Identification of a novel intronic variant in COL4A2 gene associated with fetal severe cerebral encephalomalacia and subdural hemorrhage

Abstract

Background: Genetic variants in COL4A2 are less common than those of COL4A1 and their fetal clinical phenotype has not been well described to date. We present a fetus from China with an intronic variant in COL4A2 associated with a prenatal diagnosis of severe cerebral encephalomalacia and subdural hemorrhage.

Methods: Whole exome sequencing (WES) was applied to screen potential genetic causes. Bioinformatic analysis was performed to predict the pathogenicity of the variant. In in vitro experiment, the minigene assays were performed to assess the variant's effect.

Results: In this proband, we observed ventriculomegaly, subdural hemorrhage, and extensive encephalomalacia that initially suggested cerebral hypoxic-ischemic and/or hemorrhagic lesions. WES identified a de novo heterozygous variant c.549 + 5G > A in COL4A2 gene. This novel variant leads to the skipping of exon 8, which induces the loss of 24 native amino acids, resulting in a shortened COL4A2 protein (p.Pro161_Gly184del).

Conclusion: Our study demonstrated that c.549 + 5G > A in COL4A2 gene is a disease-causing variant by aberrant splicing. This finding enriches the variant spectrum of COL4A2 gene, which not only improves the understanding of the fetal neurological disorders associated with hypoxic-ischemic and hemorrhagic lesions from a clinical perspective but also provides guidance on genetic diagnosis and counseling.

Keywords: COL4A2; Encephalomalacia; Heterozygous intronic variant; Minigene splicing assays.

Fig. 1

Brain magnetic resonance imaging (MRI) of the proband. Fetal brain MRI performed at 34 weeks: (a) T2*-weighted axial image showing hypointensities (blue arrow) due to subdural hemorrhage in the left. Lateral displacement of the brain (red arrows). Brain MRI performed at 35 weeks: (b) T1-weighted axial image showing diffuse hypointensities in cerebrum parenchyma and left subdural hemorrhage. (c)T2-weighted axial image showing diffuse hypperintensities in cerebrum parenchyma and left subdural hemorrhage. Brain MRI on the Second day after birth: (d-g) Axial and sagittal images displaying extensive cerebrumencephalomalacia, dilation of lateral ventricle, and subdural hemorrhage in left

Fig. 2

Pedigree of the studied family and the COL4A2 sequencing results. (a) Pedigree of the family. ◪ represents the affected male; □ represents the normal male; ○ represents the normal female. Arrow indicates the proband. (b) The sequencing result of the family. The rectangular symbol indicates the variant site. The mutation is might a de novo variant

Fig. 3

Results of in silico assay. (a) Result of the prediction in varSEAK online consists of these in silico assays by Human Splicing Finder (HSF). (b-d) Three patterns of RNA splicing prediction. b. Pattern one: deleting 44 bp, new splicing donor, frameshift mutation, premature termination. c. Pattern two: consistent with the wild-type RNA splicing pattern. d. Pattern three: deleting 72 bp, exon skipping. The minigene results showed that our variant of interest was consistent with pattern three. E7: exon 7; E8: exon 8; E9: exon 9; I8: intron 8

Fig. 4

Functional analysis of the COL4A2 variant effect on mRNA splicing. (a) Construction strategy of pcDNA3.1 minigene vector, * representative of the variant site. (b) The sequencing of wildtype COL4A2 gene vector and c.549 + 5G > A COL4A2 variant gene vector. (c) Gel electrophoresis of reverse transcription polymerase chain reaction products displayed a single band a (estimated 410 bp) from the wild type (wt) and a smaller band b (estimated 338 bp) in the mutant type (mut). (d) Illustration of the sequencing of band a (wild type in HEK293T and Hela cells) and band b (variant c.549 + 5G > A in HEK293T and Hela cells) products lead to a shorter transcript with deletion of exon 8 including 72 bp

Fig. 5

Predicted COL4A2 protein domains showed the loss of 24aa (p.Pro161_ Gly184del) located in collagenous domain including five (Gly-X-Y) repeats within the triple helical domain. Green denotes collagen, purple denotes collagen triple helix repeat