role of MMP21 gene in heterotaxy and congenital heart defects.