Molecular Haematopathology

Test Description and Value


Thrombophilia screening (1)

Factor V Leiden mutation screening

A single adenine-for-guanine point mutation in the gene coding for coagulation factor V results in a form of factor Va (with replacement of arginine by glutamine at position 506) that is resistant to degradation by activated protein C and leads to a relative hypercoagulable state. The test is indicated for evaluation of patients with unexplained thrombosis, particularly for those with a recurrent or positive family history of the disease. It is also useful for the evaluation of complications of pregnancy, such as pre-eclampsia or late fetal loss. Factor V Leiden mutation is found in up to 15% in selected cohorts in the western populations but extremely rare in the Chinese population. A positive result indicates presence of genetic predisposition to thrombosis development.



Dahlback B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci U S A. 1993; 90:1004-8.

Koster T, Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet. 1993; 342:1503-6.

Svensson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med. 1994; 330:517-22.

Prothrombin G20210A mutation screening

A genetic mutation in the 3' untranslated region (3'-UTR) of the prothrombin (Factor II) gene at nucleotide position 20210 involving a G to A transition is associated with higher levels of plasma factor II activity and thus an increased risk of venous thrombosis. The test is indicated for evaluation of patients with unexplained or recurrent thrombosis (or a family history of the disease) or with a heterozygous Factor V Leiden mutation. Patients with concurrent prothrombin G20210A and Factor V Leiden mutations have an early-onset and more severe thrombotic diseases. Prothrombin G20210A mutation is extremely rare in the Chinese population but may be found in up to 18% in western populations.


Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3' untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996; 55:3698-703.

Howard T, Chanell C, Marsura M, Duncan A. Massive thrombosis associated with homozygous 3'UTR prothrombin gene mutation. Blood Coagulation & Fibrinolysis. 1997; 8:316-9.

Methylenetetrahydrofolate reductase gene C677T mutation screening

Hyperhomocysteinemia is an important and independent risk factor for vascular disease and was found to be associated with increased risk of pregnancy with neural tube defects. The C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene leads to C/C, C/T and T/T genotypes, which affect the plasma homocysteine concentration in humans. The test is indicated for evaluation of selected individuals with early-onset arteriosclerotic vascular disease or thrombosis (particularly for those with hyperhomocysteinemia or a family history of the disease). It is also requested for evaluation of pregnancy at risk of neural tube defects in selected subjects. MTHFR C677T mutation is common in the Chinese population.


Kang SS, Wong PW, Susmano A, Sora J, Norusis M, Ruggie N. Thermolabile methylenetetrahydrofolate reductase: an inherited risk factor for coronary artery disease. Am J Hum Genet. 1991; 48: 536-45.

Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995; 10: 111-3.

van der Put NM, Steegers-Theunissen RP, Frosst P, Trijbels FJ, Eskes TK, van den Heuvel LP, et al. Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. Lancet. 1995; 346: 1070-1.


Hereditary Haemochromatosis Gene Mutation Screening (2)

C282Y & H63D gene mutation

A defective HLA class I gene, HFE, is responsible for hereditary haemochromatosis, one of the most frequent hereditary anomalies among whites. The gene codes for a class I \ chain that has lost its ability to bind peptides and therefore no longer participates in immunity. Instead, it has acquired a new function X an ability to form complexes with the receptor for iron-binding transferrin and thus regulate the uptake of dietary iron by cells of the intestine. The test is indicated for evaluation of individuals with increased serum ferritin levels, transferrin saturation, or liver cirrhosis. It may also be used to evaluate individuals with unexplained elevation of serum liver enzymes or non-specific compatible symptoms such as abdominal pain, fatigue, lethargy, and weight loss, which are early signs of hereditary haemochromatosis. HFE C282Y and H63D mutations are rare in the Chinese population.



Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet. 1996; 13: 399-408.

Beutler E. The significance of the 187G (H63D) mutation in hemochromatosis. Am J Hum Genet. 1997; 61: 762-4.

Beutler E, Felitti VJ, Koziol JA, Ho NJ, Gelbart T. Penetrance of 845G--> A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet. 2002; 359: 211-8.


Thalassaemia & Haemoglobinopathy Investigation (3)

a -thalassaemia genotyping

The loss of one (- a ) or both (--) of the a -globin genes on chromosome 16 is the most common cause of a -thalassaemia. In addition, a -globin genes can be inactivated by base pair substitutions such as the Hb Constant Spring and Hb Quong Sze mutations. Patients with HbH disease (--/- a ) develop chronic haemolytic anaemia of various severity, whereas fetuses with Hb Barts hydrops fetalis (--/--) die either in utero or shortly after birth as a result of severe intrauterine anaemia. Individuals with three functional a genes (- a / a a ) are clinically and haematologically silent. While carriers with a -thalassaemia trait (- a /- a or --/ a a ) only suffer from mild hypochromic microcytic anaemia, couples carrying this trait are at risk of having a hydrop baby or offspring with HbH disease. These molecular tests are employed to determine the genotype of a -thalassaemia patients and to confirm / exclude the presence of a -thalassaemia trait (e.g. the SEA deletion), especially in couples with concurrent b -thalassaemia trait in antenatal check-up.



Liu YT, Old JM, Miles K, Fisher CA, Weatherall DJ, Clegg JB. Rapid detection of alpha-thalassaemia deletions and alpha-globin gene triplication by multiplex polymerase chain reactions. Br J Haematol. 2000; 108: 295-9.

Laosombat V, Wiryyasateinkul A, Chrangtrakul Y, Fucharoen S. Rapid detection of an alpha-thalassemia variant (Hb Quong Sze). Haematologica. 2003; 8 8: ELT27.

Chui DH. Alpha-thalassemia: Hb H Disease and Hb Barts Hydrops Fetalis. Ann NY Acad Sci. 2005; 1054:25-32.



b -thalassaemia genotyping

b -thalassaemia is mainly caused by nucleotide substitutions, as well as small deletions and insertions in the b -globin gene. Due to the diversity of the mutations, the disease phenotypes are heterogeneous and can be classified into thalassaemia minor, intermedia and major depending on the clinical severity. Thalassaemia minor is related to a heterozygous mutation in b -globin gene ( b X or b +); thalassaemia intermediate often to two b + genes; and thalassaemia major to b +/ b X and b X / b X . This molecular test is employed to determine the genotype of b -thalassaemia patients and also in the clarification of thalassaemia intermedia.



Cao A, Galanello R, Rosatelli MC. Genotype-phenotype correlations in beta-thalassemias. Blood Rev. 1994; 8: 1-12.

Weatherall DJ, Clegg JB. The thalassaemia syndromes 4th ed. Blackwell Science Oxford. 2001; 846.

Tuzmen S, Schechter AN. Genetic diseases of hemoglobin: diagnostic methods for elucidating beta-thalassaemia mutations. Blood Rev 2001; 15:19-29.


Hereditary Persistence of Fetal Haemoglobin screening [HPFH-1,2,3,6 and SEA] and Chinese G g ( A g d b ) X thalassaemia

Hereditary Persistence of Fetal Haemoglobin (HPFH) and d b thalassaemia are heterogeneous disorders characterized by elevated levels of fetal haemoglobin (HbF) in adult life. HPFH heterozygotes are typically characterized by HbF levels up to 30 % with normal red blood cell indices, while d b thalassaemia heterozygotes tend to have increased HbF levels that are lower (5-20%) and accompanied by hypochromic, microcytic red cell indices. These tests are employed to evaluate the molecular mechanisms underlying increased HbF levels in adult life.



Craig JE, Barnetson RA, Prior J, Raven JL, Thein SL. Rapid detection of deletions causing delta beta thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification. Blood. 1994; 83: 1673-82.

Kosteas T, Palena A, Anagnou NP. Molecular cloning of the breakpoints of the hereditary persistence of fetal hemoglobin type-6 (HPFH-6) deletion and sequence analysis of the novel juxtaposed region from the 3' end of the beta-globin gene cluster. Hum Genet. 1997; 100: 441-5.

Forget BG. Molecular basis of hereditary persistence of fetal hemoglobin. Ann N Y Acad Sci. 1998; 850: 38-44.

Peng CT, Liu SC, Chiou SS, Kuo PL, Shih MC, Chang JY, et al. Molecular characterization of deletional forms of beta-thalassemia in Taiwan. Ann Hematol. 2003; 82: 33-6.