Clinical and Genetic Analysis of a Family With Sitosterolemia Caused by a Novel ATP-Binding Cassette Subfamily G Member 5 Compound Heterozygous Mutation

Abstract

Sitosterolemia (OMIM ##210250), also known as phytosterolemia, is a rare autosomal recessive disorder caused by mutations in the ATP-binding cassette subfamily G member 5 (ABCG5) or member 8 (ABCG8) genes. This leads to abnormal functions of the transporter sterolin-1 protein encoded by ABCG5 and sterolin-2 protein encoded by ABCG8, respectively, which can hinder the formation of stable ABCG5/G8 heterodimers, decreasing its ability to transport sterols. As a result, phytosterols in tissue or plasma are significantly increased, leading to early onset atherosclerosis-related diseases and xanthelasma of tendons and skin. In this study, whole exome sequencing was performed on a Chinese Han proband with sitosterolemia to capture the target gene and screen for suspected pathogenic mutations. Sanger sequencing of the family members was performed to verify the relationship between family genetics and phenotypes. The structural and functional changes in the transporter sterolin-1 protein after the responsible mutation were predicted using bioinformatics analysis. A novel compound heterozygous mutation in the ABCG5 gene (NM_022436) was identified in a proband with sitosterolemia, one of which was inherited from the father: c.296T >G (p.M99R), and one from the mother: c.-76 C >T. SIFT, Polyphen2, and Mutation Taster software predicted that p.M99R may be the responsible variant and a novel variant. RNAFold software predicts that c.-76 C >T may affect the transcriptional information or the binding of RNA binding proteins by regulating the structure of RNA, and ultimately affect gene transcription or RNA stability and translation. Swiss model software predicts that the amino acid sequence around p.M99R is highly conserved, and p.M99R leads to instability of the tertiary structure of the ABCG5/ABCG8 heterodimer. GPS 5.0 predicted that M99R affects the phosphorylation of nearby amino acid sequences, and DUET and VarSite software predicted that M99R affects the stability of sterolin-1 and cause disease. The p.M99R and c.-76 C >T mutations led to the formation of unstable heterodimers, which disturbed sterol absorption and excretion in vivo. The compound heterozygous variants c.296 T >G (p.m99r) and C.-76 C >T on exon 3 of ABCG5 in this family may be the molecular genetic basis of sitosterolemia.

Keywords: ABCG5; bioinformatics analysis; mutation; sitosterolemia; sterolin-1 protein.

FIGURE 1

(A) Family genetic pedigrees map for sitosterolemia. Blue indicates a carrier of the mutation of ABCG5(NM_022436) c.296 T >G (p.M99R) and green indicates a carrier of the mutation of ABCG5 c.-76 C >T. the arrow indicates the proband, the square indicates males, and circle indicates female. (B) The proband’s joint skin xanthoma. (D–E) Sanger sequencing showed that proband III1 carried the c.-76 C >T heterozygous variant inherited from mother (II4). (F–H) Sanger sequencing map showing that proband III1 carried the c.296T >G (p.M99R) heterozygous variant inherited from father (II3).

FIGURE 2

Prediction of the effect of the ABCG5 (NM_022436) c.296 T >G (p.M99R) variant on the tertiary structure of ABCG5. (A) Tertiary structure of the ABCG5/ABCG8 heterodimer (https://swissmodel.expasy.org/repository/uniprot/Q9H222?template=5do7). Green indicates ABCG5 and orange indicates ABCG8. The position marked by the arrow indicates ABCG5 p.M99R. (B) Prediction of changes in the tertiary structure of ABCG5 after p.M99R mutation (green) by Chimera (https://community.chocolatey.org/packages/chimera/1.15). (C) Protein structural information at the p.M99R site. Functional prediction of the ABCG5 M99R mutation (http://smart.embl-heidelberg.de/). (D) Conservation analysis of amino acid sequences near ABCG5 M99R. (E) GPS 5.0 predicts the effect of M99R on phosphorylation modifications of nearby amino acid sequences (http://gps.biocuckoo.cn/). (F) DUET predicts the stability effect of M99R on ABCG5 (http://structure.bioc.cam.ac.uk/duet). (G) Prediction of the pathogenicity of ABCG5 M99R by VarSite (https://www.ebi.ac.uk/thornton-srv/databases/VarSite). The pink areas are all pathogenic mutations, and the higher the score of the number of variants, the stronger the pathogenicity.

FIGURE 3

RNAFold (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold) predicts the secondary structure of ABCG mRNA fragments (−140 to + 17) in normal wild-type (Top) and C. − 76C >T mutations (Bottom). Arrow, mutation site; Underline, translation initiation codon; Ruler color, possibility of matching. The c.−76C >T mutation significantly weakens the matching possibility of the stem-loop structure, leading to the apparent loosening of the RNA at the initiation codon, which frees the initiation codon and improves the translation efficiency. This is similar to the translation regulation mode of riboswitch.