© 2004 American Thoracic Society
Familial Spontaneous Pneumothorax and FBN1 MutationsTo the Editor:Primary spontaneous pneumothorax is a common condition often affecting tall individuals with an asthenic habitus. Familial spontaneous pneumothorax is much rarer but provides an opportunity to explore the genetic substrates for primary pneumothorax. It has been suggested that familial spontaneous pneumothorax may be caused by a dominantly inherited connective tissue disorder (1, 2). The Marfan syndrome is an autosomal dominant connective tissue disorder with one of the common pulmonary manifestations of this condition being spontaneous pneumothorax. As in primary spontaneous pneumothorax, tall stature is frequent in the Marfan syndrome. The Marfan syndrome is caused by mutations in the FBN1 gene on chromosome 15, which encodes the extracellular matrix glycoprotein fibrillin-1, which polymerizes in the extracellular matrix of connective tissue to form 10- to 12-nm microfibrils. In lung, these microfibrils are found with elastin as components of elastic fibers. We hypothesized that familial spontaneous pneumothorax is caused by a connective tissue disorder that exhibits Mendelian inheritance and postulated FBN1 as the causative gene. We describe two new pedigrees exhibiting familial spontaneous pneumothorax with an autosomal dominant mode of inheritance and perform haplotype segregation analysis using microsatellite polymorphisms located within the FBN1 gene on these two pedigrees and on one further pedigree previously described (2). Microsatellites consist of repeating sequences of a short block of nucleotides, and the number of repeats may vary from person to person. Typing each family member for these polymorphic markers allows the determination of haplotypes. Observing the inheritance pattern of haplotypes through a pedigree makes it possible to establish whether a particular copy of a gene cosegregates with disease. We used this approach to assess if the phenotype of spontaneous pneumothorax cosegregates, and therefore may be due to functional changes in this gene. Four previously characterized FBN1 intragenic microsatellite polymorphisms were used to perform haplotype segregation analysis on the three pedigrees (see Figure 1) . This analysis assesses whether the pneumothorax cosegregates with a fibrillin allele. Each pedigree contained four or more affected individuals spanning a minimum of two generations. In total, 23 individuals were included for analysis: 15 affected and 8 unaffected (see Table 1) .
Pedigree A comprised a family exhibiting spontaneous pneumothorax in a father and three offspring; the mother was unaffected. Pedigree B comprised a large family from New Zealand containing eight affected individuals spanning three generations. Two of the affected individuals had single pneumothoraces, five had two episodes of pneumothorax, and one had three episodes. Pedigree C comprised a British family with five affected members. One individual had a total of seven pneumothoraces occurring over an 8-year period, two had suffered two pneumothoraces, and one had had four. Blood was obtained from each of the 23 individuals analyzed. Genomic DNA was extracted from peripheral blood leucocytes using standard procedures. For each individual, four previously characterized FBN1 intragenic microsatellite markers—mts-1, mts-2, mts-3, and mts-4—were amplified (4). Primer sequences used for amplification were as previously described (4). The products of each polymerase chain reaction (PCR) varied in size according to the number of sequence repeats between the two primers with fixed positions. PCR products were separated using an ABI prism 377 sequencer and alleles were assigned using Genescan genotype software. Each allele for the four markers was ascribed a number corresponding to the size of the amplified product in base pairs. In all three pedigrees, an autosomal dominant mode of inheritance was suggested by the pattern of transmission of the disease phenotype. For each of the three pedigrees investigated, examination of affected and unaffected family members revealed no differences in body habitus and no differentiating features indicative of connective tissue or respiratory disease. The overall ratio of male:female family members for the three pedigrees was 8:7. Genomic DNA from a total of 23 individuals (46 chromosomes) from three separate pedigrees was genotyped for four FBN1 intragenic microsatellite polymorphisms. Genotyping of the four polymorphic markers identified two distinct haplotypes in all of the 23 individuals analyzed (see Figure 1). In Pedigree A, haplotypes for the four FBN1 polymorphic markers were assigned to each of the five members of the pedigree analyzed (see Figure 1). Each member possessed two distinct haplotypes allowing both FBN1 alleles to be defined. Haplotype segregation analysis indicated that the same FBN1 allele was passed from the affected father (I.1) to two of the affected offspring (II.1 and II.6). However, the third affected offspring (son II.5) inherited the other paternal FBN1 allele. A single FBN1 allele therefore failed to cosegregate with the disease phenotype in this pedigree. In Pedigree B, haplotypes were assigned to each of the 12 members of the pedigree analyzed (see Figure 1). Again each member possessed two distinct haplotypes allowing both FBN1 alleles to be defined. Haplotype segregation analysis revealed that a single FBN1 allele failed to cosegregate with the disease phenotype throughout this pedigree. The two affected sons, II.6 and II.8, inherited different FBN1 alleles from their affected father, I.4. Finally, in Pedigree C, haplotypes sufficient to allow both FBN1 alleles to be defined were assigned to each of the six members of the pedigree analyzed. Haplotype segregation analysis indicated that the affected granddaughter, III.2, and the affected grandson, III.3, did inherit the same FBN1 allele from their affected mother, II.2. However, their affected uncle, II.3, failed to share this same FBN1 allele. This again indicates that a single FBN1 allele failed to cosegregate with the disease in this pedigree. Our findings indicate that familial spontaneous pneumothorax in these pedigrees is an autosomal dominant disorder with incomplete penetrance demonstrating variable expression. The overall sex ratio for affected individuals in our study was 1:1, supporting the finding of Lenler-Petersen (5) of equal penetrance in male and female individuals. In none of the pedigrees did a single FBN1 allele cosegregate with the disease phenotype. Mutations in the FBN1 gene therefore are not responsible for the manifestation of the autosomal dominant form of familial spontaneous pneumothorax.
a Churchill Hospital Oxford, United Kingdom FOOTNOTES Conflict of Interest Statement: C.M.C. has no declared conflict of interest; N.A.M. has no declared conflict of interest; P.A.H. has no declared conflict of interest; A.G.A. has no declared conflict of interest; R.J.O.D. has no declared conflict of interest; P.J.M. has no declared conflict of interest; P.E.T. has no declared conflict of interest. REFERENCES
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