- Email: [email protected]
A secretory clue to combined haemophilia
Literature
MOLECULAR MEDICINE TODAY, JUNE 1998
A secretory clue to combined haemophilia Mutations in the ER–Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII Nichols, W.C. et al. (1998)
Cell 93, 61–70 The coagulation factors V (FV) and VIII (FVIII) are secreted glycoproteins that are essential for maintenance of normal haemostasis. The X-linked bleeding disorder haemophilia A is due to deficiency of FVIII, while inherited FV deficiency is a rare autosomal-recessive condition that also confers a bleeding diathesis. In contrast, the molecular basis for combined FV and FVIII deficiency is unknown. Coinheritance of both haemophilia A and FV deficiency has been reported in four families, but the majority of combined deficiency patients exhibit an autosomal recessive pattern of inheritance. In this surprising study, Nichols et al. now report that the combined deficiency disorder, first reported in 1954 by Oeri et al., is the result of mutations in a gene on chromosome 18q that encodes ERGIC-53, a component of the endoplasmic reticulum (ER)–Golgi intermediate compartment. Building on previous genetic linkage studies and recombination analysis, Nichols et al. mapped the genomic region of 18q containing the combined deficiency gene and identified expressed sequence tags (ESTs) within the candidate region. One of these ESTs corresponded to the cDNA for ERGIC-53, previously described as a resident protein (function unknown) of the ER–Golgi intermediate compartment. The complete coding sequence was amplified using total RNA isolated from immortalized B-cell cultures established by treating B cells of combined deficiency patients with Epstein–Barr virus. DNA sequence analysis revealed two different mutations that accounted for all affected individuals in the nine families studied: homozygosity for a single base insertion, and homozygosity for a splice donor mutation. In all cases, the parents of the affected individuals were heterozygous carriers of the corresponding mutations, and all individuals homozygous for either mutation were clinically affected with combined FV and FVIII deficiency. This confirms complete penetrance for the disorder. Furthermore, immunofluorescence and western analysis of immortalized lymphocytes from patients homozygous for either of these two mutations demonstrated complete lack of expression of the mutated gene.
This study establishes ERGIC-53 as the gene responsible for combined FV and FVIII deficiency, and is remarkable in establishing the molecular basis for this disorder as a defect in a common pathway for intracellular trafficking of these two coagulation factors. Moreover, a protein within the ER–Golgi intermediate compartment is implicated in the efficient transport of a specific subset of secreted glycoproteins. ERGIC-53 is a type 1 transmembrane protein which has homology to leguminous lectins and exhibits mannose-selective and Ca2+-dependent binding. Factors V and VIII both contain a central B domain of conserved length. As recombinant B domain-deleted FVIII and FV retain functional activity, the role of the B domain remains unknown. The authors speculate that the B domains, which are heavily glycosylated, might provide a substrate for interaction with ERGIC-53 through a lectin-like function. Further studies based on this surprising result might lead to greater understanding of FV and FVIII biosynthesis, translating into novel approaches to coagulation factor replacement in haemophilia. Alan J. Warren MRCP, MRCPath, PhD MRC Clinician Scientist, Box 234, Dept of Haematology, Addenbrooke’s NHS Trust, Hills Road, Cambridge, UK CB2 2QQ.
The genomics era is upon us Alternative gene form discovery and candidate gene selection from gene indexing projects Burke, J., Wang, H., Hide, W. and Davidson, D.B. (1998)
Genome Res. 8, 276–290
Phylogenomics: improving functional predictions for uncharacterized genes by evolutionary analysis Eisen, J.A. (1998)
Genome Res. 8, 163–167 Genomics is viewed with trepidation by the fainthearted and boldly embraced by the brave. Somewhere in between lies a realistic expectation of the fruits we might reap from genome sequences in the absence of biological experimentation. Genomics is the study and comparison of sequenced genomes from a variety of organisms to
Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00
provide information on genes and their functions. The potential benefits of this approach, through a better understanding of the role of genes, their products and their interactions in health and disease, are enormous. Not only will genes be identified that in their mutated state cause disease, but the factors that ameliorate or exacerbate their clinical expression will be identified. Susceptible genotypes will become apparent with an opportunity to modify lifestyles to improve health and prevent disease; and the potential for rational therapies will be hugely increased. Although <5% of the human genome has been sequenced, the sequences of thousands of expressed sequence tags (ESTs), representing small portions of genes, are known. One current challenge is to organize this vast, redundant data set into a sensibly ordered collection of nonredundant sequences representing each gene only once. With this in mind, Burke et al. have written a program that aims to partition ESTs and full-length transcripts into homology classes and then divide those into subclasses based on their dissimilarity. This serves two purposes: (1) it identifies artifacts such as chimerism; and (2) it allows the researcher to identify alternatively spliced transcripts and to build a database of tissue- and developmental-stage-specific and pathologyrelated expression patterns of genes. These data will remain relevant even after the completion of the human genome sequence because they will provide gene expression profiles that can only be determined from experimental data. Another rapidly expanding area is the prediction of protein function from uncharacterized gene sequences. Eisen makes the point that although DNA sequences might be the prime determinant of function, there are exceptions where groups of genes of similar sequence perform different functions. He suggests that ‘phylogenomics’ – the investigation of the evolution of genes – will facilitate much more accurate prediction of gene functions. The power of this approach will increase dramatically as the sequences of a greater number of genomes are completed. The caveat remains, however, that somewhere along the line biological data are required to determine function accurately and to differentiate dissimilar functions of similar sequences. There will be no substitute for experimental biology in model systems and, in some cases, in humans to determine true function and the effect of gene mutation on functionality. Michèle Ramsay PhD Reader in the Department of Human Genetics, South African Institute for Medical Research and the University of the Witwatersrand, PO Box 1038, Johannesburg, 2000, South Africa.
235
MOLECULAR MEDICINE TODAY, JUNE 1998
A secretory clue to combined haemophilia Mutations in the ER–Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII Nichols, W.C. et al. (1998)
Cell 93, 61–70 The coagulation factors V (FV) and VIII (FVIII) are secreted glycoproteins that are essential for maintenance of normal haemostasis. The X-linked bleeding disorder haemophilia A is due to deficiency of FVIII, while inherited FV deficiency is a rare autosomal-recessive condition that also confers a bleeding diathesis. In contrast, the molecular basis for combined FV and FVIII deficiency is unknown. Coinheritance of both haemophilia A and FV deficiency has been reported in four families, but the majority of combined deficiency patients exhibit an autosomal recessive pattern of inheritance. In this surprising study, Nichols et al. now report that the combined deficiency disorder, first reported in 1954 by Oeri et al., is the result of mutations in a gene on chromosome 18q that encodes ERGIC-53, a component of the endoplasmic reticulum (ER)–Golgi intermediate compartment. Building on previous genetic linkage studies and recombination analysis, Nichols et al. mapped the genomic region of 18q containing the combined deficiency gene and identified expressed sequence tags (ESTs) within the candidate region. One of these ESTs corresponded to the cDNA for ERGIC-53, previously described as a resident protein (function unknown) of the ER–Golgi intermediate compartment. The complete coding sequence was amplified using total RNA isolated from immortalized B-cell cultures established by treating B cells of combined deficiency patients with Epstein–Barr virus. DNA sequence analysis revealed two different mutations that accounted for all affected individuals in the nine families studied: homozygosity for a single base insertion, and homozygosity for a splice donor mutation. In all cases, the parents of the affected individuals were heterozygous carriers of the corresponding mutations, and all individuals homozygous for either mutation were clinically affected with combined FV and FVIII deficiency. This confirms complete penetrance for the disorder. Furthermore, immunofluorescence and western analysis of immortalized lymphocytes from patients homozygous for either of these two mutations demonstrated complete lack of expression of the mutated gene.
This study establishes ERGIC-53 as the gene responsible for combined FV and FVIII deficiency, and is remarkable in establishing the molecular basis for this disorder as a defect in a common pathway for intracellular trafficking of these two coagulation factors. Moreover, a protein within the ER–Golgi intermediate compartment is implicated in the efficient transport of a specific subset of secreted glycoproteins. ERGIC-53 is a type 1 transmembrane protein which has homology to leguminous lectins and exhibits mannose-selective and Ca2+-dependent binding. Factors V and VIII both contain a central B domain of conserved length. As recombinant B domain-deleted FVIII and FV retain functional activity, the role of the B domain remains unknown. The authors speculate that the B domains, which are heavily glycosylated, might provide a substrate for interaction with ERGIC-53 through a lectin-like function. Further studies based on this surprising result might lead to greater understanding of FV and FVIII biosynthesis, translating into novel approaches to coagulation factor replacement in haemophilia. Alan J. Warren MRCP, MRCPath, PhD MRC Clinician Scientist, Box 234, Dept of Haematology, Addenbrooke’s NHS Trust, Hills Road, Cambridge, UK CB2 2QQ.
The genomics era is upon us Alternative gene form discovery and candidate gene selection from gene indexing projects Burke, J., Wang, H., Hide, W. and Davidson, D.B. (1998)
Genome Res. 8, 276–290
Phylogenomics: improving functional predictions for uncharacterized genes by evolutionary analysis Eisen, J.A. (1998)
Genome Res. 8, 163–167 Genomics is viewed with trepidation by the fainthearted and boldly embraced by the brave. Somewhere in between lies a realistic expectation of the fruits we might reap from genome sequences in the absence of biological experimentation. Genomics is the study and comparison of sequenced genomes from a variety of organisms to
Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00
provide information on genes and their functions. The potential benefits of this approach, through a better understanding of the role of genes, their products and their interactions in health and disease, are enormous. Not only will genes be identified that in their mutated state cause disease, but the factors that ameliorate or exacerbate their clinical expression will be identified. Susceptible genotypes will become apparent with an opportunity to modify lifestyles to improve health and prevent disease; and the potential for rational therapies will be hugely increased. Although <5% of the human genome has been sequenced, the sequences of thousands of expressed sequence tags (ESTs), representing small portions of genes, are known. One current challenge is to organize this vast, redundant data set into a sensibly ordered collection of nonredundant sequences representing each gene only once. With this in mind, Burke et al. have written a program that aims to partition ESTs and full-length transcripts into homology classes and then divide those into subclasses based on their dissimilarity. This serves two purposes: (1) it identifies artifacts such as chimerism; and (2) it allows the researcher to identify alternatively spliced transcripts and to build a database of tissue- and developmental-stage-specific and pathologyrelated expression patterns of genes. These data will remain relevant even after the completion of the human genome sequence because they will provide gene expression profiles that can only be determined from experimental data. Another rapidly expanding area is the prediction of protein function from uncharacterized gene sequences. Eisen makes the point that although DNA sequences might be the prime determinant of function, there are exceptions where groups of genes of similar sequence perform different functions. He suggests that ‘phylogenomics’ – the investigation of the evolution of genes – will facilitate much more accurate prediction of gene functions. The power of this approach will increase dramatically as the sequences of a greater number of genomes are completed. The caveat remains, however, that somewhere along the line biological data are required to determine function accurately and to differentiate dissimilar functions of similar sequences. There will be no substitute for experimental biology in model systems and, in some cases, in humans to determine true function and the effect of gene mutation on functionality. Michèle Ramsay PhD Reader in the Department of Human Genetics, South African Institute for Medical Research and the University of the Witwatersrand, PO Box 1038, Johannesburg, 2000, South Africa.
235