中国汉族两个马凡综合征家系FBN1基因的一个新插入突变和一个复发点突变

目的:明确两个中国北方汉族马凡综合征(Marfan syndrome,MFS)家系的临床特点,并对其进行基因诊断。方法:对两个家系进行家系调查和系谱分析,应用聚合酶链式反应-DNA测序方法对原纤维蛋白1基因(Fibrillin-1,FBN1)的所有外显子进行测序。应用Swiss-model、Polyphen-2和SIFT软件对发现的变异位点进行功能预测。结果:两个家系均呈常染色显性遗传特点,在家系1患者中发现一个新的插入突变,即第13外显子1691位碱基处插入碱基A(1691 ins A),导致蛋白在第571位氨基酸处翻译提前终止。此外,在家系2患者中发现一个已知的点突变,即第27外显子第3463位碱基由G变为A(3463 GA),导致第1155位氨基酸由天冬氨酸变为天冬酰胺。这两个变异位点在家系的健康人及50例健康对照中均未出现。功能预测发现这两个变异位点均可能会影响FBN1蛋白的结构或功能。结论:在两个MFS家系中发现一个新插入突变位点(1691 ins A)和一个已知点突变位点(3463 GA),为扩大FBN1基因的突变谱及进一步阐明FBN1基因突变在MFS中的作用提供理论依据。     

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     

Application of dHPLC for mutation detection of the fibrillin-1 gene for the diagnosis of Marfan syndrome in a National Health Service Laboratory

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the fibrillin-1 gene FBN1. Mutation detection of this 65-exon gene presents a particular challenge for the diagnostic service in cost, time constraints, and the need to maintain a stringently optimized assay procedure. Using denaturing high-performance liquid chromatography (dHPLC), we have designed a procedure for rapid mutation scanning, redesigning 50% of published primer sets, screening by Ensembl to avoid inclusion of polymorphic variations and employing a limited set of PCR conditions to allow for a high-throughput 96-well format. We have screened 262 unrelated patients with MFS or Marfan-like phenotypes and detected 103 (39.3%) mutations including 93 different mutations, 72 of which are novel. The mutations include 55 missense (53.4%) 19 splice site (18.5%), 17 frameshift (16.5%), 11 nonsense (10.7%) and 1 in-frame deletion/insertion.     

汉族马凡综合征(MFS)患者FBN1基因两种新发突变分析

为调查马凡综合征(Marfan syndrome, MFS)患者的原纤维蛋白-1(Fibrillin-1, FBN1)基因突变情况, 应用聚合酶链反应(PCR)和变性高效液相色谱法(Denaturing high-performance liquid chromatography, DHPLC)对MFS患者的FBN1基因进行突变筛查, 对DHPLC初筛异常的DNA片段进行测序分析。结果在两个MFS家系中发现FBN1基因两种新的突变: 一种为复合突变包含第55号外显子的缺失突变c.6862_6871delGGCTGTGTAG (p.Gly2288MetfsX109)、同义突变c.6861A>G和内含子的突变c.[6871+1_6871+11delGTAAGAGGATC; 6871+34dupCATCAGAAGTGACAGTGGACA]; 另一种为第20号外显子的错义突变c.2462G>A(p.Cys821Tyr)。研究表明, FBN1基因的缺失突变c.[6862_6871delGGCTGTGTAG; 6871+1_6871+11delGTAAGAGGATC] (p.Gly2288MetfsX109)和错义突变c.2462G>A(p.Cys821Tyr)可能分别是这两个家系患者的致病原因。     

Molecular analysis of eight mutations in FBN1

Mutations in the gene encoding extracellular glycoprotein fibrillin-1 (FBN1) cause Marfan syndrome (MFS) and other related connective tissue disorders. In this study, eight mutations have been detected in MFS patients by heteroduplex analysis. These comprise two missense mutations, C1835Y and C2258Y in calcium-binding epidermal growth factor-like domains, two nonsense mutations, R1541X and R2394X in transforming growth factor beta1-binding protein-like domains, one splice site mutation, which has been detected previously, and three small insertions or deletions resulting in a frameshift. Fibroblast cells have been established from seven of the MFS patients and the biochemical effects of the mutations on fibrillin-1 synthesis and secretion assessed by pulse-chase analysis. Each cysteine mutation resulted in the delayed secretion of fibrillin-1 and both nonsense and frameshift mutations caused reduced levels of synthesis and/or deposition of fibrillin-1. Indirect immunofluorescence and rotary shadowing electron microscopy analysis of fibrillin microfibrils revealed no major differences between normal and patient samples. We discuss the relative merits of the biochemical techniques used in this study.     

Microenvironmental regulation by fibrillin-1

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.     

Novel non-synonymous mutation in the transforming growth factor beta binding protein-like (TB) domain of the fibrillin-1 (FBN1) gene in a Han Chinese family with Marfan syndrome (MFS)

In order to further understand the role of fibrillin-1 (FBN1, OMIM 134797) perturbations in the pathogenesis of Marfan syndrome (MFS, OMIM 154700) we studied a Han Chinese family in which MFS was segregating. In the Chinese family with 5 affected members, mutation screening for FBN1 was performed using direct sequencing. A novel non-synonymous mutation in the transforming growth factor beta binding protein-like (TB) domain of the FBN1 gene was found. The missense mutation c.3022T>C (C1008R) located in exon 24. This mutation was present in the proband and in two other affected family members, but in neither unaffected family members nor unrelated control subjects. The novel non-synonymous mutation, c.3022T>C (C1008R) in the TB domain of FBN1 gene, may be involved in the pathogenesis of MFS in a Han Chinese family.     

Differential effect of FBN1 mutations on in vitro proteolysis of recombinant fibrillin-1 fragments

Mutations in the fibrillin-1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant disorder of connective tissue with highly variable clinical manifestations. FBN1 contains 47 epidermal growth factor (EGF)-like modules, 43 of which display a consensus sequence for calcium binding (cbEGF). Calcium binding by cbEGF modules is thought to be essential for the conformation and stability of fibrillin-1. Missense mutations in cbEGF modules are the most common mutations found in MFS and generally affect one of the six highly conserved cysteines or residues of the calcium-binding consensus sequence. We have generated a series of recombinant fibrillin-1 fragments containing six cbEGF modules (cbEGF nos. 15-20) with various mutations at different positions of cbEGF module no. 17, which is known to contain a cryptic cleavage site for trypsin. A mutation affecting a residue of the calcium-binding consensus sequence (K1300E) found in a patient with relatively mild clinical manifestations of classic MFS caused a modest increase in susceptibility to in vitro proteolysis by trypsin, whereas a mutation affecting the sixth cysteine residue of the same cbEGF module (C1320S) reported in a severely affected patient caused a dramatic increase in susceptibility to in vitro proteolysis by trypsin. A mutation at the cryptic cleavage site for trypsin abolished sensitivity of wild-type fragments and fragments containing K1300E to trypsin proteolysis. Whereas the relevance of in vitro proteolysis to the in vivo pathogenesis of MFS remains unclear, our findings demonstrate that individual mutations in cbEGF modules can affect these modules differentially and may suggest an explanation for some genotype-phenotype relationships in MFS.     

Effect of mutation type and location on clinical outcome in 1,013 probands with Marfan syndrome or related phenotypes and FBN1 mutations: an international study

Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and have been associated with a wide range of overlapping phenotypes. Clinical care is complicated by variable age at onset and the wide range of severity of aortic features. The factors that modulate phenotypical severity, both among and within families, remain to be determined. The availability of international FBN1 mutation Universal Mutation Database (UMD-FBN1) has allowed us to perform the largest collaborative study ever reported, to investigate the correlation between the FBN1 genotype and the nature and severity of the clinical phenotype. A range of qualitative and quantitative clinical parameters (skeletal, cardiovascular, ophthalmologic, skin, pulmonary, and dural) was compared for different classes of mutation (types and locations) in 1,013 probands with a pathogenic FBN1 mutation. A higher probability of ectopia lentis was found for patients with a missense mutation substituting or producing a cysteine, when compared with other missense mutations. Patients with an FBN1 premature termination codon had a more severe skeletal and skin phenotype than did patients with an inframe mutation. Mutations in exons 24-32 were associated with a more severe and complete phenotype, including younger age at diagnosis of type I fibrillinopathy and higher probability of developing ectopia lentis, ascending aortic dilatation, aortic surgery, mitral valve abnormalities, scoliosis, and shorter survival; the majority of these results were replicated even when cases of neonatal MFS were excluded. These correlations, found between different mutation types and clinical manifestations, might be explained by different underlying genetic mechanisms (dominant negative versus haploinsufficiency) and by consideration of the two main physiological functions of fibrillin-1 (structural versus mediator of TGF beta signalling). Exon 24-32 mutations define a high-risk group for cardiac manifestations associated with severe prognosis at all ages.     

A Gly1127Ser mutation in an EGF-like domain of the fibrillin-1 gene is a risk factor for ascending aortic aneurysm and dissection.

Ascending aortic disease, ranging from mild aortic root enlargement to aneurysm and/or dissection, has been identified in 10 individuals of a kindred, none of whom had classical Marfan syndrome (MFS). Single-strand conformation analysis of the entire fibrillin-1 (FBN1) cDNA of an affected family member revealed a G-to-A transition at nucleotide 3379, predicting a Gly1127Ser substitution. The glycine in this position is highly conserved in EGF-like domains of FBN1 and other proteins. This mutation was present in 9 of 10 affected family members and in 1 young unaffected member but was not found in other unaffected members, in 168 chromosomes from normal controls, and in 188 chromosomes from other individuals with MFS or related phenotypes. FBN1 intragenic marker haplotypes ruled out the possibility that the other allele played a significant role in modulating the phenotype in this family. Pulse-chase studies revealed normal fibrillin synthesis but reduced fibrillin deposition into the extracellular matrix in cultured fibroblasts from a Gly1127Ser carrier. We postulate that the Gly1127Ser FBN1 mutation is responsible for reduced matrix deposition. We suggest that mutations such as this one may disrupt EGF-like domain folding less drastically than do substitutions of cysteine or of other amino acids important for calcium-binding that cause classical MFS. The Gly1127Ser mutation, therefore, produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes to ascending aortic aneurysm and dissection later in life.     

Identification of an FBN1 mutation in bovine Marfan syndrome-like disease

Mutations in the gene encoding fibrillin-1 (FBN1), a component of the extracellular microfibril, cause Marfan syndrome (MFS). Frequent observation of cattle with a normal withers height, but lower body weight than age-matched normal cattle, was recently reported among cattle sired by phenotypically normal Bull A, in Japanese Black cattle. These cattle also showed other characteristic features similar to the clinical phenotype of human MFS, such as a long phalanx proximalis, oval face and crystalline lens cloudiness. We first screened a paternal half-sib family comprising 36 affected and 10 normal offspring of Bull A using the BovineSNP50 BeadChip (illumina). Twenty-two microsatellite markers mapped to a significant region on BTA10 were subsequently genotyped on the family. The bovine Marfan syndrome-like disease (MFSL) was mapped onto BTA10. As FBN1 is located in the significant region, FBN1 was sequenced in Bull A, and three affected and one normal cattle. A G>A mutation at the intron64 splicing accepter site (c.8227-1G>A) was detected in 31 of 36 affected animals (84.7%). The c.8227-1G>A polymorphism was not found in 20 normal offspring of Bull A or in 93 normal cattle unrelated to Bull A. The mutation caused a 1-base shift of the intron64 splicing accepter site to the 3' direction, and a 1-base deletion in processed mRNA. This 1-base deletion creates a premature termination codon, and a 125-amino acid shorter Fibrillin-1 protein is produced from the mutant mRNA. We therefore conclude that the c.8227-1G>A mutation is causative for MFSL. Furthermore, it was suggested that Bull A exhibited germline mosaicism for the mutation, and that the frequency of the mutant sperm was 14.9%.     

A Point Mutation Creating an ExtraN-Glycosylation Site in Fibrillin-1 Results in Neonatal Marfan Syndrome

Fibrillin-1 is a large cysteine-rich glycoprotein of the 10-nm microfibrils in the extracellular matrix. A spectrum of mutations in the fibrillin-1 gene (FBN1) have been identified in patients with Marfan syndrome (MFS), and the majority of mutations resulting in the neonatal and often lethal form of MFS have been identified in the restricted region of exons 24–32 of theFBN1gene. Here we report a novel point mutation in exon 25 of theFBN1gene in a patient with lethal MFS. The mutation resulted in a molecular defect rarely encountered in human diseases, the creation of an extra consensus sequence forN-glycosylation. Metabolic labeling of the patient fibroblast culture andin vitroexpression of the mutagenized cDNA construct suggest that this novelN-glycosylation site is actually utilized. Immunohistochemical and ultrastructural analyses of the fibroblast cultures of the patient show that this excessiveN-glycosylation severely affects microfibril formationin vitro;this finding emphasizes the importance of correct posttranslational modifications of fibrillin molecules for correct aggregation into microfibrillar structures.     

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A–12→G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb

Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A^–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A^–12→G mutation caused GSD IIIb in this patient. Received: 1 August 1997 / Accepted: 22 September 1997     

Multiple Molecular Mechanisms Underlying Subdiagnostic Variants of Marfan Syndrome

Mutations in the FBN1 gene, which encodes fibrillin-1, cause Marfan syndrome (MFS) and have been associated with a wide range of milder, overlap phenotypes. The factors that modulate phenotypic severity, both between and within families, remain to be determined. This study examines the relationship between the FBN1 genotype and phenotype in families with extremely mild phenotypes and in those that show striking clinical variation among apparently affected individuals. In one family, clinically similar but etiologically distinct disorders are segregating independently. In another, somatic mosaicism for a mutant FBN1 allele is associated with subdiagnostic manifestations, whereas germ-line transmission of the identical mutation causes severe and rapidly progressive disease. A third family cosegregates mild mitral valve prolapse syndrome with a mutation in FBN1 that can be functionally distinguished from those associated with the classic MFS phenotype. These data have immediate relevance for the diagnostic and prognostic counseling of patients and their family members.