Pedigree of Family 2.

<p>The index patient of Family 2 (III:1, arrow) carries the <i>BMPR2</i> promoter mutation c.-669G>A [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133042#pone.0133042.ref021" target="_blank">21</a>] and a unclassified variant in the <i>ENG</i> gene (c.1633G>A, p.(G545S)). Mut: mutation, UV: unknown variant, UTR: untranslated region, WT: wild type, X 11: exon 11 <i>ENG</i>. The horizontal line separates the two loci in <i>BMPR2</i> promoter and <i>ENG</i> exon 11.</p

Clinical characteristics of index patients at diagnosis.

<p>Clinical characteristics of index patients at diagnosis.</p

Clinical characteristics of family members.

<p>*Mean is based on 16 individuals; while maximal blood pressure, peak VO₂, EqCO₂, VO₂ at anaerobic threshold and oxygen pulse are based on 10 individuals</p><p>EqCO₂: ventilatory equivalent for carbon dioxide, max: value at maximal workload, VO₂/kg: oxygen consumption/kg.</p><p>Clinical characteristics of family members.</p

N-Terminal Pro-Brain Natriuretic Peptide Is a Useful Prognostic Marker in Patients with Pre-Capillary Pulmonary Hypertension and Renal Insufficiency

<div><p>N-terminal pro-brain natriuretic peptide (NT-proBNP) is a routinely used prognostic parameter in patients with pre-capillary pulmonary hypertension (PH). As it accumulates in the presence of impaired renal function, the clinical utility of NT-proBNP in PH patients with concomitant renal insufficiency remains unclear. In a retrospective approach, patients with pre-capillary PH (group I or IV) and concomitant renal insufficiency at time of right heart catheterization (glomerular filtration rate (GFR) ≤60 ml/min/1.73 m²) were identified out of all prevalent pre-capillary PH patients treated at a single center. Forty patients with renal insufficiency (25.8%) were identified and matched regarding hemodynamic parameters with a control group of 56 PH patients with normal renal function (GFR >60 ml/min/1.73 m²). Correlations of NT-proBNP levels with hemodynamic and prognostic parameters (time to clinical worsening and overall survival) were assessed. Overall, GFR correlated inversely with NT-proBNP and had the strongest influence on NT-proBNP levels in a stepwise multiple linear regression model including hemodynamic parameters and age (r² = 0.167). PH patients with renal insufficiency had significant higher levels of NT-proBNP (median: 1935 ng/l vs. 573 ng/l, p = 0.001). Nevertheless, NT-proBNP correlated with invasive hemodynamic parameters in these patients. Using higher cut-off values than in patients with preserved renal function, NT-proBNP levels were significantly associated with time to clinical worsening (>1660 ng/l, p = 0.001) and survival (>2212 ng/l, p = 0.047) in patients with renal insufficiency. Multivariate Cox’s proportional hazards analysis including established prognostic parameters, age and GFR confirmed NT-proBNP as an independent risk factor for clinical worsening in PH patients with renal insufficiency (hazard ratio 4.8, p = 0.007). Thus, in a retrospective analysis we showed that NT-proBNP levels correlated with hemodynamic parameters and outcome regardless of renal function. By using higher cut-off values, NT-proBNP seems to represent a valid clinical marker even in PH patients with renal insufficiency.</p></div

Overall Survival.

<p>Receiver operating characteristic (ROC) analysis to determine the cut-off value in patients with renal insufficiency (defined as glomerular filtration rate (GFR) ≤60 ml/min/1.73 m²; <b>A</b>). In Kaplan-Meier analysis, higher levels of n-terminal pro-brain natriuretic peptide (NT-proBNP) were significantly associated with poor survival (<b>B;</b> p = 0.047, log-rank).</p

Patients’ characteristics.

<p>Data are presented as mean ± SD or numbers.</p><p>Comparison of means between GFR-groups are performed by Student’s T, Mann-Whitney-U^& or Chi-Square test^§. CTD = connective tissue disease. ^# Other includes HIV, porto-pulmonary hypertension and congenital heart diseases.</p

Glomerular filtration rate (GFR).

<p>Distribution of GFR (ml/min/1.73 m²) among patients’ groups. Mean GFR was 45±11 ml/min/1.73 m² (range: 10 to 58 ml/min/1.73 m²) and 78±14 ml/min/1.73 m² (ranging from 61 to 136 ml/min/1.73 m²).</p

Multivariate Cox’s proportional hazards analysis assessing the predictive value of n-terminal pro-brain natriuretic peptide (NT-proBNP) levels on clinical worsening in 40 PH patients with concomitant renal insufficiency (defined as glomerular filtration rate (GFR) ≤60 ml/min/1.73 m²) in a model with further established non-invasive parameters, age and renal function.

<p>Median of age, 6MWD and GFR in patients with renal insufficiency were used as cut-off values.</p

Time to clinical worsening (TTCW).

<p>Receiver operating characteristic (ROC) analysis to determine the cut-off value in patients with renal insufficiency (defined as glomerular filtration rate (GFR) ≤60 ml/min/1.73 m²; <b>A</b>). In Kaplan-Meier analysis, higher levels of n-terminal pro-brain natriuretic peptide (NT-proBNP) were significantly associated with early clinical worsening (<b>B;</b> p = 0.001, log-rank).</p

Genomic analysis of the intronic region.

<p>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 <i>BMPR2</i> 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.</p