Genetic analyses

Once a factor deficiency has been diagnosed using functional assays, the underlying molecular genetic defect can then be characterised in specialist laboratories.

Once FVII CD has been confirmed based on chromogenic and/or clot-based FVII(a) activity assays, characterisation of the underlying molecular genetic defect can be performed in specialist laboratories. Genotyping is usually performed by amplification of DNA using methods based on the polymerase chain reaction (PCR) and subsequent sequencing to detect mutations. A large number of polymorphisms and mutations in the FVII gene have been described, including point, missense, nonsense and splice-site mutations, small deletions and rearrangements.¹

Once a diagnosis of haemophilia A has been confirmed based on chromogenic and/or clot-based FVIII activity assays, characterisation of the underlying molecular genetic defect can be performed in specialist laboratories. Genotyping is usually performed by amplification of DNA using methods based on the polymerase chain reaction (PCR) and subsequent sequencing to detect mutations and structural variants.

Based on other individuals with the same mutation, genotyping results can be used to predict bleeding severity and the risk of inhibitor development. Once the genetic mutation has been defined in the haemophilia A index patient, the carrier status among female relatives can also be determined and used for genetic family planning counselling and prenatal assessments. At least 2500 mutations have been identified in the FVIII gene, although not all mutations may impact FVIII expression or function. Furthermore, additional novel functional mutations may remain to be discovered.²

Once a diagnosis of haemophilia B has been confirmed based on chromogenic and/or clot-based FIX activity assays, characterisation of the underlying molecular genetic defect can be performed in specialist laboratories. Genotyping is usually performed by amplification of DNA using methods based on the polymerase chain reaction (PCR) and subsequent sequencing to detect mutations and structural variants.

Based on other individuals with the same mutation, genotyping results can be used to predict bleeding severity and the risk of inhibitor development. Once the genetic mutation has been defined in the haemophilia B index patient, the carrier status among female relatives can also be determined and used for genetic family planning counselling and prenatal assessments. Although not all genetic variants may impact FIX expression or function, at least 1000 mutations have been identified in the FIX gene, and additional novel functional mutations may remain to be discovered.³

Once FXI CD has been confirmed based on clot-based or chromogenic FXI activity assays, specialist laboratories can characterise the underlying genetic anomaly. Mutations in patients from populations with a known FXI CD founder effect can be characterised by amplification of DNA from the gene region(s) expected to harbour the known mutation(s) using methods based on the polymerase chain reaction (PCR) and subsequent sequencing. In the absence of a family history of FXI CD or the absence of a potential known founder mutation, a full molecular sequence of the FXI gene, including non-coding regions that affect transcription or splicing, may be required.⁴

Once FXIII CD has been confirmed based on chromogenic activity assays and the subtype classified using FXIII antigen assays, characterisation of the underlying molecular genetic defect can be performed in specialist laboratories for research purposes only.^5,6 Molecular analysis can identify causative genetic variants by complete sequencing of exonic and regulatory regions of the F13A1 and F13B genes.⁷ Genotyping is usually performed by amplification of DNA using methods based on PCR and subsequent sequencing to detect mutations.

Once a diagnosis of GT has been achieved based on platelet function assays, confirmation can be provided by a characterisation of the underlying molecular genetic defect. Genotyping is usually performed by amplification of genomic DNA coding for the ITGA2B and ITGB3 genes, including all 45 exons and the associated splice sites, using methods based on PCR and subsequent sequencing to detect mutations. A large number of polymorphisms and mutations have been described and include missense, nonsense and splice-site mutations, small deletions, insertions and inversions. Identified mutations should be confirmed using a second DNA analysis.^8,9

Polymerase chain reaction (PCR)

The repeated copying of a selected region of a DNA molecule can be performed using PCR. DNA with the target sequence is mixed with DNA polymerase, two oligonucleotide primers and nucleotides. A single starting molecule of target DNA is sufficient for the PCR to amplify large quantities of the DNA segment between the oligonucleotide primers. The primers are designed to attach to the target DNA at either side of the segment that should be amplified and are required to initiate DNA synthesis by the DNA polymerase, which incorporates the nucleotides complementary to the DNA template.

Overview of the amplification of a defined DNA sequence using PCR.

Sequencing

Although DNA sequencing is often an automated process, the method upon which it is usually based is the Sanger chain termination method. Identical single-stranded DNA molecules containing the genetic segment to be sequenced, for example amplified by PCR, are used as a template to which a short oligonucleotide primer is annealed at the same position on each molecule. The primer is the starting point for DNA polymerase to synthesise a complementary DNA strand by incorporating deoxyribonucleotide triphosphates (dNTPs). In addition to the four dNTPs, a small amount of the four dideoxynucleotides (ddNTPs), each labelled with a different fluorophore, is added to the mixture. These lead to the termination of DNA synthesis when they are incorporated into the growing chain at various positions because they lack the 3’-hydroxyl group required for subsequent polymerisation of the next nucleotide. This results in a set of chains with different lengths, all ending with one of the four fluorescently-labelled ddNTPs. The different chains are separated from one another using polyacrylamide gel electrophoresis, which can separate DNA molecules that differ in length by only a single nucleotide. The DNA sequence can be established from the sequence of the fluorescent bands on the electrophoresis gel.

1. Lapecorella M, Mariani G, International Registry on Congenital Factor VIID. Factor VII deficiency: defining the clinical picture and optimizing therapeutic options. Haemophilia 2008;14:1170-5.

2. Johnsen JM, Fletcher SN, Huston H, et al. Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative. Blood Adv 2017;1:824-34.

3. Johnsen JM, Fletcher SN, Huston H, et al. Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative. Blood Adv 2017;1:824-34.

4. Duga S, Salomon O. Congenital factor XI deficiency: an update. Semin Thromb Hemost 2013;39:621-31. 

5. Bolton-Maggs PH, Favaloro EJ, Hillarp A, Jennings I, Kohler HP. Difficulties and pitfalls in the laboratory diagnosis of bleeding disorders. Haemophilia 2012;18 Suppl 4:66-72.

6. Kohler HP, Ichinose A, Seitz R, et al. Diagnosis and classification of factor XIII deficiencies. J Thromb Haemost 2011;9:1404-6.

7. Biswas A, Ivaskevicius V, Thomas A, Oldenburg J. Coagulation factor XIII deficiency. Diagnosis, prevalence and management of inherited and acquired forms. Hamostaseologie 2014;34:160-6.

8. Nurden AT, Pillois X, Wilcox DA. Glanzmann thrombasthenia: state of the art and future directions. Semin Thromb Hemost 2013;39:642-55.

9. Solh T, Botsford A, Solh M. Glanzmann's thrombasthenia: pathogenesis, diagnosis, and current and emerging treatment options. J Blood Med 2015;6:219-27.