Scientific background:

Summary: Antithrombin 3 deficiency, a an autosomal dominant disorder causing hypercoagulability and recurrent thrombosis. Although easily measured in plasma, further subclassification and family counselling is available through molecular genetic analysis.

Gene: The gene spans about 14kb on chromosome 1 (1q23-q25) and consists of seven exons.

Molecule: The gene product is a glycoprotein that belongs to the alpha-2-globuline fraction. The mature protein of 432 amino acids is secreted by the liver after splitting off the signal peptide (32 amino acids) and further biochemical processing in the endoplasmatic reticulum.
   Two functional active sites can be separated in the mature protein. The reactive center, Arg393-Ser394, cleaves thrombin, and the heparin binding site at the amino terminus. Binding heparin increases inactivating function of antithrombin by 4000 fold.

Molecular anatomy: Antithrombin is a plasma protein that exists in two forms: the active monomer and an inactive heterodimer. One active molecule can inactivate an other by cleaving it and forming a heterodimer, which is also called the latent form.

Pathophysiology: Independent of vitamin K, antithrombin is synthesized and secreted by the liver. It plays a major role in inactivation of thrombin and other activated coagulation serine proteases, including factor Xa and IXa.
   In the absence of heparin, antithrombin slowly inactivates thrombin. This process is called progressive antithrombin activity, and as opposed to that, heparin cofactor activity of antithrombin account for rapid thrombin inactivation in the presence of heparin.

Clinical signs: Antithrombin deficiency is classified in type 1, with functional activity and concentration reduced simultaneously, and type 2, in which the immunologically measurable concentration of the protein is normal despite a significantly reduced functional activity. Type 2 might be further divided into heparin binding site defects (Type 2c), thrombin binding and cleavage site defects (2b), and pleiotropic defects (2c). Heparin binding site defects account for most of antithrombin deficiencies. In these patients, pharmacological reaction to standard heparin therapy is altered, while the effect of low molecular weight heparin therapy is unchanged. Also, the risk of thromboembolic diseases at physiological conditions appears not to be increased.
   Furthermore there exist unstable variants of antithrombin that in fever, acid base disorders, or other pathophysiological conditions exhibit an accelerated antithrombin activity without heparin, which explains thrombophilia in these patients, when seriously ill.

Epidemiology: Initially, the prevalence of antithrombin deficiency in the general population was estimated one in 2000 to 5000. These estimated were based on immunological measurements of the circulating protein. Then, functional assays revealed a much higher prevalence of antithrombin deficiency, one in 150 to 500. These assays also allowed for a further subclassification of antithromin deficiency.
   In patients with venous thromboembolism, the prevalence of antithrombin deficiency is about 0.5%.

Interpretation: In type 1 antithrombin deficiency the underlying mutation might be nonsense or splice mutations in one of the first exons. These mutations can be found by direct sequencing the entire coding region, but a significant portion might be caused by large deletions, duplications or rearrangement, which usually cannot be detected by sequencing.
   Direct sequencing allows to differentiate among type 2 subtypes. As most often point mutations at the specific sites of the molecule account for these functional alterations, the detection rate is much higher in these cases.

Methodology:

	clinical test	Method		Genomic sequencing of the entire coding region
		Turn-around time		25 working days
		Effort		little
		Specimen		DNA
		Quality assessment		Internal quality control only
	All known and new missense, nonsense and splice mutations can be detected.
	clinical test	Method		Multiplex Ligation-Dependent Probe Amplification
		Turn-around time		25 working days
		Effort		little
		Specimen		DNA
		Quality assessment		Internal quality control only
	clinical test	Method		Carrier testing
		Turn-around time		5 working days
		Effort		little
		Specimen		DNA
		Quality assessment		Internal quality control only
	The test is only specific about the mutation already known in this kindred.

Literature: 

Picard V et al. (2006) Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene.
Perry DJ et al. (1996) Molecular genetics of human antithrombin deficiency.
Odegård OR et al. (1978) Antithrombin III: critical review of assay methods. Significance of variations in health and disease.
Meade TW et al. (1990) Antithrombin III and procoagulant activity: sex differences and effects of the menopause.
Tait RC et al. (1994) Prevalence of antithrombin deficiency in the healthy population.
Mateo J et al. (1997) Laboratory evaluation and clinical characteristics of 2,132 consecutive unselected patients with venous thromboembolism--results of the Spanish Multicentric Study on Thrombophilia (EMET-Study).