Figure 1.
Pedigree of the large German family.
This figure represents the pedigree tree of the German family analysed in this study. The index patient of the family is marked by an arrow. All family members with manifest PAH are shown in black. Healthy family members have open symbols and those who were heterozygous for the identified mutation are marked with “Alu”. Those family members with hypertensive response due to exercise have half-filled symbols. Family members with unknown hemodynamic status have open symbols with a question mark inside. A question mark below the pedigree ID indicates that the genetic status in this family member is unknown. An open blue square marks the family members which presented at the beginning of the follow-up with hypertensive response due to exercise and changed their status to manifest PAH (II:12 and III:28). All family members who participated in the clinical and/or genetic analysis are marked by a star. The numbering of the individuals in the pedigree corresponds to the IDs of the family members in table 1.
Figure 2.
Genotype-phenotype correlations of Alu carriers and PASP during exercise.
This figure shows the genotype-phenotype correlation between Alu insertion and PASP during exercise, classified as HR and NR. Only patients who carried both HR and Alu insertion developed manifest PAH during follow-up. Patients with the Alu insertion in the BMPR2 gene show significantly more frequent Hypertensive response to exercise (10/13 vs. 8/25; Fishers exact test p = 0.016).
Table 1.
Clinical and genetic assessment in family members.
Figure 3.
Results of the analysis on RNA level.
A) Schematic representation of the genomic region from 5′-untranslated Region (5′-UTR) to exon 7 of BMPR2. Indicated are the identified polymorphisms (c.-212_-211insC and c.600A>C) used as markers for the analysis of the BMPR2 transcript in the index patient, the primer used for the amplification, the location of the inserted Alu element, the duplicated 13 bp region and a potential cryptic splice site (marked by a star with four spikes). B) The upper part of this figure shows the amplified product using the forward primer located in exon 5 with the reverse primer located at the beginning of exon 6. The PCR with primers F1 and R1 resulted in amplification of a single transcript with the polymorphism in exon 5 in apparently homozygous state (c.600A). The other PCR with the same forward primer in exon 5 as above but with another reverse primer located in exon 7 (R2) resulted in amplification of at least five products of different length. Product #3 corresponds to the normally spliced wild type sequence with an apparently homozygous c.600A polymorphism in exon 5; the two larger products (#1 and #2) contained a complete and partial (only left arm) incorporated AluYb8 element, respectively. Furthermore, two smaller products were identified in which the 5′ part of exon 6 (product #4) and the complete exon 6 (product #5) are missing. All aberrant spliced products (#1, 2, 4, and 5) contained apparently homozygous the C-allele of the polymorphism located in exon 5 (c.600C).
Figure 4.
Genomic analysis of the intronic region.
The genomic amplification with forward primer located in intron 5 and a reverse primer located in intron 6 resulted in two products of different length identified in several family members (shown is the result of 15 family members). Sequencing analysis of the larger products revealed the insertion of an AluYb8 element with an adjacent duplicated sequence motive in antisense orientation to BMPR2 in intron 5 (adjacent to exon 6). A schematic representation of the region and the insertion of the Alu element on one allele and the complete sequence of the Alu element, the duplicated sequence and the beginning of exon 6 are shown.