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(Circulation. 1997;95:2603-2606.)
© 1997 American Heart Association, Inc.

Articles

Mapping of Familial Primary Pulmonary Hypertension Locus (PPH1) to Chromosome 2q31-q32

Jane H. Morse, MD; Alison C. Jones, MRCP; Robyn J. Barst, MD; Susan E. Hodge, DSc; Kirk C. Wilhelmsen, MD, PhD; Torbjoern G. Nygaard, MD

From the Departments of Medicine (J.H.M.), Neurology (A.C.J., T.G.N.), Pediatrics (R.J.B.), and Psychiatry (S.E.H.), Columbia University College of Physicians and Surgeons, New York, NY; Columbia University School of Public Health and New York State Psychiatric Institute, New York, NY (S.E.H.); and Department of Neurology, University of California, San Francisco (K.C.W.).

Correspondence to Dr Jane H. Morse, Columbia University College of Physicians and Surgeons, 630 W 168th St, PH8W-879, New York, NY 10032. E-mail jhm4{at}columbia.edu

	Abstract

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Background The pathogenesis of primary pulmonary hypertension (PPH) is unknown, although in some instances families with multiple affected members suggest a genetic etiology.

Methods and Results We used microsatellite markers and linkage analysis in a large family with PPH to determine the chromosomal location of their disease gene. We tested a second, ethnically distinct, family for cosegregation of disease with markers from the linked region. We mapped the disease locus PPH1; GDB/HUGO designation (GDB:1381541; July 1996), approved when this work was accepted for publication in abstract form (Circulation. 1996;94[suppl I]:I-49.), in these families to a 27-cM region on chromosome 2q31-q32, with a maximum lod score of 3.87 associated with markers D2S350 and D2S364.

Conclusions Cosegregation of this region with disease in different ethnic groups suggests that we mapped an important locus in familial PPH. Careful study of additional families and sporadic cases will be required to confirm this localization of PPH1 and characterize its overall role.

Key Words: hypertension, pulmonary • genetics • mapping

	Introduction

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Families with multiple affected members suggest a genetic etiology in some instances of primary pulmonary hypertension (PPH). We used linkage analysis in two families with PPH to map the disease locus (PPH1) to a 27-cM region on chromosome 2q31-q32, containing several candidate genes.

PPH is a rare disease in which distinctive vascular changes elevate pulmonary artery pressure, leading to right ventricular failure and death. Without intervention, median survival is <3 years.¹ The pathogenesis of PPH is unknown. Histopathological studies demonstrate extensive vascular remodeling and in situ thrombosis.¹ Reported abnormalities in the production and clearance of vasoactive agents, such as endothelin and prostacyclin, have suggested dysregulation of complex endothelial interactions.¹ An underlying autoimmune process has been suggested by associations with autoantibodies and specific HLA alleles.²

PPH occurs as both sporadic and familial disease. It has been proposed that the familial form is an autosomal dominant trait with high, but incomplete penetrance,³ although segregation analyses have not been performed. Women are affected twice as often as men.³ Genetic anticipation has been proposed in some families.⁴ Two families have been reported in which hemoglobin ß-chain mutations appear to cosegregate with PPH,^{5 6} but no formal linkage study has been done. We performed a study to map the locus causing PPH.

	Methods

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The study protocol was approved by the Institutional Review Board of Columbia University College of Physicians and Surgeons. Participating family members gave informed consent. PPH was diagnosed using the algorithm developed at the National Institutes of Health PPH registry.¹

DNA for genotyping was extracted, through the use of standard protocols, from whole blood or formalin-fixed, paraffin-embedded tissue.⁷ Simple-sequence repeat polymorphisms, with mapped sets of genetic markers,^{8 9} were determined using standard protocols.

For complex diseases with unknown inheritance, using both autosomal dominant (AD) and autosomal recessive (AR) models increases the power of a study to detect true linkage.¹⁰ We analyzed the data using three models: Model 1 was AD and considered only affected members of the families ("affecteds-only" analysis). Model 2 was AD with disease penetrances assigned according to age- and gender-related incidence data⁴ (Table) and phenocopy rate (penetrance for normal homozygotes) of 0.000001. The disease allele frequency for both AD models was set at 0.00001. Model 3 was AR with penetrances that were age and gender related (Table) for disease homozygotes, 0.01 for disease heterozygotes, and 0.000001 for normal homozygotes; disease allele frequency was set at 0.003. Gene frequencies were chosen to be consistent with an estimated disease incidence of 1/100,000 to 1/1 million p.a.¹¹ Because there was male-to-male transmission in both families, we considered only autosomal inheritance. Marker allele frequencies were based on family data. Two-point (marker-to-disease) analyses used MLINK from LINKAGE 5.1¹² and multipoint analyses used VITESSE.¹³

View this table: [in this window] [in a new window]   Table 1. Age- and Gender-Related Penetrance Values for Models 2 and 3

For a genomic screen, we analyzed data from 22 living members of family 1, using 260 evenly distributed autosomal markers. In final analyses, we considered both families, including genotypes from 2 deceased members of family 1, and nine markers in the region of interest (D2S1776, D2S324, D2S350, D2S364, D2S1391, D2S152, D2S318, D2S311, and D2S1384).

	Results

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Family 1 (Fig 1a) has been previously reported² and has 1 additional affected member (IV.1). Their ancestors were of European descent. Genomic DNA was available from 5 affected (III-3 and IV-2 deceased) and 19 unaffected family members. Family 2 (Fig 1b) is Hispanic and comprises an unaffected man with 3 affected and 3 unaffected children. There was no known consanguinity in either family.

View larger version (29K): [in this window] [in a new window]   Figure 1. Pedigrees for (a) family 1 and (b) family 2. Genotyped individuals are indicated by pedigree designations II-2 to V-4 (a) and I-1 to II-6 (b). Shaded symbols represent affected individuals. Genotypes for D2S324, D2S350, D2S364, D2S1391, and D2S152, arranged by inferred haplotype, appear below individual symbols. Those in parentheses are inferred from available family data.

The 22 living members of family 1 were genotyped using 260 polymorphic markers distributed across the autosomes. For each inheritance model, the likelihoods of an observed pattern of genotypes occurring (1) by chance and (2) in the event of linkage between disease and test loci were calculated and compared. A lod score (logarithm of the ratio of these likelihoods) of 3.0 indicates 1000:1 odds in favor of linkage, and a lod score of -2.0 indicates 100:1 odds against linkage. Multipoint analyses, using the conservative affecteds-only approach (model 1), excluded >40% of the genome. The hemoglobin ß-chain gene region^{5 6} (11p15) and the HLA region² (6p21.3) were both excluded. Regions with two-point lod scores of >0.5 were investigated using additional markers, and all except one were eliminated from further analysis. Analyses with model 3 (AR inheritance) did not suggest any separate areas of interest.

Final analyses that included all members of both families and saturated the candidate region with closely spaced markers mapped the disease locus, PPH1, to a 27-cM region on chromosome 2q31-q32 flanked by recombination events at D2S1776 and D2S1384. Analysis under the AD disease model reflecting age- and gender-related penetrances (model 2) led to a maximal pairwise lod score of 3.21 at D2S1391 and multipoint lod scores of 3.87 at D2S350 and D2S364 (Fig 2). Using model 1, the maximum four-point lod score was 2.87 at D2S350, D2S364, and D2S1391; model 3 (AR) also yielded lower scores.

View larger version (12K): [in this window] [in a new window]   Figure 2. Multipoint lod scores for markers D2S1776, D2S324, D2S350, D2S364, D2S1391, D2S152, D2S318, D2S311, and D2S1384 mapped across a 27-cM region on 2q31-q32.9 On the basis of model 2 and both families, the maximal lod score was 3.87 for D2S350 and D2S364. Lod scores <-2.0 at D2S1776 and D2S1384 indicate recombination between these loci and PPH1.

	Discussion

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This study provides good evidence for a PPH1 locus mapping to a 27-cM region on chromosome 2q31-q32, although as the first report of linkage in PPH, it should be regarded as a provisional localization until confirmed in additional families. The highest lod score of 3.87 occurred in a four-point analysis, with the assumption of model 2 described above. Lod scores for the second family, although not significant in themselves, indicated cosegregation of PPH with markers for this region and added to the evidence for linkage. Although familial PPH has been thought to account for a small minority of all PPH cases,¹ detailed analyses in apparently sporadic instances suggest the role of a genetic form may be underappreciated.^{3 14} The absence of clinical or pathological distinctions between sporadic and familial forms of PPH^{1 3} also suggests our finding may have broader implications.

The PPH1 region on 2q31-q32 contains a number of candidate genes. In particular, a cluster of immunoglobulin superfamily genes encode integrin subunits v,¹⁵ 4,¹⁶ and ß6.¹⁷ The and ß integrin subunits combine to form heterodimeric signaling molecules involved in a wide variety of physiological processes,¹⁸ including angiogenesis, immune regulation, and hemostasis. Abnormalities in the function or regulation of integrin subunits could clearly play a role in the vascular remodeling seen in PPH, which is currently thought to result from dys-regulation of normal endothelial interactions.¹

Identification of the PPH1 locus and the basis of its pathogenicity may provide insight into the etiology of not only sporadic PPH but also secondary pulmonary hypertension. Long-term vasodilator therapy and transplantation have already improved outcome in PPH, but earlier diagnosis and the development of specific therapies could be facilitated by identifying the genetic defect in these families.

Note Added in Proof
Nichols et al recently described linkage of PPH to 2q31-q32 (Nat Genet. 1997;15:277-280). Their dataset apparently includes some members of family 1. This family, first reported by Morse et al,² was originally analyzed for linkage by Morse et al.¹⁹

On routine follow-up of family 2, II.4 has fulfilled diagnostic criteria for PPH, increasing maximal two- and four-point lod scores (for the same markers as previously) to 3.61 and 4.27.

	Acknowledgments

 
This work was supported by the Stephen I. Morse Fellowship (A.C.J.); grant HL-48333 and RGK and Culpeper Foundations (J.H.M.); 1996 Scientific Progress Award for the PPH Cure Foundation (J.H.M., R.J.B.); grants MH-44858, MH-52841, DK-31813, MH-28274, and MH-36197 (S.E.H.); grant NS-31212 (K.C.W.); and grant NS-32035 (T.G.N.). We thank the patients, their family members and physicians, and the General Clinical Research Center of Columbia University (grant RR-00645). We also thank Maria DeVera and Alice DiBenedetto for technical assistance.

	Footnotes

 
J.H.M. and A.C.J. contributed equally to this work.

Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10-13, 1996, and published in abstract form in Circulation. 1996;94(suppl I):I-49.

Received November 25, 1996; revision received February 3, 1997; accepted March 25, 1997.

	References

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