Benign familial hematuria associated with a novel COL4A4 mutation.

We describe a father and three offspring with hematuria. The father and one girl also complained of flank pain. Renal function tests and ophthalmological examinations were normal in all. The father had very mild neural deafness. The renal biopsy samples of two affected siblings showed changes compatible with thin basement membrane disease. Genetic analysis revealed a novel missense mutation in exon 32 of COL4A4 to be responsible for the phenotype in this family. We suggest that thin basement membrane disease may have overlapping clinical features with other causes of hematuria; genetic analysis may help in the differential diagnosis and help us further understand the disease processes.

Diffuse esophageal leiomyomatosis with perirectal involvement mimicking Hirschsprung disease.

We describe a 25-year-old woman with diffuse esophageal leiomyomatosis. During childhood, achalasia was mistakenly diagnosed in this patient. Subsequently, she underwent cardiomyotomy and developed symptoms of Hirschsprung disease. These symptoms were caused by infiltration of the esophageal and rectal walls by benign muscular hypertrophy. The pseudo-Hirschsprung disorder was manifested by chronic severe constipation, with consistent manometric findings. Clitoral hypertrophy and vulvar and periurethral leiomyoma were also present. Genetic analysis demonstrating deletion of the COL4A5/COL4A6 locus and the discovery of microscopic hematuria implied that the patient could transmit both diffuse leiomyomatosis and the Alport syndrome.

Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome.

Autosomal recessive Alport syndrome is a progressive hematuric glomerulonephritis characterized by glomerular basement membrane abnormalities and associated with mutations in either the COL4A3 or the COL4A4 gene, which encode the alpha3 and alpha4 type IV collagen chains, respectively. To date, mutation screening in the two genes has been hampered by the lack of genomic structure information. We report here the complete characterization of the 48 exons of the COL4A4 gene, a comprehensive gene screen, and the subsequent detection of 10 novel mutations in eight patients diagnosed with autosomal recessive Alport syndrome. Furthermore, we identified a glycine to alanine substitution in the collagenous domain that is apparently silent in the heterozygous carriers, in 11.5% of all control individuals, and in one control individual homozygous for this glycine substitution. There has been no previous finding of a glycine substitution that is not associated with any obvious phenotype in homozygous individuals.

Altered collagen of human pathological fibroblasts impairs the synthesis of fibronectin.

Human fibroblasts with mutated type I collagen have marked defective adhesive capacities on exogenous type I collagen and exogenous fibronectin in comparison to normal fibroblasts. This defective cell adhesion could be partly explained by the decreased level of cell surface receptors of the beta 1-integrin family, i.e., the alpha 2 integrin subunit for type I collagen and the alpha 5 integrin subunit for fibronectin, observed in pathological fibroblasts. However, it appeared that the presence of altered collagen interfered both with fibronectin biosynthesis and with its surface expression. Using a binding assay on immobilized fibronectin, we demonstrated that the mutated collagen had a weaker binding to fibronectin. In addition, the pathological fibroblasts plated on a mixture of normal exogenous type I collagen and fibronectin exhibited the same maximal level of adhesion as control fibroblasts. These results indicate that fibroblasts with the mutated collagen exhibit a decreased binding to normal fibronectin, a modification of synthesis and surface expression of fibronectin, and, finally, altered adhesive capacities.

Novel COL4A5/COL4A6 deletions and further characterization of the diffuse leiomyomatosis-Alport syndrome (DL-AS) locus define the DL critical region.

Diffuse leiomyomatosis (DL) with Alport syndrome (AS) has been shown to be associated with contiguous gene deletions of the COL4A5 and COL4A6 genes, with the COL4A6 breakpoint of the deletions invariably located in the large intron 2 of the gene. We describe four YAC clones covering the locus and a refined restriction map of the entire COL4A6 gene. These resources have allowed us to make a precise estimate of the size of COL4A6 introns 2 and 3, as well as the size of the gene itself. We also describe five novel deletions which, in conjunction with previous reports, allow the definition of a 90-kb critical region in which to search for a gene or other entity involved in the pathogenesis of DL.

Intrafamilial variable expressivity of osteogenesis imperfecta due to mosaicism for a lethal G382R substitution in the COL1A1 gene.

Fibroblasts from a 23 week old fetus affected with lethal (type II) osteogenesis imperfecta (OI) produced normal and abnormal type I procollagen molecules. The abnormal molecules were shown to contain pro alpha 1(I) chains in which the glycine at position 382 of the triple helical domain was substituted by arginine, as the result of a G-to-C transversion at nucleotide 1797 of the pro alpha (I) coding sequence. Also fibroblasts from the apparently normal father produced abnormal type I collagen but the overmodified alpha 1(I) chains tended to disappear with increasing passage number. We determined that the mutant allele accounted for approximately 36% of the COL1A1 alleles in the father's skin fibroblasts. Upon careful clinical reexamination, the man appeared to be very mildly affected with OI. The most plausible explanation for such a phenotypic variation is that the father is a mosaic for a mutation that is lethal in the heterozygous son. This finding confirms previous observations that somatic mosaicism for new dominant mutations is responsible for extreme intrafamilial variability and poses some caveats in genetic counselling.

Substitution of aspartic acid for glycine at position 310 in type II collagen produces achondrogenesis II, and substitution of serine at position 805 produces hypochondrogenesis: analysis of genotype-phenotype relationships.

Two different mutations were found in two unrelated probands with lethal chondrodysplasias, one with achondrogenesis type II and the other with the less severe phenotype of hypochondrogenesis. The mutations in the COL2A1 gene were identified by denaturing gradient gel electrophoresis analysis of genomic DNA followed by dideoxynucleotide sequencing and restriction site analysis. The proband with achondrogenesis type II had a heterozygous single-base mutation that substituted aspartate for glycine at position 310 of the alpha 1(II) chain of type II procollagen. The proband with hypochondrogenesis had a heterozygous single-base mutation that substituted serine for glycine at position 805. Type II collagen extracted from cartilage from the probands demonstrated the presence of type I collagen and a delayed electrophoretic mobility, indicating post-translational overmodifications. Analysis of CNBr peptides showed that, in proband 1, the entire peptides were overmodified. Examination of chondrocytes cultured in agarose or alginate indicated that there was a delayed secretion of type II procollagen. In addition, type II collagen synthesized by cartilage fragments from the probands demonstrated a decreased thermal stability. The melting temperature of the type II collagen containing the aspartate-for-glycine substitution was reduced by 4 degrees C, and that of the collagen containing the serine-for-glycine substitution was reduced by 2 degrees C. Electron microscopy of the extracellular matrix from the chondrocyte cultures showed a decreased density of matrix and the presence of unusually short and thin fibrils. Our results indicate that glycine substitutions in the N-terminal region of the type II collagen molecule can produce more severe phenotypes than mutations in the C-terminal region. The aspartate-for-glycine substitution at position 310, which was associated with defective secretion and a probable increased degradation of collagen, is the most destabilizing mutation yet reported in type II procollagen.

Substitution of an aspartic acid for glycine 700 in the alpha 2(I) chain of type I collagen in a recurrent lethal type II osteogenesis imperfecta dramatically affects the mineralization of bone.

We describe a new dominant mutation of type I collagen responsible for a recurrent lethal osteogenesis imperfecta. Dermal cultured fibroblasts of the proband produced both normal and overmodified type I collagen chains. Previous results (Cohen-Solal, L., Bonaventure, J., and Maroteaux, P. (1991) Hum. Genet. 87, 297-301) and cyanogen bromide peptide mapping after non-equilibrium pH gradient gel electrophoresis indicated that the anomaly was a charge mutation localized in the alpha 2CB3-5A. The mutation was identified as a G to A transition in the COL1A2 gene, which converts glycine 700 to aspartic acid in the alpha 2I chain. This mutation caused the abolition of a ScrFI site, which was also absent in the suspected mosaic father. Pulse-chase experiment showed intracellular retention and increase of the degradation of the synthesized collagen. To understand more directly the tissue defect in osteogenesis imperfecta, skin and especially bone were studied with biochemical and transmission electron microscopy techniques. Collagen matrix of both tissues was dramatically decreased and presented a retarded migration, showing that abnormal molecules were incorporated during the fibrillogenesis. The abnormal collagen mostly remained within the fibroblasts and osteoblasts, which presented typical features of intracellular retention. We observed the presence of spheritic aggregates of mineral, unrelated to the scarce and thin collagen fibrils, in bone. Such abnormal mineralization could be the consequence not only of the decrease of the collagen content but more importantly of the inability of the abnormal molecules to form an organized network necessary to the deposition of apatite crystallites.

Reexpression of cartilage-specific genes by dedifferentiated human articular chondrocytes cultured in alginate beads.

We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoretic analysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (pro alpha 1II, pc alpha 1II, and pn alpha 1II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the alpha 1(II) and alpha 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly alpha 1(II) mRNA, whereas alpha 1(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no alpha 1(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. However it was unable to restore the chondrocytic phenotype in virally transformed cells.

Achondrogenesis type IB (Fraccaro): study of collagen in the tissue and in chondrocytes cultured in agarose.

A lethal chondrodysplasia characterized by extreme micromelia was diagnosed by ultrasound examination in two sibs whose nonconsanguineous parents were healthy. Radiographic and histopathologic data indicated that the two foetuses (18 and 21 weeks old) had achondrogenesis type IB (Fraccaro). Quantitation of total collagen extractable from dried cartilage samples demonstrated a 50% decrease when compared to an age-related control. This decrease was essentially related to type II collagen. Nevertheless, the alpha chains and the CB peptides of type II collagen had a normal electrophoretic mobility. A significant amount of collagen type I was also detected. The electrophoretic pattern of collagens type IX and XI did not differ significantly from control sample. The extracellular matrix elaborated by patient chondrocytes cultured in agarose for 10-12 days, contained less collagen type II than normal cells. Labelling with 14C-proline of cultured cells showed the presence of procollagen and type II collagen chains with a normal electrophoretic mobility, but an alpha 2(I) chain was detectable in the patient material, indicating the presence of collagen type I which supported the tissue findings. The significance of the type II collagen reduction in the patient's cartilage is unclear but it is unlikely to be the primary defect in achondrogenesis type I.

A dominant mutation in the COL1A1 gene that substitutes glycine for valine causes recurrent lethal osteogenesis imperfecta.

Type I collagen chains of a proband from a family with recurrent lethal osteogenesis imperfecta (OI) migrated as a doublet when submitted to gel electrophoresis. Cyanogen bromide (CNBr) peptide mapping demonstrated that the post-translational over-modifications were initiated in alpha 1ICB7. Chemical cleavage of cDNA-RNA heteroduplexes identified a mismatch in the alpha 1I cDNA; this mismatch was subsequently confirmed by sequencing a 249-bp fragment amplified by the polymerase chain reaction. A G to T transition in the second base of the first codon of exon 41 resulted in the substitution of glycine 802 by valine. This mutation impaired collagen secretion by dermal fibroblasts. The over-modified chains were retained intracellularly and melted at a lower temperature than normal chains. Collagen molecules synthesized by parental fibroblasts had a normal electrophoretic mobility, but hybridization of genomic DNA with allele-specific oligonucleotides revealed the presence of the mutant allele in the mother's leukocytes. The mutation was not detected in her fibroblasts consistent with the protein data. These results support the hypothesis that somatic and germ-line mosaicism in the phenotypically normal mother explain the recurrence of OI.

Linkage studies of four fibrillar collagen genes in three pedigrees with Larsen-like syndrome.

We report seven children from three families who had a set of common clinical features suggestive of Larsen-like syndrome, including unusual facies, bilateral dislocations of the knees and elbows, club foot, and short stature. All of the patients originated from the island of La Réunion in the Indian Ocean. The occurrence of several affected sibs in these families and the large number of consanguineous marriages on this island are consistent with autosomal recessive inheritance of the disease. Based on this hypothesis, the pedigrees were used for linkage analysis in a candidate gene assay. Lod score calculations in a pairwise study with four different fibrillar collagen genes, COL1A1, COL1A2, COL3A1, and COL5A2, allowed us to exclude these genes as the mutant loci. Supporting this, electrophoretic analysis of collagens derived from fibroblast cultures failed to show defective molecules. We conclude that this syndrome is not a collagen disorder.

Dominant mutations in familial lethal and severe osteogenesis imperfecta.

Four families presenting with familial osteogenesis imperfecta (OI) have been studied: 2 with the lethal type II and 2 with the severe type III form. Fibroblasts of the patients, all issue from non-consanguineous parents, produced normal and abnormal alpha(I) chains. These heterozygous mutations differentiate the recurrent forms from homozygous mutations characteristic of autosomal recessive forms. Although the identity of the mutations could not be determined, such recurrence of autosomal dominant OI is probably the result of germinal mosaicism in one of the parents. Biochemical results were consistent with a somatic mosaicism in the father's fibroblasts in one family. Moreover, our studies show that not only OI type II but also severe OI type III can arise from gonadal mosaicism. We discuss the importance of such a phenomenon for genetic counseling.

A new lethal brittle bone syndrome with increased amount of type V collagen in a patient.

A new lethal brittle bone disease is described in three patients with slender long bones, thin ribs, hypomineralized calvaria, and normal facial appearance. In spite of several limb fractures this syndrome can be differentiated from the lethal forms of osteogenesis imperfecta and is better related to the thin-bone group of lethal dysplasias. Biochemical investigation of collagen from one of the patients by the use of gel electrophoresis and high-pressure liquid chromatography analyses failed to demonstrate any evident defect in the structure of type I collagen chains. Nevertheless collagen extractability from the dermis was altered owing to an increase in the proportion of acid-soluble material. Tritium-proline labeling of cultured fibroblasts confirmed the reduction in total collagen synthesis. This was attributed to a lower type I and type III amount whereas type V collagen level was markedly increased in the cell layer. RNA analysis of the three collagen types with the appropriate cDNA probes confirmed the protein data. Electron microscopic examination of bone and skin showed morphologically abnormal fibroblasts and osteoblasts with an abundant distended rough endoplasmic reticulum and an altered plasma membrane. Unexpected thin fibrils with a banding pattern and surrounding the type I fibrils were observed. They might represent type V collagen. We suggest that, in this patient, the moderate decrease in type I collagen amount is insufficient to account for the radiological findings and that type V collagen overproduction could play a role in the bone brittleness by interfering with the process of mineralization.

Localization of gamma-glutamyl-phosphate residues to the alpha 2CB3-5 peptide of type I chicken bone collagen.

alpha 2(I) chains of chicken bone collagen were isolated and purified. Analyses confirmed previous studies that the alpha 2(I) chains contained gamma-glutamyl phosphate residues. Sodium borohydride reduction of the gamma-glutamyl phosphate residues of the CaCl2 extracted and purified alpha 2(I) chains and subsequent CNBr cleavage showed that most of the gamma-glutamyl phosphate groups were located on the alpha 2CB3-5 peptides of the type I collagen molecule, suggesting a specific function for these phosphorylated residues. The reported linkage of the Ser(P) containing phosphoproteins of dentin to the alpha 2CB4 regions of collagen may indicate different biological functions for these two different, protein-bound organic phosphate residues.

[Lethal syndromes with thin bones]. / Syndromes létaux avec gracilité du squelette.

The authors report six cases from six different families of lethal brittle bone disease with narrow diaphyses and thin ribs. This phenotype should be dissociated from the lethal forms of osteogenesis imperfecta and encompass two diseases. In the first, autosomal recessive, the metaphyses of long bones are narrow, with a membranous ossification, without cartilagenous residue. Cultured fibroblasts demonstrate a marked increase in type V collagen. In the second type, the metaphyses are enlarged and the babies have a facial dysmorphism with hypoplasia of the eyebrows, frontal bossing and a small mouth.

Abnormal procollagen synthesis in fibroblasts from three patients of the same family with a severe form of osteogenesis imperfecta (type III).

Dermal fibroblast cultures from three siblings with a severe form of osteogenesis imperfecta were established in order to analyze their procollagen and collagen synthesis. Cell strains from clinically normal consanguineous parents (first cousins), were also obtained for comparison. Total collagen production in culture media was diminished by 55% in the patients fibroblasts and to a lesser extent in the parents. This decrease was specific for collagenous proteins. From polyacrylamide gel electrophoresis, it appeared that the three children had not only the same defective secretion of pro alpha 1(I) molecules but that their pro alpha 1(I) migrated slightly faster than the parental and control counterparts. Analysis of secretion confirmed a reduced rate in procollagen synthesis and the absence of intracellular storage. Upon pepsin treatment, extracellular alpha 1(I) and alpha 2(I) chains were found in the expected ratio of 2:1 and migrated normally, suggesting that the altered mobility of pro alpha 1(I) chains was related to COOH or NH2 terminal propeptides. In agreement with the reduced type I collagen production, an increase in the alpha 1(III)/alpha 1(I) ratio was also detected. Furthermore, after a 2.5-h labelling followed by alkylation with iodoacetamide, free intracellular pro alpha 2(I) and alpha 1(I) chains were detected in the absence of reduction, consistent with an abnormal intracellular ratio of pro alpha 1(I)/pro alpha 2(I) that was measured after dithiothreitol reduction. Analysis of intracellular collagen chains from parental strains following a 4-h incubation demonstrated that pro alpha 1(I) appeared as a doublet, one band with normal mobility and a less intense band migrating faster and corresponding to the defective chain found in the patients. Absence of the abnormal molecules in culture media was related to the demonstration of a defective collagen secretion by parental fibroblasts. Correlation between these biochemical findings and clinical data strongly support a recessive inheritance of the disease that could be classified as a type III form of osteogenesis imperfecta. Patients would be homozygous for the same defective allele and the asymptomatic parents would most likely be heterozygous carriers of the mutation. Although the exact location of the alteration is not yet elucidated, a splicing mutation is suggested.