(Chest. 2002;121:46S-50S.)
© 2002
American College of Chest Physicians
Genetics and Gene Expression in Pulmonary Hypertension*
Parker B. Francis Lecture
James E. Loyd, MD
*
From Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, TN.
Correspondence to: James E. Loyd, MD, Professor of Medicine, Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center, 1211 22nd Ave S, Nashville, TN; e-mail: jim.loyd{at}mcmail.vanderbilt.edu
The
prototype of clinical pulmonary hypertension is primary pulmonary
hypertension (PPH), for which interesting new information is available.
PPH is a severe and progressive disease with a mean survival of < 3
years without treatment. The central feature of PPH is widespread
obstructive lesions of small pulmonary arteries. Despite dramatic
advances in many aspects of PPH during the past decade, including
improved diagnosis, new understanding of biological mediators, and the
development of the first effective therapy, the possibility of
prevention or cure seems elusive unless the central basic mechanisms
are identified and characterized.
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Genetics
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In a large prospective study,1
6% of patients
reported a family history of PPH, but reports2
suggest that a larger percentage (
25%) has a genetic basis.
Confirmation of the speculation that most PPH is genetic will require
further evidence. Clinical and pathologic manifestations are identical
whether PPH is familial or acquired sporadically. The largest
study3
to examine the pattern of inheritance included 24
PPH families. Among a total of 429 family members, there were 124
individuals known to carry the gene, who were identified by having PPH
themselves or having progeny with PPH. Vertical transmission was
readily apparent in these pedigrees, with many cases of father-to-son
transmission, thereby excluding X-linkage. These features indicate
autosomal dominant transmission, and incomplete penetrance was present
as well. More female subjects (84 female and 40 male subjects) had the
gene (ie, they had PPH or progeny with it); among those who
had the gene, more female subjects had disease develop (72 of 84
female subjects [86%] vs 27 of 40 male subjects [68%]). The
penetrance of disease appears to be highly variable across
different PPH families.
Gender ratios of the progeny of affected members and carriers were also
analyzed, and the ages at death from PPH were analyzed by generation.
Significantly more female subjects (n = 160) than male subjects
(n = 122) were born to persons who carry the familial PPH (FPPH)
gene. This abnormal gender ratio of progeny remains to be fully
explained, but suggests the occurrence of selective loss of male
fetuses or an abnormal primary sex ratio at conception. Interestingly,
a gene recently identified to cause PPH, described later, is in a
category that has been widely recognized to be important in
development, morphogenesis, and organogenesis.4
The mean
age at death from PPH was the same for male and female subjects.
Genetic anticipation, shown as decreasing age at death in subsequent
generations, was present and statistically significant; the age at
death was 45.6 years in the oldest generation, vs 36.3 years in the
next generation, and then 24.2 years. The advances in molecular
understanding described to follow have not yet explained the phenomenon
of genetic anticipation in PPH, so it remains inconclusive at
present whether its basis is biological or artifactual.
Although women have FPPH develop twice as commonly as men, cumulative
mortality curves of each gender are similar, suggesting that the course
or severity of PPH are similar independent of gender (Fig 1
). FPPH-mortality distribution among 110 female and 51 male subjects
demonstrates that FPPH has a broad range of ages of affected patients,
such that 20% of deaths occur after the age of 50 years, and 20%
before the age of 20 years. FPPH occurs in either gender (female/male
ratio, 2:1) at any age, unlike the former clinical opinion that PPH was
predominantly a disease of young adult women. The age distribution of
PPH appears uniform across all of life, and the explanation for the
broad variability in age of onset is also needed.
Penetrance of FPPH is variable and quite low in some families, causing
skip generations in which disease is not manifested. This finding led
to early suggestions that many PPH patients who appear to have sporadic
acquired disease may in fact have a genetic basis that was
unrecognized.
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FPPH: Lung Pathology
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Pulmonary vascular pathologists categorize PPH into numerous
different pathologic classes. The most common pathologic categories are
"plexogenic" and "thromboembolic" PPH. A study to examine the
spectrum of pathologic changes in patients with FPPH was performed to
determine the number of pathologic subsets, to avoid inclusion of
heterogeneous groups in a gene search. Lung specimens from 23 affected
members of 13 families with known FPPH were examined.5
The
results demonstrated heterogeneity with coexistence of pathologic
lesions within and among families, including microthrombotic and
plexiform lesions, in addition to medial hypertrophy and eccentric
intimal fibrosis. The types and range of lesions found in these FPPH
families were not specific, so they appear to represent different
manifestations of a single pathogenetic process. The rarest subsets,
pulmonary veno-occlusive disease and pulmonary capillary
hemangiomatosis, do have unique clinical and pathologic manifestations,
which were not present in any of the families described above. Rare
instances of familial clustering of these unique entities have been
documented and these conditions probably represent distinctly different
disease processes, but their causes are not yet known.
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FPPH: Current Status
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PPH in families has been reported from countries all around the
world, including Great Britain, France, Norway, Spain, Turkey, Korea,
and others. Of the 101 US families with FPPH, most have ethnic heritage
from western Europe. FPPH in the United States appears to rarely affect
minority populations, with African-American heritage in only three
families.
Our PPH database contains records of 207 patients with FPPH, which have
occurred among > 2,000 individuals in the 101 families in the United
States. Recently, five PPH families in Tennessee were found to have a
common ancestor, and together it is now the largest reported family
with PPH (Fig 2
).6
This FPPH kindred has had 18 cases of PPH, including 16
women and 2 men. Among the obligate carriers, ie,
individuals who have transmitted disease but are not affected, there
are 9 women and 13 men. So the striking influence of gender on
expression of the gene defect is shown by the comparison in this
family: 16 women affected (9 obligates) and 2 men affected (13
obligates).
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Microsatellite Search Linked a Gene for FPPH in 1997
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In 1997, a genome-wide search for FPPH was reported that analyzed
a set of highly polymorphic markers in 19 affected individuals from six
PPH families, and demonstrated evidence for linkage with markers on
chromosome 2q.7
All patients and available family members
were then genotyped for 19 additional markers spanning approximately 40
centimorgans on the long arm of chromosome 2. This study demonstrated a
maximum two-point logarithm of odds score of 6.97 at 
= 0 with the
marker D2S389. Multipoint-linkage analysis yielded a maximum
score of 7.86 with the marker D2S311. Haplotype analysis
established a minimum candidate interval of approximately 25
centimorgans. There was no evidence for locus heterogeneity in any of
these six families studied. Another research team8
reported linkage independently at about the same time.
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Heterozygous Mutations in Bone Morphogenetic Protein Receptor
2 CAUSE FPPH
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FPPH was recently shown to be caused by mutations in bone
morphogenetic protein receptor (BMPR) 2, which is a member of the
transforming growth factor (TGF)-ß superfamily.9
The
genomic structure of BMPR2 was defined, and seven independent mutations
were detected in a cohort of eight PPH kindreds. By comparison to
in vitro studies, the identified defects of BMPR2 in FPPH
were predicted to disrupt ligand binding, kinase activity, and
heterodimer formation. These data demonstrate a molecular basis of FPPH
and underscore the importance of the TGF-ß pathway in maintenance of
vessel integrity. Another research team10
also reported
mutations of BMPR2 in FPPH about the same time.
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Bone Morphogenetic Proteins
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Members of the TGF-ß superfamily, including bone morphogenetic
proteins (BMPs), transduce signals by binding to heteromeric complexes
of type I and type II receptors, activating serine/threonine kinases,
leading to transcriptional regulation by phosphorylated Smads. For
TGF-ß, sequential binding of the ligand to a type II receptor, which
recruits and activates a type I receptor. The intracellular domain of
the serine-threonine kinase type I receptor then initiates downstream
signaling through phosphorylation of the Smad cytoplasmic transcription
factors. BMPs are secreted signaling molecules. Investigation of the
> 30 BMPs known to date have demonstrated important roles in
development, cell proliferation and differentiation, morphogenesis, and
organogenesis. Current opinion suggests that a more appropriate term
for this class would be growth and differentiation
factors.11
BMPs 2, 4, 6, and 7 signal through the type II
receptor, BMPR2.12
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BMPR2 MUTATIONS WERE ALSO FOUND IN SPORADIC PPH
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Another investigation2
reported mutations in a group
of unrelated PPH patients who had no identifiable family history of
pulmonary hypertension. Direct sequencing of the entire coding region
and the boundaries of the BMPR2 gene was performed in 50 patients with
sporadic PPH. DNA from available parents was used to assess the
occurrence of spontaneous (de novo) mutations contributing
to sporadic PPH. A total of 13 heterozygous germline mutations of the
BMPR2 gene were found among the 50 PPH subjects studied, including
missense (n = 3), nonsense (n = 3), and frameshift (n = 7)
mutations, each predicted to alter the cell-signaling response to
specific ligands. No differences in clinical features or disease
progression were seen in PPH patients with or without the germline
mutations. The sporadic form of PPH was associated with germline
mutations of the gene encoding the receptor protein BMPR2 in at least
26% of these patients.
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A Mutation in BMPR2 HAS BEEN IDENTIFIED IN HALF OF
THE PPH FAMILIES
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In this study,13
samples from 45 additional families
with PPH were analyzed and the entire coding region of the BMPR2 gene
and all intron/exon junctions were sequenced in affected individuals. A
total of 22 different mutations were identified in 23 of 45 families.
All of these mutations were shown to segregate with disease, and none
were detected in a panel of 150 normal chromosomes. Most of the
mutations (13 of 22 mutations [59%]) are either frameshift (n = 8)
or nonsense (n = 5) mutations that would be predicted to lack normal
BMPR2 function. These mutations are dispersed throughout the gene with
the most 5' frameshift mutation occurring in exon 1 and the most 3'
frameshift found in exon 12. The exon 1 frameshift mutation would
predict a protein containing only the 15 N-terminal amino acids of
BMPR2 with an additional 30 amino acids until the next stop codon. All
eight of the frameshift mutations appear to result from slippage or
stutter of the polymerase during replication as they occur in regions
of small mononucleotide or dinucleotide repeats.
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What Causes PPH in Families in Whom a Mutation in
BMPR2 IS NOT IDENTIFIED?
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Mutations of BMPR2 have not been reported for nearly half of the
known PPH families. It is possible that another gene or genes may cause
PPH, or that other mutations, perhaps intronic, in BMPR2 have not yet
been identified. Another presentation at this conference suggests that
a second locus may exist on the long arm of chromosome 2, and
independent confirmation of this finding is awaited with great
interest.
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PPH Gene Expression: Microarray Analysis
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A recent study14
employed oligonucleotide
microarray to examine the expression pattern in lung tissue obtained
from six patients with PPH, including two patients with a known family
history (FPPH) and six patients with normal lungs. For the data
analysis, gene clusters were generated and the gene-expression pattern
differences between PPH and normal lung tissue, and between PPH and
FPPH lung tissue were compared. Several genes were differentially
expressed, and this allowed characterization of the PPH gene-expression
pattern as one where genes coding for proteins involved in control of
cell growth and apoptosis were abnormal. Importantly, pattern
comparison of gene expression also distinguished between sporadic PPH
and FPPH, with 39 different genes that were demonstrated to be
differentially expressed between sporadic PPH and FPPH. Microarray gene
expression appears to be a useful technique that provides better
characterization and understanding of the pathobiology of the clinical
phenotypes of pulmonary hypertension.
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Is the Plexiform Lesion a Tumorlet? Cell Growth and Molecular
Changes in Plexiform Lesions
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Plexiform lesions, which are composed of proliferating endothelial
cells in clusters are characteristic lesions of PPH. The technology to
assess monoclonality has recently developed, and it may help to
distinguish cellular proliferation of neoplasia from that in reactive
nonneoplastic tissue. To determine whether the endothelial cell
proliferation in plexiform lesions of PPH is monoclonal or polyclonal,
the methylation pattern of the human androgen-receptor gene by
polymerase chain reaction was assessed in proliferative endothelial
cells from plexiform lesions from female patients with PPH compared to
patients with secondary pulmonary hypertension.15
In
patients with PPH, 17 of 22 lesions (77%) were monoclonal. However, in
patients with secondary pulmonary hypertension, all 19 lesions examined
were polyclonal. Smooth-muscle cell hyperplasia in pulmonary vessels
(n = 11) in PPH and secondary pulmonary hypertension was polyclonal
in all but one of the examined vessels. It appears that monoclonal
expansion of endothelial cells distinguishes between PPH and secondary
pulmonary hypertension. Monoclonal endothelial cell proliferation in
patients with PPH suggests that a somatic genetic alteration similar to
neoplastic processes may contribute to pathogenesis of PPH.
These findings contributed to a hypothesis that endothelial cells
within PPH plexiform lesions harbor mutations permissive for clonal
cell growth. In another study,16
endothelial cells of PPH
plexiform lesions demonstrated microsatellite instability within the
human MutS homolog 2 gene (10 of 20 lesions) and displayed
microsatellite site mutations and reduced protein expression of
TGF-ß–receptor type II (6 of 19 lesions) and Bax (4 of 19 lesions).
These results suggest that proliferative endothelial cells of PPH
plexiform lesions have genetic alterations associated with
microsatellite instability and concomitant perturbation of growth and
apoptosis gene expression, similar to pathogenetic features of
neoplasia.
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Summary
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The identification of BMPR2 as a gene responsible for PPH, in both
familial and sporadic disease, provides the first opportunity to
understand its central pathogenesis, as well as many other
opportunities, such as genetic testing and counseling, and directing
the development of novel therapies. In aggregate, the studies described
in the discussion above provide convincing evidence that proliferative
processes are the central pathogenetic event of PPH. Future research
efforts will be directed by many investigators to understand by what
mechanisms BMPR2 mutations cause occlusive disease in such a limited
segment of the circulation. Other goals for the immediate future should
include the identification of genes or conditions that modify the
clinical expression of BMPR2 mutations, and possibly other primary
genes, which are of enormous interest and importance and surely are
realistic at this time.
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Footnotes
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Abbreviations: BMP = bone morphogenetic protein; BMPR =
bone morphogenetic protein receptor; FPPH = familial primary
pulmonary hypertension; PPH = primary pulmonary hypertension;
TGF = transforming growth factor
Supported by National Institutes of Health grant HL-48164.
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References
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Genetics
FPPH: Lung Pathology
