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Keywords:

  • FIX;
  • genetics;
  • guidelines;
  • haemophilia B;
  • mutation analysis;
  • UKHCDO

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. References

Summary.  Haemophilia B is one of the most common inherited bleeding disorders and has a well understood pathophysiology. Our understanding of the molecular genetics of the disease has allowed the development of comprehensive carrier and prenatal diagnosis for this single gene disorder. Continuing technological developments improve our ability to provide genetic analysis in a rapid and cost-effective manner. This guideline aims to provide advice on current best laboratory practice when approaching genetic diagnosis of haemophilia B.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. References

Haemophilia B or Christmas disease is a recessively inherited X-linked bleeding disorder which results from deficiency of procoagulant factor IX (FIX). FIX deficiency is characterised by prolonged oozing after injuries, tooth extractions, or surgery, renewed bleeding after initial bleeding has stopped, and delayed bleeding. Severely affected males suffer from spontaneous joint and muscle bleeds and easy bruising. The age of diagnosis and frequency of bleeding episodes are related to the FIX clotting activity. Haemophilia severity is defined by FIX:C level in plasma, where severely affected individuals have <1 i.u. dL−1 (<1% of normal); moderate 1–5 i.u. dL−1 (1–5% of normal); and mild >5 to <40 i.u. dL−1 (>5 to <40% of normal) [1]. It is less common than haemophilia A with a frequency of approximately 1 in 25 000 males worldwide [2].

New cases of haemophilia B may be referred as a result of prior family history of the disease. In such cases male cord blood may be tested at birth to determine FIX:C. However, approximately one third of cases have no prior history of haemophilia B, these are referred to as having sporadic disease. In these cases the age of diagnosis and frequency of bleeding episodes are often related to the FIX clotting activity [2]. Patients with severe haemophilia B are usually diagnosed during the first year of life. Without treatment, they have an average of two to five spontaneous bleeding episodes each month. Patients with moderately severe haemophilia B seldom have spontaneous bleeding; however, they do have prolonged or delayed oozing after relatively minor trauma and are usually diagnosed before the age of 5–6 years. The frequency of bleeding episodes varies from once a month to once a year. Patients with mild haemophilia B do not have spontaneous bleeding; however, without treatment, abnormal bleeding occurs with surgery, tooth extraction, and major injuries. The frequency of bleeding may vary from once a year to once every 10 years. Patients with mild haemophilia B are often not diagnosed until later in life. In any patient, bleeding episodes may be more frequent in childhood and adolescence than in adulthood. Female relatives may request carrier analysis when a male relative is first diagnosed as having haemophilia, when they wish to start a family, or (frequently), when in early pregnancy. Genetic analysis is required to reliably determine female carrier status because the majority of female carriers have normal plasma FIX levels [2–4]. Carrier females with FIX clotting activity <30% are at risk for bleeding (approximately 10% of carrier females, independent of severity of disease in their family).

Genetic counselling should be performed by suitably qualified health professionals with in-depth knowledge of haemophilia. Ideally a professional with experience of managing and treating patients with haemophilia and their families should be involved. It is recommended that genetic testing for haemophilia in the UK should be performed in a member laboratory of the UKHCDO Haemophilia Genetics Laboratory Network, details of which are hosted by the British Society for Haematology (http://www.b-s-h.org.uk/DirectoryV2.June2003.pdf). This is a consortium of laboratories, mostly within Comprehensive Care Haemophilia Centres, which work to agreed peer-reviewed standards of quality.

Aims of the guideline

This guideline aims to provide advice on a rational approach to genetic diagnosis in haemophilia B using currently available laboratory techniques and best practice.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. References

This guideline document was prepared on behalf of the UKHCDO Haemophilia Genetics Laboratory Network in response to a workshop meeting held at the UKHCDO annual meeting, 9 October 2003, Newcastle upon Tyne, UK. The workshop was attended by a range of UK based health care scientists working in the field of haemophilia genetic diagnosis. Diagnostic approaches and issues were discussed within the group. The guideline-writing group, consisting of UK based scientific experts in the field of haemophilia genetic diagnosis, used the discussion as a basis for the preparation of draft guidelines. This draft was prepared with reference to relevant literature reports, reviews and web-based resources relating to the subject. The draft was circulated to the workshop attendees for comment and the final document approved by the UKHCDO Advisory Committee. The resulting guidelines are hosted by the Clinical Molecular Genetics Society (CMGS) website (http://www.cmgs.org/new_cmgs/) from where this document is derived.

The FIX gene

The FIX gene, located on the long arm of the X chromosome at Xq27, spans 33.5 kb of DNA and comprises eight exons [5]. FIX mRNA is 2.8 kb and encodes a mature protein of 415 amino acids. Haemophilia B results from heterogeneous mutations throughout the FIX gene. Unlike haemophilia A, no common repeat mutation has been identified. However, 20–30% of cases of mild haemophilia B are due to a small number of founder mutations. Exon 8 is the largest FIX exon, being 1.9 kb in length and representing almost half of the FIX coding region. Half of all FIX mutations are found in this exon [6]. Mutations in the promoter of the FIX gene are relatively rare (∼2% of the total) but important because they can give rise to the unique haemophilia B Leyden phenotype, where symptoms typically ameliorate at puberty from severe to mild or even asymptomatic [7]. For a review on the molecular aspects of haemophilia B see [8]. Key information resources for the FIX gene are given in Table 1.

Table 1.  Key nomenclature and reference sequence identifiers for the FIX gene.
Gene name (HUGO nomenclature)FIX (F9)
  1. FIX, factor IX.

OMIM number306900
Gene cards IdF9
Ensembl gene IDENSG00000101981
Chromosomal locationXq27.1–q27.2
Medline MESH termHaemophilia B, FIX
NCBI LocusLinkHsF9 (Locus ID 2158)

Diagnostic strategy

Previously characterized mutations

The Division of Medical and Molecular Genetics at Guy's Hospital, London, has characterized the mutation(s) responsible for haemophilia B in a large proportion of UK families. However, this programme was never intended as a diagnostic service and is no longer active. Many laboratories are now screening their patients for mutations in the FIX gene (see the UKHCDO Directory of Molecular Diagnostic Services for Inherited Bleeding Disorders, hosted on the British Society for Haematology website (http://www.b-s-h.org.uk/DirectoryV2.June2003.pdf). The UKHCDO [9] haemophilia patient database, an annually updated listing of all UK registered patients, notes whether a mutation has been detected in a particular patient. Details of the mutation characterized in a patient of interest are only available from their Haemophilia Centre Director (see listing on UK Haemophilia Society website (http://www.haemophilia.org.uk/).

Unknown mutation detection

Mutations are generally sought in affected males and then confirmed or excluded in female relatives. The method selected will be dependent on resources and expertise available in a particular laboratory [9,10]. Current methods which have been applied by many centres performing mutation prescreening in the UK haemophilia B population rely on heteroduplex formation and subsequent detection of mismatched heteroduplexes. There are two major heteroduplex formation methods in current use; Conformation sensitive gel electrophoresis (CSGE) and Denaturing high performance liquid chromatography (dHPLC).

Conformation sensitive gel electrophoresis

Conformation sensitive gel electrophoresis (CSGE) [11] is a variant of heteroduplex analysis and has been applied to screening the FIX gene for mutations [12]. It has the advantages of being simple and relatively rapid to perform and does not require the use of radiolabel. Despite this apparent simplicity, the technique requires a great deal of skill, both technical and interpretive, to achieve good sensitivity. PCR products subjected to CSGE should optimally be no greater than 500 bp and have a high degree of overlap (≥100 bp) when amplifying large exons such as exon 8. Given careful design and application, mutation detection sensitivity of >90% can be achieved.

Denaturing high performance liquid chromatography

Denaturing high performance liquid chromatography (DHPLC) [13,14] separates hetero- and homoduplexes due to their differences in melting behaviour and subsequent retention time on a non-porous polystyrene-divinylbenzene matrix. The dHPLC requires specialist equipment (most commonly used is the Transgenomic Wave System) but is otherwise a technically straightforward and rapid way to screen for mutations in the FIX gene [15].

Care should be taken with assay design; good primer design assisted by software analysis of amplicon melting characteristics is essential, if high detection sensitivity is to be achieved.

An experienced scientist should expect >95% sensitivity (150–500 bp). Fragments less than 110–120 bp in size are liable to loss of detection sensitivity. Larger fragments (up to 1–1.2 kb) can be studied, but this invariably results in the need to accommodate more melt domains and hence more injections per sample at a range of oven temperatures.

Best Practice Guidelines have been produced for dHPLC and can be found on the CMGS website (http://cmgs.org/BPG/Guidelines/2002/dhplc.htm). Other mutation prescreening methods may be used, e.g. CMMC [16] or DGGE [17] but are not currently employed by the network.

FIX deletions

Patients having partial or complete FIX gene deletions are relatively rare, comprising only ∼3% of patients [6]. Deletions may be detected by lack of PCR amplification of particular exons or by apparent non-inheritance of a polymorphic allele within a family. Detection of large deletions is often difficult in heterozygous carriers. Currently, methodologies measuring gene dosage, e.g. Quantitative PCR/RT–PCR or loss of heterozygosity are the best option, although not infallible. Newer methodologies are available for exploration, e.g. Multiplex Ligation-dependent Probe Amplification (MLPA) [18].

Direct sequencing

DNA sequencing is considered the gold standard for mutation detection. The FIX gene is small enough to contemplate sequencing the coding region, splice junctions, and the 5′ and 3′ regions for previously unknown mutations [19], within the time constraints of a diagnostic service. Normally a candidate mutation would be identified in a hemizygous male haemophilic before applying DNA sequencing to determine the presence or absence of a nucleotide alteration in at risk family members. Failing this, a known obligate carrier female can be used for initial mutation identification. Once a mutation is identified in a family, direct sequencing is the preferred methodology with which to confirm or exclude its’ presence in other family members. Direct sequencing is not infallible when detecting heterozygous base changes. ‘Preferential incorporation’ may lead to heterozygotes being missed. ‘Heterozygote sequencing’ should always be performed both 5′ (forward) and 3′ (reverse). Design of a ‘mutation specific test’, e.g. restriction digest or allele specific PCR is an alternative approach to consider.

DNA sequencing best practice

Refer to the CMGS website Sequencing Best Practice Guidelines (http://cmgs.org/BPG/Guidelines/2002/sequence.htm) for guidance on minimum sequence quality and interpretation standards. It is recommended that that the following points be given particular attention:

  • 1
    Software analysis tools (e.g. tools which facilitate comparative sequence analysis such as the Staden Package (http://staden.sourceforge.net/) should be employed when analysing large quantities of DNA sequence data.
  • 2
    Sequence analysis should always be performed on both forward and reverse strands and any sequence change used for diagnosis should be confirmed by repeat sequencing in relevant family members. As a minimum this should be done with recourse to the original DNA (or stored blood) sample and reamplification from the original sample. Some centres may wish to issue an interim report until they have been able to verify a base change in an independent sample from relevant individuals.

Mutation validation

When a novel nucleotide change is found, caution should be exercised before deciding that it is the one responsible for disease. Whereas termination, deletion and insertion mutations may obviously be causative, missense and other changes may not. The haemophilia B Database maintained by Dr Peter Green at King's College, London (http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html) acts as an online repository of information of interest to those involved in haemophilia B genetic analysis. This should be consulted to determine whether the change has been previously reported. Entries on the database need to be interpreted with a degree of caution. Minimum checks should include the following questions:

  • 1
    Does the previously reported disease severity agree with the FIX:C level in the patient being analysed?
  • 2
    Has the candidate mutation been reported previously as a polymorphism?
  • 3
    Is the amino acid change likely to be pathogenic, e.g. is the change non-conservative in nature or is it within a recognized functionally important region of the protein?
  • 4
    Is the changed amino acid conserved across species?
  • 5
    Could the candidate mutation affect splicing? Software tools, e.g. StrataSplice (http://www.sanger.ac.uk/Software/analysis/stratasplice/) or the NetGene2 server (http://www.cbs.dtu.dk/services/NetGene2/) can be used to allow alternative splice site prediction.
  • 6
    Could the ethnic origin of the patient affect interpretation of polymorphism/candidate mutation status for a given base change?

Published data on structure-function relationships in FIX, etc. may assist in the assignment of possible cause to a particular base change [20–24]. Where uncertainty remains, the family should be tested to determine whether the nucleotide alteration tracks with the disease and a panel of normal DNA samples of the same ethnic origin (where possible) examined to rule out a polymorphic change. Wherever possible, candidate mutations should be confirmed in affected, or excluded in unaffected males on the maternal side of the family. Candidate mutations can still be used as bespoke genetic markers if they track appropriately within the family, irrespective of their disease association.

Linkage analysis

Historically, linkage analysis was the method most commonly used to determine female carrier status and perform prenatal diagnosis in families with haemophilia B [4]. Linkage studies are being superseded by direct mutation analysis protocols (see above). However, intragenic linked markers are still useful and may be of particular value under certain circumstances, such as:

  • 1
    Where a family has previously been investigated by linked markers and the mutation has not been identified.
  • 2
    Where a mutation has not been verified.
  • 3
    Where a mutation has not been found.

Linked markers  The use of intragenic markers only should be considered. The entire ∼35 kb of the FIX gene has been sequenced, but no short tandem repeat polymorphisms have been found. However, the DdeI polymorphism in intron 1 is a complex repeat [25] having two common and several rare alleles and a Caucasian heterozygosity rate of 35%, making it a useful component of a panel of markers for linkage analysis.

There are 11 diallelic polymorphisms throughout the FIX gene which can also be used in linkage analysis. Their rate of heterozygosity varies markedly between different ethnic groups. In Caucasians, a combination of just three markers, MseI in the 5′ untranslated region [26], DdeI [27] and HhaI [28] in the 3′ flank of FIX give a combined heterozygosity rate of ∼80%. A combination of markers [10] can achieve informativity in ∼95% of Afro-Caribbean families, 85–90% of Caucasian families and ∼60% of Asian families. As all of these polymorphisms lie within or very closely flank the FIX gene, the rate of recombination between any marker and the mutation is well under 1%. FIX polymorphisms may be analysed very simply. Naturally occurring or introduced restriction enzyme sites are available for each polymorphism, so each marker can be PCR amplified, restriction enzyme digested, electrophoresed and visualized rapidly. PCR primer sequences for commonly analysed FIX polymorphisms are listed in Table 2.

Table 2.  Commonly analysed FIX polymorphisms.
Primer designation Sequence 5′–3′ PCR product size/bp* Heterozygosity†Ref.
  1. *+, presence of restriction site; −, absence.

  2. †Caucasian heterozygosity rate, see Goodeve [10] or Goodeve and Peake [3] for other ethnic groups.

HhaI forwardACA GGC ACC TGC CAT CAC TT−230 bp0.48Winship et al. [28]
HhaI reverseAGA TTT CAA GCT ACC AAC AT+150 and 80 bp  
DdeI forwardGGG ACC ACT GTC GTA TAA TGT GG369 bp large common allele0.36Bowen et al. [27]
DdeI reverseCTG GAG GAT AGA TGT CTC TAT CTG319 bp small common allele  
MseI forwardGAT AGA GAA ACT GGA AGT AGA CCCUndigested = 369 bp0.44Winship et al. [26]
  −176, 83, 73 and 37 bp  
MseI reverseTTA GGT CTT TCA CAG AGT AGA ATT T+176, 57, 26, 73 and 37 bp  
SalI forwardCTC GTT GTG CAC ATG TAC CC−317 bp0.49Toyozumi et al. [30]
SalI reverseCAA TAC CAC CCT ATC CTT CGT CGA+295 and 22 bp  
MnlI forwardAAG TGA CAA GGA TGG GCC TCA ATCUndigested = 422 bp0.44Tsang et al. [31]
  −333, 62 and 27 bp  
MnlI reverseGAA ACT TGC CTA AAT ACT TCT C+214, 119, 62 and 27 bp  

Linkage analysis problems

Linkage analysis fails in a number of families for one of the following reasons:

  • 1
    Lack of prior family history of haemophilia.
  • 2
    Key pedigree members not available.
  • 3
    Polymorphisms uninformative in key female(s).
  • 4
    Non-paternity.

In these families, mutation detection should be used. Linkage analysis cannot determine the carrier status of the mother of a haemophilic. When direct mutation detection is not possible, linkage analysis provides an acceptable alternative, which offers a high degree of diagnostic confidence.

Prenatal diagnosis

Prenatal diagnosis is generally performed by chorionic villus sampling at between 11 and 13 weeks of gestation. Direct karyotype analysis may be performed to determine foetal sex and to ensure that there are no chromosomal abnormalities. Rapid PCR based sexing protocols using amelogenin (AMXY) specific primer sets are in common usage. Female foetuses sexed by this method require confirmation that no maternal contamination is present in the sample. Female foetuses require no further analysis. The carrier status of females may be determined later in life if indicated or desired. Male haemophilia status can be determined by analysis of a previously determined familial mutation or informative marker. As all of these analyses for FIX involve PCR amplification, results should be provided within 2–3 days of the CVS sample being taken. Detailed discussion of issues of PND can be found in the UKHCDO document, Clinical Genetics Services for Haemophilia [29].

Wording of reports

Reports must be clear, concise, accurate, fully interpretive, credible and authoritative. For general guidance on report writing refer to the CMGS web site for the Report Writing Best Practice Guidelines (http://www.cmgs.org/BPG/Default.htm). Suggested wordings are given below:

Mutation analysis reporting  Wording will depend on the confidence placed in the interpretation of any candidate mutation, as discussed above. Suggested wording for a mutation which has a high confidence attached to it may include:

Mutation analysis in malesx has a FIX mutation (No.nt>nt, aaNo.aa), previously reported in the FIX gene/not previously reported. The mutation is consistent with the severity of haemophilia B in x.

A brief explanation as to why a novel mutation is considered causative should be included, especially for a missense mutation. For example, the altered amino acid is conserved across X (a number of) species, and/or is structurally/functionally important; this base change has been excluded as a common polymorphism by analysis of >100 normal alleles, etc.

Mutation analysis in femalesy carries a FIX mutation (No.nt>nt, aaNo.aa) which is consistent with the severity of haemophilia B seen in male relative x.

Mutation nomenclature

For guidance on nomenclature conventions when reporting mutations refer to the Human Genome Variation Society (http://www.genomic.unimelb.edu.au/mdi/mutnomen/).

Nucleotides and amino acids should be numbered as per Yoshitake et al. [5]. Nucleotide numbering starts from ‘the proposed transcription initiation site’ +1 and is continuous through introns. Amino acid numbering starts at +1 for the first codon of the mature protein (tyrosine). The signal peptide is numbered from −46 (initiator methionine) to −1 (arginine). The intron or exon containing the mutation should be stated. Conventionally FIX exons have been labelled A–H and the introns have been numbered one to seven. A standard numbering system of one to eight for exons may be used instead. The use of lower case letters for intronic sequence is recommended.

To avoid potential confusion between single letter amino acid codes and nucleotides, the following convention is recommended: Nucleotide position 17711G>A, corresponding amino acid position C99Y.

In the body of the report, the full name of each amino acid should be specified to avoid confusion between single letter amino acid and nucleotide codes, e.g. ‘Cysteine (C) 1234 to Alanine (A), or C1234A’.

Linkage analysis using intragenic markers  The female can be diagnosed as a carrier/excluded from being a carrier, with a risk of error due to meiotic recombination of <1%.

Mosaicism

Germline and somatic mosaicism may complicate any genetic diagnosis in haemophilia B. Particular attention should be given to the possibility of mosaicism in sporadic haemophilia where the mother of an affected male does not appear to carry the mutation in her leucocyte DNA, particularly (although not exclusively) where the apparent de novo mutation is a point mutation. It is recommended not to state that the mother of a haemophilic is not a carrier, even when the mutation is not identified in her somatic DNA. A specific reference to the possibility of germline mosaicism should be added.

Reference samples for test optimization and validation

Reference samples have yet to be established for haemophilia B.

Disclaimer

The information and advice contained within this guideline are believed to be true and accurate at the time of going to press. Neither the authors nor the publishers can accept any legal responsibility for any errors or omissions that may have occurred.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. References
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