Parental somatic and germ-line mosaicism for a multiexon deletion with unusual endpoints in a type III collagen (COL3A1) allele produces Ehlers-Danlos syndrome type IV in the heterozygous offspring.

Ehlers-Danlos syndrome (EDS) type IV is a dominantly inherited disorder that results from mutations in the type III collagen gene (COL3A1). We studied the structure of the COL3A1 gene of an individual with EDS type IV and that of her phenotypically normal parents. The proband was heterozygous for a 2-kb deletion in COL3A1, while her father was mosaic for the same deletion in somatic and germ cells. In fibroblasts from the father, approximately two-fifths of the COL3A1 alleles carried the deletion, but only 10% of the COL3A1 alleles in white blood cells were of the mutant species. The deletion in the mutant allele extended from intron 7 into intron 11. There was a 12-bp direct repeat in intron 7 and intron 11, the latter about 60 bp 5' to the junction. At the breakpoint there was a duplication of 10 bp from intron 11 separated by an insertion of 4 bp contained within the duplicated sequence. The father was mosaic for the deletion so that the gene rearrangement occurred during his early embryonic development prior to lineage allocation. These findings suggest that at least some of the deletions seen in human genes may occur during replication, rather than as a consequence of meiotic crossing-over, and that they thus have a risk for recurrence when observed de novo.     

Haploinsufficiency for one COL3A1 allele of type III procollagen results in a phenotype similar to the vascular form of Ehlers-Danlos syndrome, Ehlers-Danlos syndrome type IV

Mutations in the COL3A1 gene that encodes the chains of type III procollagen result in the vascular form of Ehlers-Danlos syndrome (EDS), EDS type IV, if they alter the sequence in the triple-helical domain. Although other fibrillar collagen-gene mutations that lead to allele instability or failure to incorporate proalpha-chains into trimers-and that thus reduce the amount of mature molecules produced-result in clinically apparent phenotypes, no such mutations have been identified in COL3A1. Furthermore, mice heterozygous for Col3a1 "null" alleles have no identified phenotype. We have now found three frameshift mutations (1832delAA, 413delC, and 555delT) that lead to premature termination codons (PTCs) in exons 27, 6, and 9, respectively, and to allele-product instability. The mRNA from each mutant allele was transcribed efficiently but rapidly degraded, presumably by the mechanisms of nonsense-mediated decay. In a fourth patient, we identified a point mutation, in the final exon, that resulted in a PTC (4294C-->T [Arg1432Ter]). In this last instance, the mRNA was stable but led to synthesis of a truncated protein that was not incorporated into mature type III procollagen molecules. In all probands, the presenting feature was vascular aneurysm or rupture. Thus, in contrast to mutations in genes that encode the dominant protein of a tissue (e.g., COL1A1 and COL2A1), in which "null" mutations result in phenotypes milder than those caused by mutations that alter protein sequence, the phenotypes produced by these mutations in COL3A1 overlap with those of the vascular form of EDS. This suggests that the major effect of many of these dominant mutations in the "minor" collagen genes may be expressed through protein deficiency rather than through incorporation of structurally altered molecules into fibrils.     

Exclusion of COL1A1, COL1A2, and COL3A1 genes as candidate genes for Ehlers-Danlos syndrome type I in one large family

Summary Ehlers-Danlos syndrome (EDS) type I is a generalized connective tissue disorder, the major manifestations of which are soft, velvety hyperextensible skin and moderately severe joint hypermobility. The gene defect or defects causing EDS type I have not yet been defined, but previous observations suggested that the syndrome may be caused by mutations in the genes for type-I collagen (COL1A1 and COL1A2) or type-III collagen (COL3A1). Here, we performed linkage studies for these three genes in large Azerbaijanian family with EDS type I. Three polymorphisms in the COL3A1 gene, two in the COL1A1 gene, and one in the COL1A2 gene were tested using the polymerase chain reaction. The data obtained excluded linkage of any of the three genes to EDS type I in the family.On leave of absence from Institute of Human Genetics, National Research Center of Medical Genetics, Moskvorechie St., 1. Moscow 115478, USSR     

Linkage of a polymorphic marker for the type III collagen gene (COL3A1) to atypical autosomal dominant Ehlers-Danlos syndrome type IV in a large Belgian pedigree

Summary We have examined a large family in which eleven members have a form of autosomal dominant Ehlers-Danlos syndrome type IV. Analysis of fibroblast cultures from affected individuals showed a partial deficiency of type III collagen production. The protein produced was, however, normal in all aspects examined. Using a restriction site polymorphism associated with the structural gene for human type III collagen (COL3A1), we have found tight linkage between the low frequency polymorphic allele and the clinical expression of the disease (lod=3.86 at =0), identifying the type III collagen gene as the disease locus.     

Molecular defects of type III procollagen in Ehlers-Danlos syndrome type IV

Summary Fibroblasts from most patients with Ehlers-Danlos syndrome (EDS) type IV, a disorder characterized by fragility of skin, blood vessels, and internal organs, secrete reduced amounts of type III procollagen. In 7 of 8 cell strains analyzed, we found evidence of structural defects in half of the type III procollagen chains synthesized, such as deletions or bona fide amino acid substitutions, which cause delayed formation and destabilization of the collagen triple helix and, as a consequence, reduced secretion of the molecule. The data suggest that EDS type IV is often caused by heterozygosity for mutations at the COL3A1 locus, which affect the structure of type III procollagen. The triple-helical region of the molecule, like the homologous region of type I procollagen, appears to be particularly vulnerable.Parts of this work have been presented at the 2nd International Conference on Molecular Biology and Pathology of Matrix, Philadelphia, June 15–18, 1988     

Vascular Ehlers-Danlos syndrome

Vascular Ehlers-Danlos syndrome, also known as Ehlers-Danlos syndrome type IV, is a life-threatening inherited disorder of connective tissue, resulting from mutations in the COL3A1 gene coding for type III procollagen. Vascular EDS causes severe fragility of connective tissues with arterial and gastrointestinal rupture, and complications of surgical and radiological interventions. As for many rare orphan diseases, delay in diagnosis is common, even when the clinical features are typical, leading to inadequate or inappropriate treatment and management. In childhood many individuals with vascular EDS are first thought to have coagulation disorders. In adulthood, four main clinical findings, including a striking facial appearance, easy bruising, translucent skin with visible veins and rupture of vessels, gravid uterus or intestines, contribute to the diagnosis, which can be confirmed by SDS-PAGE studies of type III procollagen molecules synthesis by cultured fibroblasts or by the identification of a mutation in the COL3A1 gene coding for type III procollagen. Vascular EDS is inherited as an autosomal dominant trait. Varied molecular mechanisms have been observed and, of the mutations described to date, most have been unique to each family or "private", with no correlation between genotype and phenotype. Vascular EDS is of particular importance to surgeons, radiologists, obstetricians and geneticists since, although there is currently no specific treatment for the condition, knowledge of the diagnosis may help in the management of visceral complications, pregnancy and genetic counseling.     

Identification of novel pro-alpha2(IX) collagen gene mutations in two families with distinctive oligo-epiphyseal forms of multiple epiphyseal dysplasia.

Multiple epiphyseal dysplasia (MED) is a genetically heterogeneous disorder with marked clinical and radiographic variability. Traditionally, the mild "Ribbing" and severe "Fairbank" types have been used to define a broad phenotypic spectrum. Mutations in the gene encoding cartilage oligomeric-matrix protein have been shown to result in several types of MED, whereas mutations in the gene encoding the alpha2 chain of type IX collagen (COL9A2) have so far been found only in two families with the Fairbank type of MED. Type IX collagen is a heterotrimer of pro-alpha chains derived from three distinct genes-COL9A1, COL9A2, and COL9A3. In this article, we describe two families with distinctive oligo-epiphyseal forms of MED, which are heterozygous for different mutations in the COL9A2 exon 3/intron 3 splice-donor site. Both of these mutations result in the skipping of exon 3 from COL9A2 mRNA, but the position of the mutation in the splice-donor site determines the stability of the mRNA produced from the mutant COL9A2 allele.     

Splicing Defects in the COL3A1 Gene: Marked Preference for 5′ (Donor) Splice-Site Mutations in Patients with Exon-Skipping Mutations and Ehlers-Danlos Syndrome Type IV

Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis.     

Marfan syndrome: exclusion of genetic linkage to five genes coding for connective tissue components in the long arm of chromosome 2

Summary Marfan syndrome represents a heterogeneous connective tissue disease, the symptoms arising in several tissues and organs. The defective gene(s) behind this autosomal dominant condition has not been found despite considerable research. The main targets of the research have been the genes coding for connective tissue components. Several of the candidate genes suspected to be defective in Marfan syndrome are located on the long arm of chromosome 2. These genes include a cluster of two genes coding for fibrillar collagens COL3A1 and COL5A2, and a third member of the collagen gene family: COL6A3. Furthermore, genes for elastin (ELN) and fibronectin (FN) are also located in this area of chromosome 2. We studied this chromosomal area using restriction fragment length polymorphism (RFLP) linkage analysis in five Finnish Marfan families with affected members in three generations. In two point linkage analyses, Lod scores of –3.192 ( = 0.1) to COL3A1, –1.683 ( = 0) to COL6A3 and –2.664 ( = 0.01) to FN were obtained, whereas the linkage analysis between elastin and the disease was non-informative (Lod score 0.444, = 0). With the multipoint linkage analysis that permits simultaneous examination of several loci and more efficient use of family data, we obtained an exclusion of all these loci as the site of the mutation leading to Marfan syndrome in these families.     

The human tyrosine aminotransferase gene: characterization of restriction fragment length polymorphisms and haplotype analysis in a family with tyrosinemia type II

Summary Deficiency in hepatic tyrosine aminotransferase (TAT) causes tyrosinemia type II, an autosomal recessively inherited disorder. Using a TAT cosmid clone, we have identified an MspI restriction fragment length polymorphism (RFLP) 5 to the TAT gene, with allele frequencies of 0.63 and 0.37. Analysis of the cloned maternal and paternal TAT alleles from patient with tyrosinemia type II led to the identification of a HaeIII RFLP at the 3 end of the TAT gene, with allele frequencies of 0.94 and 0.06. The two RFLPs are 27 kb apart and in no allelic association. From haplotype frequencies, a polymorphism information content (PIC) value of 0.44 was obtained. The two RFLPs have allowed the unambiguous identification of the mutant TAT alleles in the patient's pedigree by haplotype analysis.     

Genetic heterogeneity of severe von Willebrand disease type III in the German population

The genetic heterogeneity of severe von Willebrand disease (vWd) type III was estimated by analysing extended haplotypes of eleven intragenic restriction fragment length polymorphisms and one variable number of tandem repeat polymorphism in 32 patients from 28 families from Germany or of German origin. All patients were screened for gross deletions and for mutations at potential hot spot regions of the von Willebrand factor (vWf) gene. Disease-associated haplotypes were established in 24 families. Only a few, apparently unrelated families shared common haplotypes suggesting a considerable genetic heterogeneity in the German population of vWd type III patients. Defects causing vWd type III were identified on 14 out of 56 chromosomes (25%). Gross deletions were detected in two families. A complete homozygous deletion of the vWf gene was displayed in one patient. Another patient was compound heterozygous for a large deletion of at least 100 kb of the vWf gene with an additional, as yet unidentified, defect. One homozygous missense mutation was detected in exon 10, and two non-sense mutations were detected in exon 8 and exon 45 of the vWf gene, respectively. A frameshift mutation (C) in exon 18 was identified in five families and an additional frameshift mutation (G) was found in exon 28 in one family. It appears that C is the most common molecular defect in German patients with vWd type III. Its association with a number of different haplotypes suggests repeated de novo mutations at a mutation hot spot. Evidence is presented that particular molecular defects causing vWd type III are associated with different patterns of inheritance, depending on their location within the vWf gene. Complete deletions of the gene and nonsense mutations in the pro-sequence are correlated with recessive inheritance, whereas frameshift and nonsense mutations in the gene sequence corresponding to the mature vWf subunit tend to be inherited in a dominant fashion.     

Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV

Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an alphaGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.     

Osteogenesis imperfecta type I: molecular heterogeneity for COL1A1 null alleles of type I collagen.

Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 "null" allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5' donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon, that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype.     

The Stickler syndrome: Evidence for close linkage to the structural gene for type II collagen

The Stickler syndrome is an autosomal dominant hereditary disorder of connective tissue with pleiotropic features including premature osteoarthropathy, mild spondyloepiphyseal dysplasia, vitreoretinal degeneration, and the Pierre-Robin sequence. Genetic linkage studies in two families with the Stickler syndrome have been performed using restriction fragment length polymorphisms associated with the structural gene for type II collagen, COL2A1. No recombinants between the Stickler phenotype and COL2A1 were observed. The total LOD score for linkage of the Stickler syndrome and COL2A1 at a recombination fraction (theta) of zero is 3.59. These findings suggest that, at least in some families, the mutation causing Stickler syndrome affects the structural locus for type II collagen.     

Genetic linkage analysis of hereditary arthro-ophthalmopathy (Stickler syndrome) and the type II procollagen gene.

Hereditary arthro-ophthalmopathy (AO), or Stickler syndrome, is a dominantly inherited disorder characterized by vitreo-retinal degeneration and frequently accompanied by epiphyseal dysplasia and premature degenerative joint disease. Three large families with AO were analyzed for clinical manifestations of the disease and for coinheritance of the genetic defect with RFLPs in the type II procollagen gene (COL2A1). Genetic linkage between AO and COL2A1 was demonstrated in the largest family, with a maximum LOD score of 3.52 at a recombination distance of zero. Data from a second family also supported linkage of AO and COL2A1, with a LOD score of 1.20 at a recombination distance of zero. These results are consistent with the conclusion that mutations in the COL2A1 gene are responsible for AO in these two families. In a third AO family, however, recombination between AO and COL2A1 occurred in at least one meiosis, and the data were inconclusive with respect to linkage.     

Colocalization of the genes for the alpha 3(IV) and alpha 4(IV) chains of type IV collagen to chromosome 2 bands q35-q37.

Each type of basement membrane in man contains between two and five genetically distinct type IV collagens: alpha 1(IV)-alpha 5(IV). Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been isolated. We have recently isolated partial cDNAs for the fifth member of the family, designated alpha 4(IV). On the basis of comparison of the deduced peptide sequences of all five chains, the type IV collagens can be divided into two families: alpha 1-like, comprising alpha 1(IV), alpha 3(IV), and alpha 5(IV); and alpha 2-like, comprising alpha 2(IV) and alpha 4(IV). Genes encoding the alpha 1(IV) and alpha 2(IV) chains (COL4A1 and COL4A2) both map to human chromosome 13q34 and have been shown to be transcribed from opposite DNA strands using a common bidirectional promoter that allows coordinate regulation of the two chains. Indeed, these two chains are commonly found together in basement membrane and form [alpha 1]2.[alpha 2] heterotrimers. Whereas alpha 1(IV) and alpha 2(IV) have been found in all basement membranes studied hitherto, it has been shown that alpha 3(IV) and alpha 4(IV) are found in only a subset of basement membranes. In basement membranes where either of these molecules is present, however, they are found together. In view of this relationship and the structural similarities between alpha 1(IV) and alpha 3(IV) and between alpha 2(IV) and alpha 4(IV), we hypothesized that COL4A3 and COL4A4, the genes encoding alpha 3(IV) and alpha 4(IV), respectively, have a genomic organization similar to that of COL4A1 and COL4A2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in the vitreous phenotype of Stickler syndrome can be caused by different amino acid substitutions in the X position of the type II collagen Gly-X-Y triple helix

Stickler syndrome is a dominantly inherited disorder characterized by arthropathy, midline clefting, hearing loss, midfacial hypoplasia, myopia, and retinal detachment. These features are highly variable both between and within families. Mutations causing the disorder have been found in the COL2A1 and COL11A1 genes. Premature termination codons in COL2A1 that result in haploinsufficiency of type II collagen are a common finding. These produce a characteristic congenital "membranous" anomaly of the vitreous of all affected individuals. Experience has shown that vitreous slit-lamp biomicroscopy can distinguish between patients with COL2A1 mutations and those with dominant negative mutations in COL11A1, who produce a different "beaded" vitreous phenotype. Here we characterize novel dominant negative mutations in COL2A1 that result in Stickler syndrome. Both alter amino acids in the X position of the Gly-X-Y triple-helical region. A recurrent R365C mutation occurred in two unrelated sporadic cases and resulted in the membranous vitreous anomaly associated with haploinsufficiency. In a large family with linkage to COL2A1, with a LOD score of 2.8, a unique L467F mutation produced a novel "afibrillar" vitreous gel devoid of all normal lamella structure. These data extend the mutation spectrum of the COL2A1 gene and help explain the basis for the different vitreous phenotypes seen in Stickler syndrome.     

Consistent linkage of dominantly inherited osteogenesis imperfecta to the type I collagen loci: COL1A1 and COL1A2

The segregation of COL1A1 and COL1A2, the two genes which encode the chains of type I collagen, was analyzed in 38 dominant osteogenesis imperfecta (OI) pedigrees by using polymorphic markers within or close to the genes. This was done in order to estimate the consistency of linkage of OI genes to these two loci. None of the 38 pedigrees showed evidence of recombination between the OI gene and both collagen loci, suggesting that the frequency of unlinked loci in the population must be low. From these results, approximate 95% confidence limits for the proportion of families linked to the type I collagen genes can be set between .91 and 1.00. This is high enough to base prenatal diagnosis of dominantly inherited OI on linkage to these genes even in families which are too small for the linkage to be independently confirmed to high levels of significance. When phenotypic features were compared with the concordant collagen locus, all eight pedigrees with Sillence OI type IV segregated with COL1A2. On the other hand, Sillence OI type I segregated with both COL1A1 (17 pedigrees) and COL1A2 (7 pedigrees). The concordant locus was uncertain in the remaining six OI type I pedigrees. Of several other features, the presence or absence of presenile hearing loss was the best predictor of the mutant locus in OI type I families, with 13 of the 17 COL1A1 segregants and none of the 7 COL1A2 segregants showing this feature.