Upstream Open Reading Frame-Introducing Variants in Patients with Primary Familial Brain Calcification

Neurology Biochemistry Genetics Life Sciences Medicine
Primary Familial Brain Calcification Genetic Variants 5' UTR Protein Expression Reporter Assay Pathogenic Variants Genetic Diagnosis

Research Background and Problem Statement

Primary Familial Brain Calcification (PFBC) is a rare neurological disorder characterized by microvascular calcification in the basal ganglia and other brain regions. Although at least six genes associated with PFBC have been identified (including SLC20A2, XPR1, PDGFB, PDGFRB, MYORG, and JAM2), the pathogenic cause remains unclear in over 50% of patients. These unexplained cases may contain cryptic variants in the non-coding regions of known genes.

Researchers hypothesized that variants in the 5’ untranslated region (UTR) might introduce AUG codons, initiating mRNA translation and leading to loss of function in some PFBC genes. To test this hypothesis, the research team re-annotated exome sequencing data from 113 PFBC-related probes and discovered two upstream AUG-introducing variants in the 5’UTR of the PDGFB gene.

Paper Source and Author Introduction

This article, titled “Upstream open reading frame-introducing variants in patients with primary familial brain calcification,” was written by Anne Rovelet-Lecrux, Antoine Bonnevalle, Olivier Quenez, and other scholars. The research team is primarily from institutions in France, including Univ Rouen Normandie, INSERM U1245, and CHU Rouen. The paper was published in the European Journal of Human Genetics in 2024, with Gaël Nicolas as the corresponding author.

Research Process

Research Steps and Subjects

  1. Patient Selection and Exome Sequencing:

    • The research team conducted a multi-center, national-level patient recruitment in France and performed exome sequencing. Selected patients met the following criteria: brain calcification exceeding age-related thresholds, and normal blood levels of parathyroid hormone, phosphorus, and calcium.

  2. Data Re-annotation and Variant Screening:

    • Using 5UTR and UTR annotation tools, the exome data of 113 PFBC patients without known pathogenic variants were re-annotated, identifying two variants in the 5’UTR of the PDGFB gene.

  3. Variant Functional Validation:

    • A GFP reporter gene system was used to evaluate the impact of variants on protein expression. Both variants were found to significantly reduce GFP protein expression levels.

  4. Clinical Gene Co-segregation Analysis:

    • Sanger sequencing was used to verify the genetic co-segregation of these variants in PFBC patient families.

Samples and Experimental Methods

  • Samples: 113 PFBC patients without known pathogenic variants.
  • Sequencing Coverage: Agilent v5, v5+UTR, and v6+UTR capture kits were used for sequencing, ensuring high coverage of the 5’UTR regions of all genes of interest.
  • Reporter Gene System:
    • HEK293 cells were transfected with GFP plasmids containing different PDGFB 5’UTR variants for in vitro experiments.
    • GFP expression levels were analyzed by Western Blot and quantitative PCR.

Main Results

Discovery of Two Pathogenic Variants

  • c.-373C>G variant: This variant showed co-segregation in the family and was predicted to form a functional upstream open reading frame (uORF), inhibiting normal PDGFB translation.
  • c.-318C>T variant: This variant was found in a simple case and predicted to cause an overlapping out-of-frame open reading frame (oORF), also significantly affecting protein expression.

Experimental Validation Results

  • Western Blot and qPCR Results: Variants c.-373C>G and c.-318C>T both significantly reduced protein levels in the GFP system.
    • Western Blot showed that the c.-318C>T variant resulted in a larger than expected GFP protein, indicating effective use of the new initiator AUG.

Conclusions and Significance

The study demonstrates that upstream AUG-introducing variants in the 5’UTR of the PDGFB gene are a potential pathogenic mechanism for PFBC. This mechanism inhibits translation or leads to the production of non-functional proteins by introducing functional uORFs and frame-shifted oORFs. This provides a new diagnostic direction for unexplained PFBC patients, emphasizing the importance of re-annotating and detecting variants in non-coding regions.

Research Highlights

  1. Innovative Hypothesis: Proposed a new mechanism of PFBC caused by AUG codon introduction in 5’UTR variants.
  2. Experimental Validation: Verified the functional impact of variants using a GFP reporter system, confirming their pathogenicity.
  3. Clinical Application Value: Provided a new direction for pathogenic gene screening in unexplained PFBC cases, alerting medical professionals not to overlook non-coding region variants.

Other Important Information

This article also emphasizes the importance of screening for variants by re-annotating existing exome sequencing data before conducting whole-genome sequencing. Although fewer coding region variants may have significant pathogenicity, uncommon non-coding region variants may also play important roles. Therefore, future research should further expand the sample size to confirm potential non-coding region pathogenic variants in other PFBC genes.