Lora K. Hedges

Alterations in oestrogen metabolism: implications for higher penetrance of familial pulmonary arterial hypertension in females

Mutations in bone morphogenetic protein receptor type 2 (BMPR2) cause familial pulmonary arterial hypertension (FPAH), but the penetrance is reduced and females are significantly overrepresented. In addition, gene expression data implicating the oestrogen-metabolising enzyme CYP1B1 suggests a detrimental role of oestrogens or oestrogen metabolites. We examined genetic and metabolic markers of altered oestrogen metabolism in subjects with a BMPR2 mutation. Genotypes for CYP1B1 Asn453Ser (N453S) were determined for 140 BMPR2 mutation carriers (86 females and 54 males). Nested from those subjects, a case–control study of urinary oestrogen metabolite levels (2-hydroxyoestrogen (2-OHE) and 16α-hydroxyoestrone (16α-OHE1)) was conducted in females (five affected mutation carriers versus six unaffected mutation carriers). Among females, there was four-fold higher penetrance among subjects homozygous for the wild-type genotype (N/N) than those with N/S or S/S genotypes (p = 0.005). Consistent with this finding, the 2-OHE/16α-OHE1 ratio was 2.3-fold lower in affected mutation carriers compared to unaffected mutation carriers (p = 0.006). Our findings suggest that variations in oestrogens and oestrogen metabolism modify FPAH risk. Further investigation of the role of oestrogens in this disease with profound sex bias may yield new insights and, perhaps, therapeutic interventions.

Gross BMPR2 gene rearrangements constitute a new cause for primary pulmonary hypertension

Purpose: Approximately 50% of patients with familial primary pulmonary hypertension (FPPH) have been reported to have mutations within the bone morphogenic protein receptor type 2 (BMPR2) gene. The vast majority of these mutations were identified by PCR amplification and sequencing of individual exons. The aim of our study was to determine if additional BMPR2 mutations not found by exon sequencing alone could account for a significant portion of these negative cases.Methods: We examined DNA samples from 12 families, previously found to be negative for BMPR2 mutations, to identify any large BMPR2 gene rearrangements.Results: Southern blot analysis found large gene rearrangements in four (33%) unrelated kindreds. Further analysis by reverse transcriptase PCR (RT-PCR) of BMPR2 transcripts from two of these kindreds found one to be heterozygous for a exon 10 duplication and the second to be heterozygous for a deletion of exons 4 to 5. Nonhomologous recombination is believed to be the cause of these large insertions/deletions.Conclusion: Our results demonstrate the inherent problems associated with exon-by-exon sequencing and the importance of other screening methods such as Southern blot and RT-PCR in the identification of BMPR2 mutations.

Comparison of Chromosome Telomere Integrity in Multiple Tissues from Subjects at Different Ages

Telomere DNA, at the ends of each chromosome, is conserved in nature and required for chromosome replication and stability. Reduction in telomere length has been observed in several malignancies as well as in leukocytes from healthy persons with advancing age. There is a paucity of data regarding telomere length and the effects of in vivo aging in different tissues. These data could be helpful in interpreting telomere length and understanding the role of telomere integrity and telomerase activity in malignant cells. We report telomeric DNA integrity studies of blood and skin collected from eight Caucasians of both sexes representing each decade of life from the fetus to 72 years of age without exposure to chemotherapy or radiation. In addition, telomeric data from 15 other tissues from the fetus and 8 other tissues from the 72-year-old male were examined. No significant differences were found in the shortest telomere size, the average telomere size, or telomere size variation between blood and skin from subjects at different ages. The average telomere size was 11.7 +/- 2.2 kb for blood and 12.8 +/- 3.7 for skin in all subjects studied. The shortest telomere length was 5.4 +/- 1.9 kb for blood and 4.3 +/- 0.9 kb for skin. Significant differences (P < 0.001) were found in the overall length of the DNA hybridization signal representing the shortest telomere size and the length of the DNA peak migration hybridization signal representing variation in telomere size between the 20-week fetus and the 72-year-old male. The 72-year-old male showed the shortest telomeres and the most variation (heterogeneity) in telomere size for all tissues studied, but the greatest differences were observed in blood compared with other tissues (e.g., average telomere length was 12.2 kb in the fetus and 7.2 kb in the 72-year-old male). The size of the telomere was negatively correlated with age for all tissues studied.

Penetrance of pulmonary arterial hypertension is modulated by the expression of normal BMPR2 allele.

Familial pulmonary arterial hypertension (FPAH) is a progressive, fatal disease caused by mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2). FPAH is inherited as an autosomal dominant trait, and shows incomplete penetrance in that many with BMPR2 mutations do not develop FPAH, suggesting a role for, as yet unidentified, modifier genes in disease penetrance. We hypothesized that variable levels of expression of the wild‐type (WT) BMPR2 allele could act as a modifier and influence penetrance of FPAH. WT BMPR2 levels were determined by real‐time PCR analysis in lymphoblastoid (LB) cell lines derived from normal controls and individuals with FPAH. The FPAH kindreds analyzed carried mutations that result in the activation of nonsense‐mediated decay (NMD) pathway, which leads to the degradation of the mutant RNA, thus ensuring that only the WT BMPR2 transcripts will be detected in the real‐time assay. Our data show that WT and mutant BMPR2 levels can be reproducibly measured in patient‐derived LB cell lines, and that unaffected mutation carrier‐derived LB cell lines have higher levels of WT BMPR2 transcripts than FPAH patient‐derived LB cell lines (p≤0.005). Our findings suggest that the levels of expression of WT BMPR2 allele transcripts is important in the pathogenesis of FPAH caused by NMD+ mutations. Furthermore, our study illustrates a novel application of lymphoblastoid cell lines in the study of PAH, especially important because the affected site, that is, the lung, is not available for unaffected mutation carriers. Hum Mutat 0,1–6, 2009.

Microsatellite Instability in Malignant Melanoma

Defective mismatch repair has been detected in human colorectal and endometrial carcinomas which exhibit microsatellite instability (MIN). The purpose of this study was to search for MIN in melanoma. Paraffin-embedded neoplastic and non-neoplastic control cells were obtained from 20 untreated individuals with cutaneous malignant melanoma. Breslow thickness ranged from 0.2-7.4 mm (mean 1.4). Cells were carefully scraped from glass slides so that tumor and control DNA could be isolated and then amplified by polymerase chain reaction (PCR) at seven separate microsatellites localized to specific chromosome regions: 1p22 (D1S187), 5q11.2-13.3 (D5S107), 6q21-23.3 (D6S357), 9p21 (IFNA), 11p15.2 (D11S861), 17p13.1 (D17S786), and 18q11 (D18S34). Heterozygosity indices were > or = 0.70. Loci from these chromosome regions were chosen because of cytogenetic abnormalities reported in melanoma (1p, 6q, 9p), location of common oncogenes (11p-HRAS, 17p-TP53), or use in other MIN studies (5q, 18q). Five individuals (25%) demonstrated MIN. There was no correlation with tissue thickness. One individual demonstrated MIN at two loci and one individual demonstrated loss of heterozygosity. The results indicate that MIN occurs in melanoma, albeit less frequently than reported in carcinomas.

Estrogen Metabolite 16α-Hydroxyestrone Exacerbates Bone Morphogenetic Protein Receptor Type II–Associated Pulmonary Arterial Hypertension Through MicroRNA-29–Mediated Modulation of Cellular Metabolism

Background— Pulmonary arterial hypertension (PAH) is a proliferative disease of the pulmonary vasculature that preferentially affects women. Estrogens such as the metabolite 16&agr;-hydroxyestrone (16&agr;OHE) may contribute to PAH pathogenesis, and alterations in cellular energy metabolism associate with PAH. We hypothesized that 16&agr;OHE promotes heritable PAH (HPAH) via microRNA-29 (miR-29) family upregulation and that antagonism of miR-29 would attenuate pulmonary hypertension in transgenic mouse models of Bmpr2 mutation. Methods and Results— MicroRNA array profiling of human lung tissue found elevation of microRNAs associated with energy metabolism, including the miR-29 family, among HPAH patients. miR-29 expression was 2-fold higher in Bmpr2 mutant mice lungs at baseline compared with controls and 4 to 8-fold higher in Bmpr2 mice exposed to 16&agr;OHE 1.25 &mgr;g/h for 4 weeks. Blot analyses of Bmpr2 mouse lung protein showed significant reductions in peroxisome proliferator–activated receptor-&ggr; and CD36 in those mice exposed to 16&agr;OHE and protein derived from HPAH lungs compared with controls. Bmpr2 mice treated with anti–miR-29 (20-mg/kg injections for 6 weeks) had improvements in hemodynamic profile, histology, and markers of dysregulated energy metabolism compared with controls. Pulmonary artery smooth muscle cells derived from Bmpr2 murine lungs demonstrated mitochondrial abnormalities, which improved with anti–miR-29 transfection in vitro; endothelial-like cells derived from HPAH patient induced pluripotent stem cell lines were similar and improved with anti–miR-29 treatment. Conclusions— 16&agr;OHE promotes the development of HPAH via upregulation of miR-29, which alters molecular and functional indexes of energy metabolism. Antagonism of miR-29 improves in vivo and in vitro features of HPAH and reveals a possible novel therapeutic target.

Role of BMPR2 Alternative Splicing in Heritable Pulmonary Arterial Hypertension Penetrance

Background— Bone morphogenic protein receptor 2 (BMPR2) gene mutations are the most common cause of heritable pulmonary arterial hypertension. However, only 20% of mutation carriers get clinical disease. Here, we explored the hypothesis that this reduced penetrance is due in part to an alteration in BMPR2 alternative splicing. Methods and Results— Our data showed that BMPR2 has multiple alternative spliced variants. Two of these, isoform-A (full length) and isoform-B (missing exon 12), were expressed in all tissues analyzed. Analysis of cultured lymphocytes of 47 BMPR2 mutation–positive heritable pulmonary arterial hypertension patients and 35 BMPR2 mutation–positive unaffected carriers showed that patients had higher levels of isoform-B compared with isoform-A (B/A ratio) than carriers (P=0.002). Furthermore, compared with cells with a low B/A ratio, cells with a high B/A ratio had lower levels of unphosphorylated cofilin after BMP stimulation. Analysis of exon 12 sequences identified an exonic splice enhancer that binds serine arginine splicing factor 2 (SRSF2). Because SRSF2 promotes exon inclusion, reduced SRSF2 expression would mean that exon 12 would not be included in final BMPR2 mRNA (thus promoting increased isoform-B formation). Western blot analysis showed that SRSF2 expression was lower in cells from patients compared with cells from carriers and that siRNA-mediated knockdown of SRSF2 in pulmonary microvascular endothelial cells resulted in elevated levels of isoform-B compared with isoform-A, ie, an elevated B/A ratio. Conclusions— Alterations in BMPR2 isoform ratios may provide an explanation of the reduced penetrance among BMPR2 mutation carriers. This ratio is controlled by an exonic splice enhancer in exon 12 and its associated splicing factor, SRSF2.Background Bone morphogenic protein receptor 2 (BMPR2) gene mutations are the most common cause of heritable PAH (HPAH). However only 20% of mutation carriers get clinical disease. Here we explored the hypothesis that this reduced penetrance is in part due to an alteration in BMPR2 alternative splicing.

High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression profiling of peripheral blood mononuclear cells

High-altitude pulmonary hypertension (HAPH) is a consequence of chronic alveolar hypoxia, leading to hypoxic vasoconstriction and remodeling of the pulmonary circulation. Brisket disease in cattle is a naturally occurring animal model of hypoxic pulmonary hypertension. Genetically susceptible cattle develop severe pulmonary hypertension and right heart failure at altitudes >7,000 ft. No information currently exists regarding the identity of the pathways and gene(s) responsible for HAPH or influencing severity. We hypothesized that initial insights into the pathogenesis of the disease could be discovered by a strategy of (1) sequencing of functional candidates revealed by single nucleotide polymorphism (SNP) analysis and (2) gene expression profiling of affected cattle compared with altitude-matched normal controls, with gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA). We isolated blood from a single herd of Black Angus cattle of both genders, aged 12–18 months, by jugular vein puncture. Mean pulmonary arterial pressures were 85.6±13 mmHg STD in the 10 affected and 35.3±1.2 mmHg STD in the 10 resistant cattle, P<0.001. From peripheral blood mononuclear cells, DNA was hybridized to an Affymetrix 10K Gene Chip SNP, and RNA was used to probe an Affymetrix Bovine genome array. SNP loci were remapped using the Btau 4.0 bovine genome assembly. mRNA data was analyzed by the Partek software package to identify sets of genes with an expression that was statistically different between the two groups. GSEA and IPA were conducted on the refined expression data to identify key cellular pathways and to generate networks and conduct functional analyses of the pathways and networks. Ten SNPs were identified by allelelic association and four candidate genes were sequenced in the cohort. Neither endothelial nitric oxide synthetase, NADH dehydrogenase, TG-interacting factor-2 nor BMPR2 were different among affected and resistant cattle. A 60-gene mRNA signature was identified that differentiated affected from unaffected cattle. Forty-six genes were overexpressed in the affected and 14 genes were downregulated in the affected cattle by at least 20%. GSEA and Ingenuity analysis identified respiratory diseases, inflammatory diseases and pathways as the top diseases and disorders (P<5.14×10−14), cell development and cell signaling as the top cellular functions (P<1.20×10−08), and IL6, TREM, PPAR, NFkB cell signaling (P<8.69×10−09) as the top canonical pathways associated with this gene signature. This study provides insights into differences in RNA expression in HAPH at a molecular level, and eliminates four functional gene candidates. Further studies are needed to validate and refine these preliminary findings and to determine the role of transcribed genes in the development of HAPH.

Idiopathic and heritable PAH perturb common molecular pathways, correlated with increased MSX1 expression

The majority of pulmonary arterial hypertension (PAH) is not associated with BMPR2 mutation, and major risk factors for idiopathic PAH are not known. The objective of this study was to identify a gene expression signature for IPAH. To accomplish this, we used Affymetrix arrays to probe expression levels in 86 patient samples, including 22 healthy controls, 20 IPAH patients, 20 heritable PAH patients (HPAH), and 24 BMPR2 mutation carriers that were as yet unaffected (UMC). Culturing the patient cells removes the signatures of drug effects and inflammation which have made interpretation of results from freshly isolated lymphocytes problematic. We found that gene expression signatures from IPAH patients clustered either with HPAH patients or in a single distinct group. There were no groups of genes changed in IPAH that were not also changed in HPAH. HPAH, IPAH, and UMC had common changes in metabolism, actin dynamics, adhesion, cytokines, metabolism, channels, differentiation, and transcription factors. Common to IPAH and HPAH but not UMC were an upregulation of vesicle trafficking, oxidative/nitrosative stress, and cell cycle genes. The transcription factor MSX1, which is known to regulate BMP signaling, was the most upregulated gene (4∗) in IPAH patients. These results suggest that IPAH cases have a shared molecular origin, which is closely related to, but distinct from, HPAH. HPAH and IPAH share the majority of altered signaling pathways, suggesting that treatments developed to target the molecular etiology of HPAH will also be effective against IPAH.

Transcripts from a novel BMPR2 termination mutation escape nonsense mediated decay by downstream translation re‐initiation: implications for treating pulmonary hypertension

Hamid R, Hedges LK, Austin E, Phillips III JA, Loyd JE, Cogan JD. Transcripts from a novel BMPR2 termination mutation escape nonsense mediated decay by downstream translation re‐initiation; implications for treating pulmonary hypertension.