Mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) in patients with Sorsby's fundus dystrophy.

The hereditary macular dystrophies are progressive degenerations of the central retina and contribute significantly to irreversible visual loss in developed countries. Among these disorders, Sorsby's fundus dystrophy (SFD), an autosomal dominant condition, provides an excellent mendelian model for the study of the genetically complex age-related macular degeneration (AMD), the most common maculopathy in the elderly. Recently, we mapped the SFD locus to 22q13-qter. This same region contains the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which is known to play a pivotal role in extracellular matrix remodeling. We have now identified point mutations in the TIMP3 gene in affected members of two SFD pedigrees. These mutations are predicted to disrupt the tertiary structure and thus the functional properties of the mature protein.

[Mild bleeding diathesis in a 62-year-old woman with hereditary thrombocytopenia]. / Milde Blutungsneigung bei einer 62-Jährigen mit hereditärer Thrombozytopenie.

A 62-year-old woman presented with severe, isolated thrombocytopenia. Due to the positive family history and normal thrombocyte morphology ANKRD26-associated thrombocytopenia 2 (THC2) was suspected. The diagnosis was confirmed by DNA sequencing. Although this is the first case report on THC2 in Germany, we anticipate that THC2 might be a frequent cause of hereditary thrombocytopenia. A specific therapy was not necessary, but would consist of platelet supplementation.

Congenital macrothrombocytopenia associated with a combination of functional polymorphisms in the TUBB1 gene.

Congenital thrombocytopenia in childhood and adolescence requires an extensive diagnostic workup to find the underlying reason. We report on a 13-year-old female patient who was incidentally found to have moderate thrombocytopenia which was also diagnosed in her father and brother. Within the microscopic evaluation of a peripheral blood smear macrothrombocytes were found. Immunofluorescence microscopy of the patient's platelets detected the lack of ß1-tubulin. Analysis of the TUBB1 gene revealed three known missense variants in heterozygous state which in combination might explain the ß1-tubulin defect.

Large deletions play a minor but essential role in congenital coagulation factor VII and X deficiencies.

UNLABELLED: Congenital factor VII (FVII) and factor X (FX) deficiencies belong to the group of rare bleeding disorders which may occur in separate or combined forms since both the F7 and F10 genes are located in close proximity on the distal long arm of chromosome 13 (13q34). We here present data of 192 consecutive index cases with FVII and/or FX deficiency. 10 novel and 53 recurrent sequence alterations were identified in the F7 gene and 5 novel as well as 11 recurrent in the F10 gene including one homozygous 4.35 kb deletion within F7 (c.64+430_131-6delinsTCGTAA) and three large heterozygous deletions involving both the F7 and F10 genes. One of the latter proved to be cytogenetically visible as a chromosome 13q34 deletion and associated with agenesis of the corpus callosum and psychomotor retardation. CONCLUSIONS: Large deletions play a minor but essential role in the mutational spectrum of the F7 and F10 genes. Copy number analyses (e. g. MLPA) should be considered if sequencing cannot clarify the underlying reason of an observed coagulopathy. Of note, in cases of combined FVII/FX deficiency, a deletion of the two contiguous genes might be part of a larger chromosomal rearrangement.

Allelic heterogeneity of alkaptonuria in Central Europe.

Defects of the homogentisate 1,2 dioxygenase (HGO; E.C. No. 1.13.11.5) have been identified as the molecular cause of alkaptonuria in humans (AKU) and the aku mouse. Here, we report on the genetic basis of 30 AKU patients from Central Europe. In addition to five mutations described previously, we have detected five novel HGO mutations. Recombinant expression of mutated HGO enzymes in E. coli demonstrates the inactivating effect of three of these mutations. A genetic epidemiologic study in Slovakia, the country with the highest incidence of alkaptonuria, demonstrates that two recurrent mutations (c.183-1G > A and Glyl61Arg) are found on more than 50% of AKU chromosomes. An analysis of the allelic association with intragenic DNA markers and of the geographic origins of the AKU chromosomes suggests that several independent founders have contributed to the gene pool, and that subsequent genetic isolation is likely to be responsible for the high prevalence of alkaptonuria in Slovakia.

Generation and degradation of human endostatin proteins by various proteinases.

The angiogenesis inhibitor endostatin is a fragment of the NC1 domain of collagen XVIII. The generation of endostatin has been investigated only in murine hemangioendothelioma cell cultures and was ascribed to cathepsin L. Distinct endostatin-like fragments were detected in human tissues and serum. To identify proteinases able to generate such fragments, we incubated human NC1 with proteinases of all classes, including cathepsin L. Eleven out of 12 generate fragments with an N-terminus within the same 15 residue stretch as those occurring physiologically, indicating that this region is sensitive to many proteinases. None correspond to mouse endostatin. However, the efficiencies of these proteinases differed markedly. Some proteinases also proved to degrade endostatin, pointing to another regulatory loop of angiogenesis.

The rare human fragile site 16B.

The rare human fragile site 16B (FRA16B) has been found to occur spontaneously. Its expression in lymphocyte cultures can also be induced or greatly enhanced by addition of chemicals which are known to bind to AT-rich DNA regions. Following optimal treatment with 150 microg/ml berenil 24 h prior to fixation, the heterozygote frequency of FRA16B is found to be about 5% in populations of European descent. Thus, FRA16B represents the most common of the rare fragile sites. Consistent with cytogenetic observations, the molecular characterization of FRA16B revealed that it is an amplified 33-base pair AT-rich minisatellite repeat. These interindividually variable, extremely large repeat expansions of 15-70 kb in size do not seem to interfere with the expression of genes essential for human development since heterozygotes and homozygotes for FRA16B are normal.

Mosaicism for an ectopic NOR at 8pter and a complex rearrangement of chromosome 8 in a patient with severe psychomotor retardation.

We describe a 3-year-old girl with severe delays in mental and motor skills, a history of generalized seizures, and subtle dysmorphic features. Conventional cytogenetics revealed a mosaic karyotype. A de novo ectopic NOR at the telomeric region of the short arm of one chromosome 8 (8ps) was found in 90% of lymphocyte and in 98% of fibroblast metaphases. A small NOR-bearing marker chromosome and a large derivative chromosome 8 without short arm satellites (der(8)) were present in the remaining cells. FISH with a probe specific for centromeres 14 and 22 labeled both the telomeric region of 8ps and the small marker centromere. Der(8) included an inverted duplication of 8p and a rearranged duplication of 8q but lacked a second centromere. A subtelomeric probe for 8p revealed a cryptic deletion in 8ps and der(8). Thus, the karyotype represents a combination of submicroscopic partial monosomy 8pter and mosaic trisomy 8.

Evaluation of the gene encoding the tissue inhibitor of metalloproteinases-3 in various maculopathies.

PURPOSE: Mutations in the gene encoding the tissue inhibitor of metalloproteinases-3 (TIMP3) have been shown previously to cause Sorsby's fundus dystrophy, an autosomal-dominant disorder characterized by extracellular matrix irregularities in Bruch's membrane. To assess the involvement of TIMP3 in a variety of other macular dystrophies, the authors have screened this gene for disease-causing mutations in age-related macular degeneration (AMD), adult vitelliform macular dystrophy (AVMD), central areolar choroidal dystrophy (CACD), syndrome-associated macular dystrophies, cone-rod dystrophy, and a group with unspecified macular degeneration. METHODS: Single-stranded conformational analysis of the entire coding region was performed using the polymerase chain reaction and oligonucleotide primers flanking the five exons of the TIMP3 gene as well as the putative promotor region and a highly conserved fragment of the 3'-untranslated region. The authors analyzed a total of 217 patients, including 143 patients with AMD, 28 patients with AVMD, 21 patients with CACD, and 25 patients with other forms of macular dystrophy. RESULTS: In the 217 patients analyzed, the authors have identified one sequence alteration (a G-to-C base change) in the 5'-untranslated region in a patient with AMD. However, the functional consequences of this mutation are not clear. No other disease-causing mutations were found. The authors have characterized a frequent intragenic polymorphism in exon 3 of the TIMP3 gene (heterozygosity = 0.57) that will be useful for genetic linkage or allele sharing analyses or both. CONCLUSIONS: The authors' results suggest that TIMP3 is not a major factor in the cause of AMD, AVMD, and CACD. Thus far, Sorsby's fundus dystrophy appears to be the only phenotype known to be associated with mutations in TIMP3.

Sorsby fundus dystrophy: reevaluation of variable expressivity in patients carrying a TIMP3 founder mutation.

Interfamilial phenotypic variations in Sorsby fundus dystrophy (SFD) have given rise to controversy as to whether SFD constitutes more than 1 nosologic entity. The recent identification of the tissue inhibitor of metalloproteinases-3 (TIMP3) as the gene causing SFD has made it possible to readdress the question of genetic and clinical heterogeneity. In this study, we have extended previous findings on a Ser181Cys founder mutation in SFD families from the British Isles and show that carriers of this mutation residing in Canada, the United States, and South Africa likewise are descendants of the British ancestor. In addition, we have reevaluated the question of variable SFD phenotypes by analyzing the available clinical data on carriers of the Ser181Cys mutation.

Ocular ochronosis in alkaptonuria patients carrying mutations in the homogentisate 1,2-dioxygenase gene.

AIMS: To assess the involvement of the recently identified human homogentisate 1,2-dioxygenase gene (HGO) in alkaptonuria (AKU) in two unrelated patients with ochronosis of the conjunctiva, sclera, and cornea. METHODS: A mutation screen of the entire coding region of the HGO gene was performed using single stranded conformational analysis after polymerase chain reaction with oligonucleotide primers flanking all 14 exons of the HGO gene. Fragments showing aberrant mobility were directly sequenced. RESULTS: Two homozygous missense mutations, L25P and M368V, were identified, each of which leads to the replacement of a highly conserved amino acid in the HGO protein. CONCLUSIONS: The authors describe a novel mutation, L25P, in the German population and bring to 18 the total number of known HGO mutations.

[Sorsby's fundus dystrophy. A genetically homogeneous disease]. / Sorsbys Fundusdystrophie. Eine genetisch homogene Erkrankung.

BACKGROUND: The recent identification of the tissue inhibitor of metalloproteinases-3 (TIMP3) as the gene underlying SFD pathology has made it possible to address the question of genetic heterogeneity in this disorder. In addition, it now has become feasible to clarify whether SFD is directly involved in other maculopathies and, in particular, may represent a genetic model for age-related macular degeneration. PATIENTS: Genetic analysis were performed in five unrelated and 18 related British SFD pedigrees as well as in 143 patients affected with age-related macular degeneration, 28 patients with adult vitelliform macular dystrophy, 21 patients with central areolar choroidal dystrophy and 25 individuals with other forms of macular dystrophies. RESULTS: Molecular genetic analyses confirmed the autosomal dominant mode of inheritance in SFD. In all five unrelated SFD pedigrees individual TIMP3 mutations were identified introducing an additional cysteine residue into the C-terminal region of the mature protein. Affected individuals from 18 SFD families residing in Great Britain, Canada, Oregon and South Africa were found to carry a common ancestral Ser181Cys mutation. The clinical variability of this Ser181Cys mutation was reevaluated. A mutational screen in 217 patients with various maculopathies revealed no disease-causing mutations in the TIMP3 gene. CONCLUSION: So far, TIMP3 mutations have exclusively been associated with SFD. Therefore, this disorder appears to be genetically homogeneous with complete penetrance but variable expressivity.

Large germline deletions and duplication in isolated cerebral cavernous malformation patients.

Cerebral cavernous malformations (CCM) are vascular lesions that predispose to headaches, seizures, and hemorrhagic stroke. Hereditary CCMs are usually associated with the occurrence of multiple CCMs and occur with a frequency of 1:2,000 to 1:10,000. In this study, eight isolated cases with multiple CCMs but no CCM1-3 point mutation were analyzed using the multiplex ligation-dependent probe amplification assay. Four genomic rearrangements were identified including a previously unreported large duplication within the CCM1 gene and a novel deletion involving the entire coding region of the CCM2 gene. Consequently, systematic screening for CCM deletions/duplications is recommended.

Genetics of cerebral cavernous angioma.

Cerebral cavernous malformations (CCM) are hamartomatous vascular anomalies characterized by densely packed, grossly enlarged immature capillaries without intervening neural tissue. Depending on their location and size (ranging from a few millimeters to several centimeters), the biologically dynamic lesions become symptomatic during the second to fourth decade of life. Clinical symptoms include recurrent headaches, seizures, intracranial hemorrhage, and stroke. There are sporadic and autosomal dominantly inherited forms of CCM. Causal mutations have been demonstrated in three genes, KRIT1, MGC4607, and PDCD10, but additional genes are likely to be discovered. These genes are therefore thought to play a role in angiogenesis. Their specific modes of actions, their contribution to and their likely penetrance in the genesis of CCM are the subject of current investigations. Genetic counseling is strongly advisable for patients with a positive family history and for seemingly sporadic cases with multiple lesions, and genetic testing should be considered on an individual basis. The identification of a mutation enables precise genetic testing of relatives. Given the 50 % a priori risk of autosomal dominant inheritance, the benefits of genetic testing are twofold: a positive test result in a presymptomatic carrier permits close neuroradiological surveillance and timely neurosurgical intervention; a negative test result relieves the proband of unwarranted anxiety and unnecessary medical supervision.

Adult vitelliform macular dystrophy is frequently associated with mutations in the peripherin/RDS gene.

Mutations in the peripherin/RDS gene, which encodes a photoreceptor-specific membrane glycoprotein, have been identified in a variety of retinal phenotypes. However, the mechanisms by which specific mutations in this gene can cause typical features of retinal dystrophies clinically as distinct as retinitis pigmentosa or macular degeneration are still unknown. Recently, a single case of adult vitelliform macular dystrophy (AVMD) has been associated with a Y258Stop mutation. To assess the frequency of peripherin/RDS mutations in the clinically heterogeneous group of AVMD, we analyzed the entire coding region of the gene in 28 unrelated patients. We identified five novel mutations including two presumed null allele mutations. Thus, our results demonstrate that a significant portion of AVMD patients (18%) carry point mutations in peripherin/RDS, suggesting that this gene is frequently involved in the pathogenesis of this macular disorder. In addition, this study shows that the variable phenotypes in AVMD are due, at least in part, to genetic heterogeneity and are likely to be caused by mutations in disease genes thus far unknown.

Genomic organization of the human tissue inhibitor of metalloproteinases-3 (TIMP3)

The tissue inhibitors of metalloproteinases (TIMPs) play a crucial role in the physiological turnover of the extracellular matrix (ECM) by tightly regulating matrix metalloproteinase (MMP) activities. Disturbances in the TIMP/MMP system have been implicated in many disease processes where loss of ECM integrity is a principal feature. More recently, we have shown that mutations in TIMP3 cause the autosomal dominant disorder Sorsby's fundus dystrophy (SFD). This is a macular degeneration disorder with characteristic ECM irregularities in Bruch's membrane. To further facilitate mutational analysis and to provide a basis for functional studies, we now report the genomic organization of the human TIMP3 gene.

A second independent Tyr168Cys mutation in the tissue inhibitor of metalloproteinases-3 (TIMP3) in Sorsby's fundus dystrophy.

Sorsby's fundus dystrophy (SFD) is a rare autosomal dominant macular disorder with age of onset usually in the fourth decade. It is characterised by loss of central vision owing to subretinal neovascularisation and disciform macular degeneration. In an effort to identify the SFD gene, the disease locus was first mapped to chromosome 22q13-qter by genetic linkage analysis, the same chromosomal region as the gene encoding the tissue inhibitor of metalloproteinases-3 (TIMP3). Subsequently, two separate mutations in TIMP3 were found in affected members of two unrelated SFD pedigrees (Tyr168Cys and Ser181Cys). More recently, two additional SFD related mutations, Ser156Cys and Gly167Cys, have provided further confirmation that heterozygous mutations in TIMP3 are causally responsible for the SFD phenotype. We now report the occurrence of the Tyr168Cys mutation in an SFD patient of Austrian descent and show that this mutation found earlier in an American SFD family arose independently. The new findings add to an emerging pattern of SFD mutations which all seem to affect the C-terminal region of the mature TIMP3 protein. In addition, all known mutations cause a change of an amino acid to a cysteine residue. This suggests a critical role for the additional C-terminal free thiol group in SFD pathogenesis.