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.     

Marfan syndrome: whole-exome sequencing reveals de novo mutations,second gene and genotype–phenotype correlations in the Chinese population

Marfan syndrome (MFS) is a dominant monogenic disease caused by mutations in fibrillin 1 (FBN1). Cardiovascular complications are the leading causes of mortality among MFS. In the present study, a whole-exome sequencing of MFS in the Chinese population was conducted to investigate the correlation between FBNI gene mutation and MFS. Forty-four low-frequency harmful loci were identified for the FBN1 gene in HGMD database. In addition, 38 loci were identified in the same database that have not been related to MFS before. A strict filtering and screening protocol revealed two patients of the studied group have double mutations in the FBN1 gene. The two patients harboring the double mutations expressed a prominent, highly pathological phenotype in the affected family. In addition to the FBN1 gene, we also found that 27 patients had mutations in the PKD1 gene, however these patients did not have kidney disease, and 16 of the 27 patients expressed aortic related complications. Genotype-phenotype analysis showed that patients with aortic complications are older in the family, aged between 20 and 40 years.     

Fifteen novel FBN1 mutations causing Marfan syndrome detected by heteroduplex analysis of genomic amplicons.

Mutations in the gene encoding fibrillin-1 (FBN1), a component of the extracellular microfibril, cause the Marfan syndrome (MFS). This statement is supported by the observations that the classic Marfan phenotype cosegregates with intragenic and/or flanking marker alleles in all families tested and that a significant number of FBN1 mutations have been identified in affected individuals. We have now devised a method to screen the entire coding sequence and flanking splice junctions of FBN1. On completion for a panel of nine probands with classic MFS, six new mutations were identified that accounted for disease in seven (78%) of nine patients. Nine additional new mutations have been characterized in the early stages of a larger screening project. These 15 mutations were equally distributed throughout the gene and, with one exception, were specific to single families. One-third of mutations created premature termination codons, and 6 of 15 substituted residues with putative significance for calcium binding to epidermal growth factor (EGF)-like domains. Mutations causing severe and rapidly progressive disease that presents in the neonatal period can occur in a larger region of the gene than previously demonstrated, and the nature of the mutation is as important a determinant as its location, in predisposing to this phenotype.     

Rapid and efficient FBN1 mutation detection using automated sample preparation and direct sequencing as the primary strategy

Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and the other type-1 fibrillinopathies. Finding these mutations is a major challenge considering that the FBN1 gene has a coding region of 8,600 base pairs divided into 65 exons. Most of the more than 600 known mutations have been identified using a mutation scanning method prior to sequencing of fragments with a suspected mutation. However, it is not obvious that these screening methods are ideal, considering cost, efficiency, and sensitivity. We have sequenced the entire FBN1 coding sequence and flanking intronic sequences in samples from 105 patients with suspected MFS, taking advantage of robotic devices, which reduce the cost of supplies and the quantity of manual work. In addition, automation avoids many tedious steps, thus reducing the opportunity for human error. Automated assembling of PCR, purification of PCR products, and assembly of sequencing reactions resulted in completion of the FBN1 sequence in half of the time needed for the manual protocol. Mutations were identified in 69 individuals. The mutation detection rate (76%), types, and genetic distribution of mutations resemble the findings in other MFS populations. We conclude that automated sequencing using the robotic systems is well suited as a primary strategy for diagnostic mutation identification in FBN1.     

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.     

A Novel Mutation of the Fibrillin Gene Causing Ectopia Lentis

Ectopia lentis (EL), a dominantly inherited connective tissue disorder, has been genetically linked to the fibrillin gene on chromosome 15 (FBN1) in earlier studies. Here, we report the first EL mutation in the FBN1 gene confirming that EL is caused by mutations of this gene. So far, several mutations in the FBN1 gene have been reported in patients with Marfan syndrome (MFS). EL and MFS are clinically related but distinct conditions with typical manifestations in the ocular and skeletal systems, the fundamental difference between them being the absence of cardiovascular involvement in EL. We report a point mutation, cosegregating with the disease in the described family, that displays EL over four generations. The mutation changes a conserved glutamic acid residue in an EGF-like motif, which is the major structural component of the fibrillin and is repeated throughout the polypeptide. In vitro mutagenetic studies have demonstrated the necessity of an analogous glutamic acid residue for calcium binding in an EGF-like repeat of human factor IX. This provides a possible explanation for the role of this mutation in the disease pathogenesis.     

The roles of two novel FBN1 gene mutations in the genotype-phenotype correlations of Marfan syndrome and ectopia lentis patients with marfanoid habitus

Mutations in the fibrillin-1 (FBN1) gene have been identified in patients with Marfan syndrome (MFS) and Marfan-like connective tissue disorders. In this study, two Chinese families were recruited. The patients in family 1 were well characterized with MFS, while those in family 2 displayed Marfan-like disorders such as ectopia lentis (EL) and marfanoid habitus, but did not develop cardiovascular diseases. We aimed to analyze the pathogenic mutations and their relationships with phenotypes in these two Chinese families. All participants underwent complete physical, ophthalmic, and cardiovascular examinations. The 65 exons and flanking intronic sequences of FBN1 were amplified by polymerase chain reaction, and screened for mutations by denaturing high-performance liquid chromatography and sequencing. One hundred and fifteen unrelated controls were analyzed using the same methods to confirm the mutations. In family 1, we identified the mutation p.C499S in the calcium-binding epidermal growth factor (cbEGF)-like domain 3 of FBN1. In family 2, the mutation p.C908Y was identified in an interdomain region of the hybrid motif 2 linked to the cbEGF-like domain 10. It can be concluded that FBN1 mutations involving cysteine substitutions are usually associated with MFS and EL with some MFS features. Moreover, pathology seemed more serious when the mutations disrupted the three disulfide bridges in the cbEGF-like domains, which was more likely to cause typical MFS than if the mutations occurred in the hybrid motifs. Our data preliminarily establish a genotype-phenotype correlation in the diagnostic process of MFS and predominant EL with Marfan-like features.     

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。     

Recurrence of Marfan syndrome as a result of parental germ-line mosaicism for an FBN1 mutation

Mutations in the FBN1 gene cause Marfan syndrome (MFS), a dominantly inherited connective tissue disease. Almost all the identified FBN1mutations have been family specific, and the rate of new mutations is high. We report here a de novo FBN1mutation that was identified in two sisters with MFS born to clinically unaffected parents. The paternity and maternity were unequivocally confirmed by genotyping. Although one of the parents had to be an obligatory carrier for the mutation, we could not detect the mutation in the leukocyte DNA of either parent. To identify which parent was a mosaic for the mutation we analyzed several tissues from both parents, with a quantitative and sensitive solid-phase minisequencing method. The mutation was not, however, detectable in any of the analyzed tissues. Although the mutation could not be identified in a sperm sample from the father or in samples of multiple tissue from the mother, we concluded that the mother was the likely mosaic parent and that the mutation must have occurred during the early development of her germ-line cells. Mosaicism confined to germ-line cells has rarely been reported, and this report of mosaicism for the FBN1 mutation in MFS represents an important case, in light of the evaluation of the recurrence risk in genetic counseling of families with MFS.     

Left Ventricular Systolic Dysfunction in Asymptomatic Marfan Syndrome Patients Is Related to the Severity of Gene Mutation: Insights from the Novel Three Dimensional Speckle Tracking Echocardiography

Background

In asymptomatic Marfan syndrome (MFS) patients we evaluated the relationship between the types of fibrillin-1 (FBN1) gene mutation and possible altered left ventricular (LV) function as assessed by three-dimensional speckle tracking echocardiography (3D-STE).

Methods and Results

Forty-five MFS patients (mean age 24±15 years) and 40 age-matched healthy controls were studied. Genetic evaluation for the FBN1 gene was carried on 32 MFS patients. Gene mutation (n = 15, 47%) was classified as mild when the mutation resulted in nearly normally functioning protein, while mutations resulting in abnormally function protein were considered to be severe (n = 17, 53%). All patients and controls underwent 3D-STE for evaluation of LV function by an echocardiographer blinded to the results of the genetic testing. Compared to controls, MFS patients had significantly lower 3D-STE derived LV ejection fraction (EF, 57.43±7.51 vs. 62.69±4.76%, p = 0.0001), global LV longitudinal strain (LS, 14.85±2.89 vs. 17.90±2.01%, p = 0.0001), global LV circumferential strain (CS, 13.93±2.81 vs. 16.82±2.17%, p = 0.0001) and global LV area strain (AS, 25.76±4.43 vs. 30.51±2.61%, p = 0.0001). Apart from the global LV LS all these parameters were significantly lower in patients with severe gene mutation than in those with mild mutation (p<0.05). In the multivariate linear regression analysis only the type of mutation had a significant influence on the 3D-STE derived LVEF (p = 0.017), global CS (p = 0.005) and global AS (p = 0.03).

Conclusions

In asymptomatic MFS patients latent LV dysfunction can be detected using 3D STE. The LV dysfunction is mainly related to the severity of gene mutation, suggesting possible primary cardiomyopathy in MFS patients.     

Marfan Database (second edition): 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. These mutations are private, essentially missense, generally non-recurrent and widely distributed throughout the gene. To date no clear genotype/phenotype relationship has been observed excepted for the localization of neonatal mutations in a cluster between exons 24 and 32. The second version of the computerized Marfan database contains 89 entries. The software has been modified to accomodate new functions and routines.     

The pathogenicity of the Pro1148Ala substitution in the FBN1 gene: causing or predisposing to Marfan syndrome and aortic aneurysm, or clinically innocent?

In individuals with the Marfan syndrome (MFS), mutations have been identified in the fibrillin-1 gene (FBN1) at 15q21.1. A proline-to-alanine change at position 1148 in exon 27 (Pro1148Ala) has been reported in probands with MFS, aortic aneurysm or Marfanoid-craniosynostosis. It was suggested that this mutation could be a risk factor for aortic dilatation, since it was rarely observed in control populations. To investigate further the pathogenicity of this substitution, we screened 416 unrelated control individuals by allele-specific oligonucleotide (ASO) hybridization. We found 16 individuals who carried the alanine allele (3.8%), 3 of whom were homozygous. Five were of Latin American and eight were of Asian extraction. We also screened 133 probands with MFS, aortic aneurysm or related connective tissue disorders and found 4 (3%) that were heterozygous for the 1148Ala allele. All positive results were confirmed by DNA sequencing. In 20 individuals with 1148Ala, we confirmed the association with the rarer A allele at the IVS27-5G→A polymorphism. Our results suggest that the Pro1148Ala change is a polymorphism of ancient evolutionary origin that is more prevalent in Asian and Latin American than in Caucasian or African populations. Received: 4 October 1996 / Revised: 3 December 1996     

Solution structure and dynamics of a calcium binding epidermal growth factor-like domain pair from the neonatal region of human fibrillin-1

Fibrillin-1 is a mosaic protein mainly composed of 43 calcium binding epidermal growth factor-like (cbEGF) domains arranged as multiple, tandem repeats. Mutations within the fibrillin-1 gene cause Marfan syndrome (MFS), a heritable disease of connective tissue. More than 60% of MFS-causing mutations identified are localized to cbEGFs, emphasizing that the native properties of these domains are critical for fibrillin-1 function. The cbEGF12-13 domain pair is within the longest run of cbEGFs, and many mutations that cluster in this region are associated with severe, neonatal MFS. The NMR solution structure of Ca(2+)-loaded cbEGF12-13 exhibits a near-linear, rod-like arrangement of domains. This observation supports the hypothesis that all fibrillin-1 (cb)EGF-cbEGF pairs, characterized by a single interdomain linker residue, possess this rod-like structure. The domain arrangement of cbEGF12-13 is stabilized by additional interdomain packing interactions to those observed for cbEGF32-33, which may help to explain the previously reported higher calcium binding affinity of cbEGF13. Based on this structure, a model of cbEGF11-15 that encompasses all known neonatal MFS missense mutations has highlighted a potential binding region. Backbone dynamics data confirm the extended structure of cbEGF12-13 and lend support to the hypothesis that a correlation exists between backbone flexibility and cbEGF domain calcium affinity. These results provide important insight into the potential consequences of MFS-associated mutations for the assembly and biomechanical properties of connective tissue microfibrils.     

Structural signatures and membrane helix 4 in GLUT1: inferences from human blood-brain glucose transport mutants

Exon IV of SLC2A1, a multiple facilitator superfamily (MFS) transporter gene, is particularly susceptible to mutations that cause GLUT1 deficiency syndrome, a human encephalopathy that results from decreased glucose flux through the blood-brain barrier. Genotyping of 100 patients revealed that in a third of them who harbor missense mutations in the GLUT1 transporter, transmembrane domain 4 (TM4), encoded by SLC2A1 exon IV, contains mutant residues that have the periodicity of one face of a kinked alpha-helix. Arg-126, located at the amino terminus of TM4, is the locus for most of the mutations followed by other arginine and glycine residues located elsewhere in the transporter but conserved among MFS proteins. The Arg-126 mutants were constructed and assayed for protein expression, targeting, and transport capacity in Xenopus oocytes. The role of charge at position 126, as well as its accessibility, was investigated in R126H by determining its activity as a function of extracellular pH. The results indicate that intracellular charges at the MFS TM2-3 and TM8-9 signature loops and flanking TMs 3, 5, and 6 are critical for the structure of GLUT1 as are TM glycines and that TM4, located at the catalytic core of MFS proteins, forms a helix that surfaces into the extracellular solution where another proton facilitates transport.     

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.     

Application of next-generation sequencing to screen for pathogenic mutations in 123 unrelated Chinese patients with Marfan syndrome or a related disease

Marfan syndrome(MFS) is a systemic connective tissue disease principally affecting the ocular, skeletal and cardiovascular systems. This autosomal dominant disorder carries a prevalence of 1:3,000 to 1:5,000. This study aims to define the mutational spectrum of MFS related genes in Chinese patients and to establish genotype-phenotype correlations in MFS. Panel-based targeted next-generation sequencing was used to analyze the FBN1, TGFBR1 and TGFBR2 genes in 123 unrelated Chinese individuals with MFS or a related disease. Genotype-phenotype correlation analyses were performed in mutation-positive patients. The results showed that 97 cases/families(78.9%; 97/123) harbor at least one(likely) pathogenic mutation, most of which were in FBN1; four patients had TGFBR1/2 mutations; and one patient harbored a SMAD3 mutation. Three patients had two FBN1 mutations, and all patients showed classical MFS phenotypes. Patients with a dominant negative-FBN1 mutation had a higher prevalence of ectopia lentis(EL). Patients carrying a haploinsufficiency-FBN1 mutation tended to have aortic dissection without EL. This study extends the spectrum of genetic backgrounds of MFS and enriches our knowledge of genotype-phenotype correlations.