Unilateral microfibrillar abnormalities in a case of asymmetric Marfan syndrome.

The Marfan syndrome is a dominantly inherited connective-tissue disorder characterized by ocular, cardiovascular, and musculoskeletal abnormalities. Although the underlying biochemical and molecular defect(s) of this pleiotropic disease is currently unknown, we have consistently observed apparent diminished content of elastin-associated microfibrillar fibers accumulating in skin, or produced by cultured fibroblasts, from patients with the Marfan syndrome and have documented the cosegregation of these immunofluorescent abnormalities of microfibrillar fibers with the Marfan syndrome phenotype in family studies. Recently, an unusual patient has been described with unilateral phenotypic features of the Marfan syndrome, providing an unique opportunity to compare microfibrillar fibers and other connective-tissue components between the affected and nonaffected sides. In the present report, we demonstrate striking differences in apparent content of microfibrillar fibers, as determined by indirect immunofluorescence of skin and fibroblast cultures, that are revealed when multiple homologous samples derived from different sides of the patient's body are compared. In contrast, no differences in apparent content of type III collagen or in the biosynthesis and apparent structure of types I and III (pro)collagens were found. HLA types and chromosome heteromorphisms were identical in fibroblasts from both sides of the body, eliminating the formal possibility of chimerism and suggesting that a postzygotic mutation accounts for the asymmetric manifestation of the Marfan syndrome in this patient. The observation of striking decreases in microfibrillar fibers on the affected side of the body provides further evidence that abnormalities of this component of the elastic fiber system may be central to the pathogenesis and possibly the etiology of the Marfan syndrome.     

Genetic disorders of the elastic fiber system.

Over the last decade, a considerable amount of new information has emerged describing the protein components of elastic fibers. It is now evident that elastic fibers are complex extracellular matrix polymers, composed of at least 19 different proteins that comprise both the microfibrillar and the amorphous components of elastic fibers. Mutations in three of the genes encoding the most abundant of these elastic fiber proteins result in a broad spectrum of elastic tissue phenotypes, ranging from skeletal and skin abnormalities to vascular and ocular defects. The following disorders will be discussed in this review: supravalvular aortic stenosis; Williams-Beuren syndrome; cutis laxa; Marfan syndrome; ectopia lentis; familial thoracic aortic aneurysms and dissections; MASS syndrome; isolated skeletal features of Marfan syndrome; Shprintzen-Goldberg syndrome; and congenital contractural arachnodactyly.     

Decreased extracellular deposition of fibrillin and decorin in neonatal Marfan syndrome fibroblasts

Abnormalities of the microfibrillar protein fibrillin (Fib) have been reported in Marfan syndrome (MFS). The so-called neonatal Marfan syndrome (nMFS) is a lethal phenotype displaying features that are not seen in classical MFS. We have therefore studied the biosynthesis and extracellular deposition of Fib and decorin in fibroblasts from a patient with nMFS and controls. Immunofluorescence of the patient's cell cultures showed an almost complete absence of Fib and a marked reduction of decorin in the extracellular matrix (ECM). The nMFS skin revealed Fib on subbasal microfibrillar bundles in the papillary dermis, and Fib associated with elastic fibers in the reticular dermis; the bundles and fibers were fragmented and thinner than normal. Pulse-chase labeling of cells with [³⁵S]Met/Cys revealed moderately reduced secretion, but a diminished deposition of Fib in the ECM; this was more apparent at a longer chase time. Fib mRNA and synthesis appeared to be normal, where-as both decorin mRNA and biosynthesis were reduced. We therefore assume a structural Fib defect in this patient causing reduced deposition into and/or enhanced removal from the ECM, whereas the reduced decorin biosynthesis may be a secondary regulatory phenomenon. The clinical relevance of this remains unclear. Our findings imply that Fib defects may be responsible for the severe, complex phenotype of nMFS.     

Abnormal fibrillin assembly by dermal fibroblasts from two patients with Marfan syndrome

The microfibrillar glycoprotein fibrillin is linked to the Marfan syndrome, an autosomal dominant connective tissue disorder. In this study, fibrillin synthesis, deposition and assembly has been investigated in Marfan dermal fibroblast lines from two unrelated patients for whom distinct mutations in the fibrillin gene FBN1 have been identified. In patient NB, a point mutation has occurred which causes an amino acid substitution and the other patient (GK) has a deletion in one allele. The two cell lines were broadly comparable with respect to de novo fibrillin synthesis and its distribution between medium and cell layer compartments. Electrophoresis of fibrillin immunoprecipitates confirmed the presence of fibrillin in medium and cell layers. GK cells secreted an additional higher relative molecular mass fibrillin-immunoreactive component. The time-course of fibrillin secretion was similar for the two lines, but differences in fibrillin aggregation were apparent. Rotary shadowing electron microscopy of extracted cell layers demonstrated the presence of abundant and extensive microfibrils in NB cell layers. These were abnormal in their gross morphology in comparison to microfibrils isolated from control cultures. No periodic microfibrillar structures were isolated from GK cell layers. These studies underline the need to classify fibrillin defects in terms of biochemical and ultrastructural criteria. Examination of the effects of individual mutations on microfibril organization will be particularly informative in elucidating the relationship between microfibril dysfunction and the complex clinical manifestations of Marfan patients.     

Elastic and collagenous networks in vascular diseases

Supravalvular aortic stenosis (SVAS), Marfan syndrome (MFS) and Ehlers-Danlos syndrome type IV (EDS IV) are three clinical entities characterized by vascular abnormalities that result from mutations of structural components of the extracellular matrix (ECM). Analyses of naturally occurring human mutations and of artificially generated deficiencies in the mouse have provided insights into the pathogenesis of these heritable disorders of the connective tissue. SVAS is associated with haploinsufficiency of elastin, one of the two major components of the elastic fibers. SVAS is characterized by narrowing of the arterial lumen due to the failure of regulation of cellular proliferation and matrix deposition. Mutations in fibrillin 1 are the cause of dissecting aneurysm leading to rupture of the ascending aorta. Fibrillin-1 is the building block of the microfibrils that span the entire thickness of the aortic wall and are a major component of the elastic fibers that reside in the medial layer. The vascular hallmark of EDS IV is rupture of large vessels. The phenotype is caused by mutations in type III collagen. The mutations ultimately affect the overall architecture of the collagenous network and the biomechanical properties of the adventitial layer of the vessel wall. Altogether, these genotype-phenotype correlations document the diversified contributions of distinct extracellular macroaggregates to the assembly and function of the vascular matrix.     

Elucidation of the gene defect in Marfan syndrome. Success by two complementary research strategies.

Marfan syndrome, which is characterized by manifestations in the skeletal, ocular and cardiovascular systems, is one of the most common inherited connective-tissue disorders. The independently performed genetic assignment of the Marfan locus and classical biochemical and immunohistochemical analyses complemented each other in the search for the Marfan gene defect and in 1991 the fibrillin gene in chromosome 15 was identified as the Marfan gene. So far, three mutations leading to the Marfan phenotype have been reported in this gene coding for a microfibrillar protein. The available data suggests a wide spectrum of different mutations of fibrillin and although mutations of the fibrillin gene account for the majority of Marfan cases, evidence also exists for locus heterogeneity in a minority of Marfan cases.     

Natural and recombinant interferons provide different protection of human cells with impaired repair system (Marfan syndrome) against mutagens

It was shown that pretreatment of human cells with interferons (IF) of different origin has an unequal protective effect under the action of various mutagens with different activity. The protective effect of IF was estimated using the test of sister chromatid exchanges. Natural leucocyte alpha IF is highly effective in healthy human cells and in those of patients having Marfan syndrome. The latter are characterised by disorder in DNA repair under the action of 4-nitroquinoline-1-oxide (4-NQO), 8-methoxypsoralen and gamma-rays. Recombinant interferon (alpha 2) displayed no activity against gamma-rays in cells of healthy donors and patients with Marfan syndrome. Nor was it effective in the cells of patients in the experiments with 4-NQO. The absence of correlation between the ability of IF to protect the cells and their influence on the rate of cell proliferations was established.     

A novel de novo mutation in exon 14 of the fibrillin-1 gene associated with delayed secretion of fibrillin in a patient with a mild Marfan phenotype

The Marfan syndrome, an autosomal dominant heritable disorder of connective tissue, is caused by mutations in the gene for fibrillin-1, FBN1. A novel FBN1 mutation was identified using temperature-gradient gel electrophoresis of a reverse-transcribed polymerase chain reaction product spanning exons 14 to 16. The mutation, G1760A, is predicted to result in the amino acid substitution C587Y and thus to disrupt one of the disulfide bonds of the calcium-binding epidermal growth factor-like module encoded by exon 14. C587Y was found to be a de novo mutation in a relatively mildly affected 15-year-old girl whose clinical phenotype was characterized mainly by ectopia lentis and thoracic scoliosis. Metabolic labeling of cultured dermal fibroblasts from the affected patient demonstrated delayed secretion of fibrillin with normal synthesis and no decrease in incorporation into the extracellular matrix compartment. Fibrillin immunostaining of confluent dermal fibroblast cultures revealed no visible difference between the patient’s cells and control cells. Characterization of many different FBN1 mutations from different regions of the gene may provide a better understanding of clinical and biochemical genotype-phenotype relationships. Received: 31 October 1996 / Accepted: 3 March 1997     

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.     

Software and database for the analysis of mutations in the human FBN1 gene.

Fibrillin is the major component of extracellular microfibrils. Mutations in the fibrillin gene on chromosome 15 (FBN1) were described at first in the heritable connective tissue disorder, Marfan syndrome (MFS). More recently, FBN1 has also been shown to harbor mutations related to a spectrum of conditions phenotypically related to MFS and many mutations will have to be accumulated before genotype/phenotype relationships emerge. To facilitate mutational analysis of the FBN1 gene, a software package along with a computerized database (currently listing 63 entries) have been created.     

Molecular genetics of pseudoxanthoma elasticum: a metabolic disorder at the environment-genome interface?

Pseudoxanthoma elasticum (PXE) is a relatively rare heritable disorder affecting the skin, eyes and cardiovascular system, with considerable morbidity and mortality. The disease affects the elastic fibers of affected organs, which become progressively calcified. Thus, PXE has been considered as a prototypic heritable connective tissue disorder affecting the elastic fiber system. Recently, PXE has been linked to mutations in the MRP6/ABCC6 gene, a member of the ABC transporter family, expressed primarily in the liver and the kidneys. This information, together with clinical observations suggesting environmental, hormonal and/or dietary modulation of the disease, raises the intriguing possibility that PXE is a primary metabolic disorder at the environment-genome interface.     

Abnormalities in cartilage and bone development in the Apert syndrome FGFR2(+/S252W) mouse

Apert syndrome is an autosomal dominant disorder characterized by malformations of the skull, limbs and viscera. Two-thirds of affected individuals have a S252W mutation in fibroblast growth factor receptor 2 (FGFR2). To study the pathogenesis of this condition, we generated a knock-in mouse model with this mutation. The Fgfr2(+/S252W) mutant mice have abnormalities of the skeleton, as well as of other organs including the brain, thymus, lungs, heart and intestines. In the mutant neurocranium, we found a midline sutural defect and craniosynostosis with abnormal osteoblastic proliferation and differentiation. We noted ectopic cartilage at the midline sagittal suture, and cartilage abnormalities in the basicranium, nasal turbinates and trachea. In addition, from the mutant long bones, in vitro cell cultures grown in osteogenic medium revealed chondrocytes, which were absent in the controls. Our results suggest that altered cartilage and bone development play a significant role in the pathogenesis of the Apert syndrome phenotype.     

Mutations in CTC1, encoding the CTS telomere maintenance complex component 1, cause cerebroretinal microangiopathy with calcifications and cysts

Cerebroretinal microangiopathy with calcifications and cysts (CRMCC) is a rare multisystem disorder characterized by extensive intracranial calcifications and cysts, leukoencephalopathy, and retinal vascular abnormalities. Additional features include poor growth, skeletal and hematological abnormalities, and recurrent gastrointestinal bleedings. Autosomal-recessive inheritance has been postulated. The pathogenesis of CRMCC is unknown, but its phenotype has key similarities with Revesz syndrome, which is caused by mutations in TINF2, a gene encoding a member of the telomere protecting shelterin complex. After a whole-exome sequencing approach in four unrelated individuals with CRMCC, we observed four recessively inherited compound heterozygous mutations in CTC1, which encodes the CTS telomere maintenance complex component 1. Sanger sequencing revealed seven more compound heterozygous mutations in eight more unrelated affected individuals. Two individuals who displayed late-onset cerebral findings, a normal fundus appearance, and no systemic findings did not have CTC1 mutations, implying that systemic findings are an important indication for CTC1 sequencing. Of the 11 mutations identified, four were missense, one was nonsense, two resulted in in-frame amino acid deletions, and four were short frameshift-creating deletions. All but two affected individuals were compound heterozygous for a missense mutation and a frameshift or nonsense mutation. No individuals with two frameshift or nonsense mutations were identified, which implies that severe disturbance of CTC1 function from both alleles might not be compatible with survival. Our preliminary functional experiments did not show evidence of severely affected telomere integrity in the affected individuals. Therefore, determining the underlying pathomechanisms associated with deficient CTC1 function will require further studies.     

Mismatch Repair Genes on Chromosomes 2p and 3p Account for a Major Share of Hereditary Nonpolyposis Colorectal Cancer Families Evaluable by Linkage

Two susceptibility loci for hereditary nonpolyposis colo-rectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis.