Identification of fibrillin-1 gene mutations in Marfan syndrome by high-resolution melting analysis

Marfan syndrome has been associated with approximately 562 mutations in the fibrillin-1 (FBN1) gene. Mutation scanning of the FBN1 gene with DNA direct sequencing is time-consuming and expensive because of its large size. This study analyzed the diagnostic value of high-resolution melting analysis as an alternative method for scanning of the FBN1 gene. A total of 75 polymerase chain reaction (PCR) amplicons (179-301 bp, average 256 bp) that covered the complete coding regions and splicing sites were evaluated on the 96-well LightCycler system. Melting curves were analyzed as fluorescence derivative plots (−dF/dT vs. temperature). To determine the sensitivity of this method, a total of 82 samples from patients with Marfan syndrome and 50 unaffected individuals were analyzed. All mutations reported in this study had been confirmed previously by direct sequencing analysis. Melting analysis identified 48 heterozygous variants. The variant c.3093 G>T (exon 25) was incorrectly identified by melting curve analysis. The sensitivity of the technique in this sample was 98.78% (81/82). This study demonstrated that high-resolution melting analysis is a reliable gene scanning method with greater speed than DNA sequencing. Our results support the use of this technology as an alternative method for the diagnosis of Marfan syndrome as well as its suitability for high-throughput mutation scanning of other large genes.     

Analysis of the fibrillin-1 gene (FBN1) in patients with Marfan syndrome

Thirty exons of the fibrillin-1 gene (FBN1) were screened in patients with Marfan syndrome (MFS), and eleven point mutations, insertions, and deletions were detected. These included two missense mutations resulting in conformational changes of the calcium-binding EGF-like domains of FBN1 and nine polymorphisms located in both coding and noncoding regions of FBN1. Three intragenic polymorphic microsatellite loci—MTS-1, MTS-2, and MTS-4—were analyzed in MFS patients and unrelated healthy controls, and significant differences between these two groups were found for the MTS-2 and MTS-4 allele frequency distributions. Haplotype frequency distributions on wild-type and mutant chromosomes of MFS patients were also significantly different. The predominant haplotype was 2-11-8 on wild-type chromosomes, and 2-2-8 on mutant chromosomes. These data are a prerequisite to working out DNA diagnosis of MFS.     

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.     

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     

Neonatal lethality of mouse A/J-7SM consomic strain is caused by an insertion mutation in the Dchs1 gene

Mammalian Genome - Homosomic mice of the A/J-7SM consomic mouse strain that introduced the entire chromosome 7 (Chr 7) of SM/J into the A/J strain exhibited neonatal lethality. We tentatively...     

Classical and neonatal Marfan syndrome mutations in fibrillin-1 cause differential protease susceptibilities and protein function

Mutations in fibrillin-1 give rise to Marfan syndrome (MFS) characterized by vascular, skeletal, and ocular abnormalities. Fibrillins form the backbone of extracellular matrix microfibrils in tissues including blood vessels, bone, and skin. They are crucial for regulating elastic fiber biogenesis and growth factor bioavailability. To compare the molecular consequences of mutations causing the severe neonatal MFS with mutations causing the milder classical MFS, we introduced representative point mutations from each group in a recombinant human fibrillin-1 fragment. Structural effects were analyzed by circular dichroism spectroscopy and analytical gel filtration chromatography. Proteolytic susceptibility was probed with non-physiological and physiological proteases, including plasmin, thrombin, matrix metalloproteinases, and cathepsins. All mutant proteins showed a similar gross secondary structure and no differences in heat stability as compared with the wild-type protein. Proteins harboring neonatal mutations were typically more susceptible to proteolytic cleavage compared with those with classical mutations and the wild-type protein. Proteolytic neo-cleavage sites were found both in close proximity and distant to the mutations, indicating small but significant structural changes exposing cryptic cleavage sites. We also report for the first time that cathepsin K and V cleave non-mutated fibrillin-1 at several domain boundaries. Compared with the classical mutations and the wild type, the group of neonatal mutations more severely affected the ability of fibrillin-1 to interact with heparin/heparan sulfate, which plays a role in microfibril assembly. These results suggest differential molecular pathogenetic concepts for neonatal and classical MFS including enhanced proteolytic susceptibility for physiologically relevant enzymes and loss of function for heparin binding.     

AFBN1 gene in patients with Marfan syndrome

30 exons have been analyzed using SSCP in patients with MFS or related phenotypes. We report 2 missense mutations occur in calcium-binding Epidermal Growth Factor-like (EGFcb) domains and 9 polymorphisms located both in coding and noncoding regions of FBN1 gene. Three intragenic microsatellite polymorphic markers MTS-1, MTS-2 and MTS-4 have been analyzed in patients with MFS and unrelated unaffected control individuals. We found significant differences in allele frequency distribution of MTS-2 and MTS-4 loci between MFS patients and unaffected individuals. Haplotype frequency distribution on normal and mutant chromosomes were significantly different. The most common haplotype was 2-11-8 which was predominant on normal chromosomes of affected individuals. Haplotype 2-2-8 was observed in 18% of cases on mutant chromosomes and in 4% of cases on normal chromosomes. These data demonstrate possibility and application of haplotype-segregation analysis with use of these intragenic markers for diagnostic purposes in affected families by Marfan's syndrome.     

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.     

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.     

Functional consequences of an in vivo mutation in exon 10 of the human GLUT1 gene

The functional consequences of an in vivo heterozygous insertion mutation in the human facilitated glucose transporter isoform 1 (GLUT1) gene were investigated. The resulting frameshift in exon 10 changed the primary structure of the C-terminus from 42 in native GLUT1 to 61 amino acid residues in the mutant. Kinetic studies on a patient's erythrocytes were substantiated by expressing the mutant cDNA in Xenopus laevis oocytes. K(m) and V(max) values were clearly decreased explaining pathogenicity. Targeting to the plasma membrane was comparable between mutant and wild-type GLUT1. Transport inhibition by cytochalasin B was more effective in the mutant than in the wild-type transporter. The substrate specificity of GLUT1 remained unchanged.     

Acute intermittent porphyria caused by a G to C mutation in exon 12 of the porphobilinogen deaminase gene that results in exon skipping

Genomic DNA from a patient with acute intermittent porphyria were analyzed by the polymerase chain reaction (PCR)-direct sequencing method. The patient was heterozygote for a point mutation G to C at the last position of exon 12 of the porphobilinogen deaminase (PBG-D) gene. Analysis of the cDNA fragments amplified by PCR revealed that the patient has the abnormal PBG-D mRNA, which does not have exon 12 and exists in an approximately equal amount to the normal mRNA.     

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%.     

Grey, a novel mutation in the murine Lyst gene, causes the beige phenotype by skipping of exon 25

The murine beige mutant phenotype and the human Chediak-Higashi syndrome are caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene and the human CHS gene, respectively. In this report we have analyzed a novel murine mutant Lyst allele, called Lyst(bg-grey), that had been found in an ENU mutation screen and named grey because of the grey coat color of affected mice. The phenotype caused by the Lyst(bg-grey) mutation was inherited in a recessive fashion. Melanosomes of melanocytes associated with hair follicles and the choroid layer of the eye, as well as melanosomes in the neural tube-derived pigment epithelium of the retina, were larger and irregularly shaped in homozygous mutants compared with those of wild-type controls. Secretory vesicles in dermal mast cells of the mutant skin were enlarged as well. Test crosses with beige homozygous mutant mice (Lyst(bg)) showed that double heterozygotes (Lyst(bg)/Lyst(bg-grey)) were phenotypically indistinguishable from either homozygous parent, demonstrating that the ENU mutation was an allele of the murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in Lyst(bg-grey) mutants, which is predicted to cause a missense D2399E mutation and the loss of the following 77 amino acids encoded by exon 25 but leave the C-terminal end of the protein intact. Analysis of the genomic Lyst locus around exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C transition point mutation. Western blot analysis suggests that the Lyst(bg-grey) mutation causes instability of the LYST protein. Because the phenotype of Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation defines a critical region for the stability of the murine LYST protein.     

Effect of an exon 1 mutation in the myostatin gene on the growth traits of the Bian chicken

Myostatin (MSTN), or growth and differentiation factor 8 (GDF8), is a member of the transforming growth factor (TGF)-β superfamily. This family functions as a negative regulator of skeletal muscle development and growth in mammals. Single-nucleotide polymorphisms in exon 1 of the Bian chicken myostatin gene were detected by polymerase chain reaction-restriction fragment length polymorphism. A mutation (c.234G>A) in exon 1 was found. Female Bian chickens of genotypes AA and GA had significantly higher body weights than those of genotype GG (P < 0.05 or P < 0.01) from 6 to 18 weeks of age. These results suggested that the mutation c.234G>A in exon 1 could be used as a genetic marker for Bian chicken growth traits.     

Detection of a novel mutation in exon 20 of the BRCA1 gene

Hereditary breast cancer constitutes 5–10% of all breast cancer cases. Inherited mutations in the BRCA1 and BRCA2 tumor-suppressor genes account for the majority of hereditary breast cancer cases. The BRCA1 C-terminal region (BRCT) has a functional duplicated globular domain, which helps with DNA damage repair and cell cycle checkpoint protein control. More than 100 distinct BRCA1 missense variants with structural and functional effects have been documented within the BRCT domain. Interpreting the results of mutation screening of tumor-suppressor genes that can have high-risk susceptibility mutations is increasingly important in clinical practice. This study includes a novel mutation, p.His1746 Pro (c.5237A>C), which was found in BRCA1 exon 20 of a breast cancer patient. In silico analysis suggests that this mutation could alter the stability and orientation of the BRCT domain and the differential binding of the BACH1 substrate.     

Familial occurrence of typical and severe lethal congenital contractural arachnodactyly caused by missplicing of exon 34 of fibrillin-2.

Genetic linkage studies have linked congenital contractural arachnodactyly (CCA), a usually mild heritable connective-tissue disorder, to FBN2, the fibrillin gene on chromosome 5. Recently, FBN2 mutations in two patients with CCA have been described. Here we report an A-->T transversion at the -2 position of the consensus acceptor splice site, resulting in the missplicing of exon 34, a calcium-binding epidermal growth factor-like repeat in fibrillin-2 in a mother and daughter with CCA. Significantly, the mother exhibited a classic CCA phenotype with arachnodactyly, joint contractures, and abnormal pinnae, whereas her daughter exhibited a markedly more severe CCA phenotype, which included cardiovascular and gastrointestinal anomalies that led to death in infancy. Analysis of cloned fibroblasts showed that the mother is a somatic mosaic for the exon 34 missplicing mutation, whereas all the daughter''s cells harbored the mutation.