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The Haemophilia A Mutation Search Test and Resource Site, Home Page of the Factor VIII Mutation Database: HAMSTeRS

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1996 Oxford University Press

100–102 Nucleic Acids Research, 1996, Vol. 24, No. 1

The haemophilia A mutation search test and resource

site, home page of the factor VIII mutation database:

HAMSTeRS

Adam I. Wacey, Geoffrey Kemball-Cook

1

, Haig H. Kazazian

2,+

, Stylianos E. Antonarakis

3

,

Rainer Schwaab

4

, Peter Lindley

5,

and E. G. D. Tuddenham

1,

*

Thrombosis Research Institute, Emmanuel Kaye Building, Manresa Road, London SW3 6LR, UK, 1Haemostasis Research Group, MRC Clinical Science Centre, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK, 2John Hopkins University, Baltimore, MD 21205, USA, 3Division of Medical Genetics, University of Geneva Medical School and Cantonal Hospital, 1211 Geneva, Switzerland, 4Institut fur

Klinische Biochemie de Universitat Bonn, Sigmund Freud Strasse 25, 53105 Bonn, Germany and 5Synchrotron

Radiation Department, Daresbury Laboratory, Daresbury, Cheshire WA4 9AD, UK

Received October 2, 1995; Accepted October 12, 1995

ABSTRACT

In order to facilitate easy access to and aid understand-ing of the causes of haemophilia A at the molecular level we have constructed HAMSTeRS, the third release of the factor VIII mutation database and the first release of this database that may be accessed and interrogated over the internet through a World Wide Web browser. The database also presents a review of the structure and function of factor VIII and the molecular genetics of haemophilia A, a real time update of the biostatistics of each parameter in the database, a molecular model of the A1, A2 and A3 domains of the factor VIII protein (based on the crystal structure of caeruloplasmin) and a bulletin board for discussion of issues in the molecular biology of factor VIII.

INTRODUCTION

The haemostatic protein factor VIII (FVIII) acts as a co-factor in the activation of factor X by factor IXa ( reviewed in 1). The FVIII gene contains 26 exons and spans 186 kb of DNA (2). Deleterious mutations in the FVIII gene have been demonstrated to either reduce the expression and/or abrogate the activity of the FVIII protein and thus cause haemophilia A (3), an X-linked bleeding disorder affecting ∼1 in 5000 males (4).

Over the last two years dramatic growth in the number of mutations identified in the FVIII gene has rendered their publica-tion in a database through tradipublica-tional media increasingly difficult. However, the recent development of electronic publishing, remote-ly accessible over the Internet, has provided a resource in which the size of the data field, its complexity and its physical location are irrelevant to its publication. Moreover, electronically published data can be searched, changed and updated in real time. This new means of communication thus provides an ideal vehicle through which to publish an interactive database of nucleotide substitu-tions, delesubstitu-tions, insertions and rearrangements of the FVIII gene.

Since 1990 the pioneering development and dissemination of HTML (HyperText Mark-up Language) Graphical User Interfaces (GUIs) by CERN has driven the evolution of the Internet, from a convoluted text-based method of communication to an intuitive ‘point and click’ resource utilizing graphics and sound to connect physically remote sites through virtual links (5). Accessing the internet through a GUI interface, more commonly known as a World Wide Web browser, provides a convenient method for the non-computer specialist to readily access and interrogate the database.

MATERIALS AND METHODS

The HTML script was written on a Silicon Graphics (Mountain View, CA) Indigo Elan R4000 using Web author 1.0 and displayed using the GUI Netscape 1.1N (retrievable via anony-mous ftp from the European UNIX archive phoenix.ic.ac.uk). The CGI script was coded in C. Image files were recorded in interlaced, background-transparent GIF format and the largest page contained some 28 k of thumbnails and images, making it easily accessible in a reasonable time via the commonly used 14.4 baud telephone link. The site is served from the UNIX systems of the Hammersmith Hospital through a 100 Mb fibre optic line (URL http://www. hamsters.rpms.ac.uk).

The system was alpha tested against the Mosaic and Netscape 1.0 and 1.1N GUI browsers and the text-based browser Lynx. However, to get the full beneficial effects of graphical links it is recommended that the site be accessed through one of the more modern browsers, such as Netscape 1.1N.

RESULTS

In order to facilitate the easiest possible navigation of the site the architecture of the database was designed to be as simple as possible (see Figs 1 and 2). All of the functions of the database may be accessed from the top level menu of the home page. To utilize multiple functions the user simply returns to the home page and selects a new function from this menu.

* To whom correspondence should be addressed

Present addresses: +Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA and CCL Daresbury Laboratory, Warrington WA4 4AD, UK

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101

Nucleic Acids Research, 1994, Vol. 22, No. 1Nucleic Acids Research, 1996, Vol. 24, No. 1 101

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Nucleic Acids Research, 1996, Vol. 24, No. 1

102

Figure 2. Schematic representation of the architecture of the HAMSTeRS home page.

DISCUSSION

As an aid to the professional scientist who is unfamiliar with the molecular genetics of haemophilia A the first function presents a review of the field (6).

The database itself may be interrogated from the second function. The user is presented with a standard format HTML script multiparameter form. The form is delimited using pull-down menus and direct input. In all of the appropriate parameters the ‘wild card’ choice (*) may be used to denote the search parameter ‘all’. Upon completion the form is automatically sent to the database, which searches for hits against its data field. The database then returns to the user a Web page containing all of the observed hits. This highly flexible system allows the user to search for either a specific nucleotide substitution or all mutants that are consistent with the user-definable search parameters (e.g. all Asn→X mutants in exon 11).

It is now becoming increasingly unusual to publish observations of single nucleotide substitutions, however, paradoxically, with the growth of bioinformatics, the larger the volume of the published database the greater its validity and potential use to the scientific community. The third function in the menu thus allows the user to submit newly defined variants to the database in essentially the same manner as the searches are made. Upon retrieval by other scientists the database will also return the details of the authors of the mutation report, thus allowing citation of the work. The database also contains two parameter fields of date submitted and user remarks. The user can thus search the database for recent mutations and any interesting observations noted for the mutants.

The fourth function in the menu presents a real time update of the biostatistics of each parameter in the database, for example codon usage, per cent CpGs, etc.

The recent elucidation through X-ray crystallography of the structure of caeruloplasmin (7) has allowed the construction of a homologous model of the A1, A2 and A3 domains of FVIII through comparative methods (8). The fifth function of the menu will email the user the PDB co-ordinates of the model. The model can then be visualized through one of the more complex molecular graphics packages, such as BIOSYM (Biosym Technologies Inc., San Diego, CA) or RIBBONS 2.5 (9). It is likely, however, that the large Unix workstations necessary to run these packages would not be accessible to all of the users interested in the structural biochemistry of the mutant proteins, particularly since most of these users are members of the molecular genetics community. However, this function also provides a link to the Protein Structure Data Bank (http: www.pdb.bnl.gov/cgi-bin/browse; 10), from where the IBM and Macintosh molecular viewer RasMol can be downloaded to allow viewing of the structure.

It was envisaged that the home page could provide a convenient site for researchers to place or answer FVIII-related questions on a dedicated bulletin board. This capacity is provide by the sixth function of the main menu. Methodological problems, the dis-cussion of novel ideas or any other point that would require a timely response from the haemostasis community could be posted.

Since this new publishing medium is potentially accessible by anyone with a computer, an on-line glossary is provided for the interested lay reader in the seventh function of the menu. It was hoped haemophilia A sufferers could keep up to date with progress in the field or that the home page could act as an educational tool to students.

The site would also welcome additional views and reviews to keep it current with the state of the art.

Citation

Users of HAMSTeRS are asked to cite this article in their publications.

ACKNOWLEDGEMENT

We wish to thank all colleagues who have submitted unpublished results to the database.

REFERENCES

1 Tuddenham,E.G.D. and Cooper,D.N. (1994) The Molecular Genetics of

Hae-mostasis and its Inherited Disorders. Oxford University Press, Oxford, UK.

2 Toole,J.J., Knopf,J.L, Wozney,J.M., Sultman,L.A., Buecker,J.L., Pittman,D.D., Kaufman,R.J., Brown,E., Shoemaker,C., Orr,E.C., Amphlett,G.W., Foster,W.B., Coe,M.L., Knuston,G.J., Fass,D.N. and Hewrick,R.M. (1984) Nature, 312, 342–347.

3 Antonarakis,S.E., Kazazian,H.H. and Tuddenham, E.G.D. (1995) Hum.

Mutat., 5, 1–22.

4 Kazazian,H.H., Tuddenham,E.G.D. and Antonarakis,S.E. (1995) In Scriver,C.R., Beaudet,A.L., Sly,W.S. and Valle,D. (eds), The Metabolic

and Molecular Bases of Inherited Disease, 7th Edn. McGraw-Hill, pp.

3241–3267.

5 Jacobson,D. (1994) Protein Sci., 3, 2159–2161.

6 Kemball-Cook,G. and Tuddenham,E.G.D. (1995) The molecular defect in haemophilia A. In Textbook of Haemophilia, Chapman and Hall, London. 7 Zaitseva, I., Zaitsev, V., Card, G., Moshkov, B., Ralph, A. and Lindley, P.F.

(1995) The nature of the copper centres in human ceruloplasmin.

Biological Inorganic Chemistry, in press.

8 Greer,J. (1990) Genetics, 7, 317–334.

9 Carson,M. (1991) Appl. Crystallogr., 24, 958–961.

Figure

Figure 1. (Left) HAMSTeRS home page. (Bottom right) Quiz database form. (Top right) RasMol rendering of factor VIII, a domain model based on caeruloplasmin.
Figure 2. Schematic representation of the architecture of the HAMSTeRS home page.

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