Literature Analysis: A Novel Mechanism of PFIC Caused by SEMA7A R148W Mutation Revealed by Multiplex Fluorescence IHC

1. Research Overview

In September 2021, Professor Jin Chai's team from Southwest Hospital, Army Medical University, published an online article entitled "A homozygous R148W mutation in Semaphorin 7A causes progressive familial intrahepatic cholestasis" in the international journal EMBO Molecular Medicine (Impact Factor: 12.137). This study for the first time proposed that a homozygous R148W mutation in Semaphorin 7A (SEMA7A) causes Progressive Familial Intrahepatic Cholestasis (PFIC) and elaborated on its underlying mechanism. Notably, the 6-Color Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50030) from the Absin product line of ANT BIO PTE. LTD. was a key technical tool employed in this research, providing reliable support for the accurate detection of target proteins.

2. Background of Progressive Familial Intrahepatic Cholestasis (PFIC)

Progressive Familial Intrahepatic Cholestasis (PFIC) is an autosomal recessive hereditary hepatocellular cholestasis syndrome caused by gene mutations that impair bile secretion or excretion. It is characterized by severe pruritus and predominantly affects children. Generally, hepatocellular primary cholestasis is present in the neonatal period, and patients often die of liver failure before adulthood. Due to the lack of clear understanding of its pathogenesis, effective therapeutic strategies are limited, making in-depth research on PFIC's genetic and molecular mechanisms crucial for improving clinical diagnosis and treatment.

3. Research Process and Key Findings

3.1 Discovery of the SEMA7A R148W Mutation

The major discovery originated from a female infant with PFIC. She exhibited prolonged jaundice and flatulence in the first two weeks after birth, recovered after treatment without sequelae. However, subsequent liver function tests showed abnormally high serum levels of ALT, AST, and TBA. Further medical examinations ruled out various known liver diseases, prompting researchers to suspect PFIC—a genetic intrahepatic cholestasis. After screening for mutations in known PFIC-related genes in the child, a homozygous (S267F) mutation was found in the sodium/taurocholate cotransporting polypeptide (NTCP/SLC10A1) of the patient. Although changes in the functions of NTCP and SLC10A1 may cause symptoms similar to those of the patient, the former does not cause liver damage, and the latter did not show elevated serum ALT, AST, and TBA levels in mouse models. Thus, this cause was excluded.

To identify the real cause, the research team continued to focus on genetic factors, using various genetic models to screen Whole-Genome Sequencing (WGS) data. Finally, they identified a potential missense variant (p.R148W) in the SEMA7A gene. Further genetic testing confirmed variations in the genomic organization, DNA, and protein of SEMA7A in the patient

Subsequently, the research team established a Sema7a homozygous mutant mouse model. Compared with wild-type mice and heterozygous mice, the homozygous mice showed elevated serum ALT, AST, and TBA levels at 4 and 8 weeks of age, accompanied by liver damage—findings highly similar to those in patients with the Sema7a homozygous mutation.

3.2 Abnormal Expression of Bsep and Mrp2 Proteins in Mutant Mice

Since PFIC is an intrahepatic cholestasis, the research team performed LC-MS/MS analysis on liver extracts from the mouse model. The results showed that the levels of markers indicating impaired hepatic BA excretion were significantly increased in the livers of Sema7a homozygous mutant mice. Further detection of mRNA transcripts of canalicular membrane bile acid transporters, basolateral membrane bile acid transporters, and bile acid synthases, as well as Western blot analysis of each protein, revealed that the expressions of Bsep and Mrp2 were particularly noteworthy. The expression levels of these two proteins in the homozygous mouse group were significantly lower than those in the wild-type and heterozygous groups, but the levels of their mRNA transcripts were not downregulated

3.3 Validation by Multiplex Fluorescence IHC Technology

To further confirm the expression of Bsep and Mrp2, the research team employed multiplex fluorescence immunohistochemistry technology, using the 6-Color Multiplex Fluorescence IHC Staining Kit (abs50030) from the Absin product line of ANT BIO PTE. LTD. Both the detection of mouse liver tissues and primary hepatocytes cultured in vitro by the collagen sandwich method showed that the contents of Bsep and Mrp2 in the tissues and cells of Sema7a homozygous mutant mice were lower than those in wild-type mice

Primary mouse hepatocytes in collagen sandwich culture were transfected with SEMA7A_WT or SEMA7A_R148W constructs at different transfection doses, followed by multiplex staining. The results showed that the Sema7a mutation could reduce the expression of hepatic Bsep and Mrp2 proteins in a dose-dependent manner—higher transfection doses led to lower expressions. The decreased expressions of these two key proteins ultimately resulted in intrahepatic cholestasis (Figure 4).

4. Research Images

Figure 1. Genetic variation analysis of SEMA7A in patients. The images show the genomic organization, DNA, and protein variations of SEMA7A in patients with the Sema7a homozygous mutation.

Western blot results of bile acid transporter proteins. The expression levels of Bsep and Mrp2 in homozygous mice are significantly lower than those in wild-type and heterozygous mice, while their mRNA transcript levels are not downregulated. (Wild-type: Wild type; Heterozygote: Heterozygous; Homozygote: Homozygous)

5. Research Significance

This study is the first to identify the SEMA7A R148W homozygous mutation as a novel cause of PFIC, revealing the underlying mechanism by which this mutation leads to intrahepatic cholestasis—specifically, reducing the expression of Bsep and Mrp2 proteins in a dose-dependent manner without affecting their mRNA transcription. These findings not only deepen the understanding of the genetic and molecular mechanisms of PFIC but also provide new targets for the clinical diagnosis and treatment of PFIC. Additionally, the successful application of ANT BIO PTE. LTD.'s multiplex fluorescence IHC technology in this research highlights its important role in validating protein expression and localizing target molecules in complex disease models.

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8. Disclaimer

This article is AI-compiled and interpreted based on the original work in document 1225.docx (IF 12——PFIC New Mechanism Proposal and Research Progress). All intellectual property (e.g., images, data) of the original publication shall belong to the journal EMBO Molecular Medicine and Professor Jin Chai's research team. For any infringement, please contact us promptly and we will take immediate action.

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