PRRX1 Loss‐of‐Function Mutations Underlying Familial Atrial Fibrillation

Figures

• 

  Figure 1. Pedigree structure and genetic mapping analysis of a 4‐generation family with atrial fibrillation. The 4‐generation family with atrial fibrillation was arbitrarily designated as family 1. Family members are recognized by generations and numbers (Roman‐Arabic numerals) given below pedigree symbols. A deceased family member is marked by a diagonal line through the symbol. A vertical bar beneath a family member means the chromosomal segment determined by genetic analysis. Microsatellite markers spanning the linkage region on chromosome 1q24.2–q25.1 are shown to the left of the pedigree, with each member’s genotypes (represented by numbers) for each marker displayed next to the chromosome bars. The filled bars denote the chromosomal segment derived from the mutation‐carrying chromosome of the affected ancestor (I‐1).

• 

  Figure 2. The PRRX1‐Gln107* mutation causally linked to atrial fibrillation. A, A representative standard 12‐lead ECG from the proband affected with atrial fibrillation. B, Sequencing chromatograms of the affected proband and an unaffected control subject, exhibiting the heterozygous PRRX1 mutation and its wild‐type control. An arrow symbol points to the heterozygous nucleotides of C/T in the affected proband (mutant type) or the homozygous nucleotides of C/C in an unaffected subject (wild type). Sequencing analysis unveiled a heterozygous mutation of c.319C>T in PRRX1, resulting in a substitution of a premature stop codon for the glutamine (Gln)–encoding codon at amino acid 107 of PRRX1 (PRRX1‐Gln107*).

• 

  Figure 3. The PRRX1‐Arg146Ile mutation responsible for atrial fibrillation. A, A representative standard 12‐lead ECG from the index patient experiencing atrial fibrillation. B, Sequencing electropherograms of the index patient and a healthy control individual, with the identified PRRX1‐Arg146Ile mutation indicated by an arrow. Sequence analysis revealed a heterozygous mutation of c.437G>T in PRRX1, leading to a transversion of arginine (Arg) into isoleucine (Ile) at amino acid 146 of PRRX1 (PRRX1‐Arg146Ile).

• 

  Figure 4. Diminished transcriptional activity of PRRX1 caused by Gln107* or Arg146Ile mutation. A, In cultured HeLa cells, PRRX1 transcriptionally activated the SHOX2 promoter in a dose‐dependent manner. B, Transactivation of the SHOX2 promoter‐driven luciferase by wild‐type PRRX1 (WT) or Gln107*‐mutant PRRX1 (Q107*) or R146I‐mutant PRRX1 (R146I), alone or in combination, showed that Gln107* or R146I had a significantly reduced transcriptional activity. C, In cultured HeLa cells, PRRX1 transcriptionally activated the ISL1 promoter in a dose‐dependent mode. D, Transactivation of the ISL1 promoter‐driven luciferase by WT or Q107* or R146I, alone or in combination, showed that Gln107* or R146I had a significantly decreased transcriptional activity. Transfection experiments were performed in triplicate, and the results are expressed as means±SD. All the letters from a to h indicate P<0.001, when compared with the same amounts of WT.

• 

  Figure 5. Decreased DNA‐binding ability of PRRX1 resulted from Gln107* or Arg146Ile mutation. A, The ability of wild‐type PRRX1 (WT), Gln107*‐mutant PRRX1 (Q107*), or R146I‐mutant PRRX1 (R146I) to bind the ISL1 promoter DNA. B, The ability of WT, Q107*, or R146I to bind the SHOX2 promoter DNA. Electrophoretic mobility shift assay showed that WT normally bound the ISL1 promoter DNA (A) or the SHOX2 promoter DNA (B), but either Q107* or R146I had a significantly decreased DNA‐binding affinity for the ISL1 promoter DNA (A) or the SHOX2 promoter DNA (B), when compared with WT.

• 

  Figure 6. Normal subcellular distribution of wild‐type and mutant PRRX1 proteins. Subcellular localizations of wild‐type PRRX1 (WT), Gln107*‐mutant PRRX1 (Q107*), and R146I‐mutant PRRX1 (R146I) were determined by immunofluorescence in HeLa cells. HeLa cells, transfected with WT, Q107*, or R146I, were stained with anti‑FLAG (red) and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). Enlarged images of FLAG and DAPI staining and merged images of HeLa cells were presented. WT, Q107*, or R146I was localized exclusively to the nuclei with normal subcellular distribution. Bar=100 μm.