Dr. István Balogh, Dr. János Kappelmayer, Dr. József Tőzsér (2011)
University of Debrecen
The previously described Duchenne/Becker muscular dystrophy and cystic fibrosis are among the most common monogenic severe diseases. In the following, examples are shown for much more rare monogenic severe disorders, providing samples for the great variability for mutational spectrum, diverse clinical picture and methodological possibilities. The common feature of the diseases described is that, as the genes responsible for the diseases are cloned, prenatal molecular diagnostic procedures are applicable, especially, when the family-specific alteration is known (Factor V Leiden, being only a risk factor for venous thrombosis is excluded from this list).
Polycystic kidney and hepatic disease gene 1 (PKHD1).
Unlike the much more benign dominant counterpart, the autosomal recessive polycystic kidney disease (ARPKD) is a significant cause of neonatal morbidity and mortality accounting for a neonatal mortality rate of 25-35%. Prevalence of the disease is 1:20,000 with a carrier frequency of 1:70. The gene that is responsible for the ARPKD encodes a large protein with unknown function (fibrocystin or polyductin), see in Figure 10.1.
PKHD1 gene lacks any mutational hot spot, therefore, direct mutation analysis by gene sequencing is the recommended molecular genetic approach in the diagnosis of ARPKD.
Niemann Pick Type C disease (NPC).
Niemann Pick Type C disease (NPC).
Figure 10.3. shows some amino acid residues affected by mutations casing Niemann-Pick type C disease. This domain is rich in cysteine amino acds, which are frequently involved in the formation of disulphide bridge. Duble arrows show the already known pathogenic alterions. Amino acid reidues in red are phylogenetically conserved, which means that they cannot be replaced by other amino acids.
Examples of inherited diseases in the blood coagulation system: multifactorial and monogenic disorders During the process of the humoral way of blood coagulation, a fibrin network is formed, which closes the wound. A key component of this process is the activation of the final effector enzyme, thrombin (T, Figure 10.4.).
Activation of the procoagulant factor V (FV). Activation is done by thrombin or by active factor X (FXa). The non-enzymatic FVa is a cofactor of FXa in the prothrombin - thrombin conversion.
The thrombin-thrombomodulin complex activates the natural anticoagulant protein C (PC).
Factor V has anticoagulant properties too.
The FXa / FVa complex in the activated membrane surface activates prothrombin in the presence of Ca2+.
Activated protein C (APC) inactivates the procoagulant active factor V (FVa) in the presence of its cofactor, protein S by limited proteolysis that involves cleavage next to several arginine residues.
Procoagulant factor V can be inactivated by thrombin too.
Activated factor VIII is inactivated by APC/PS/FVac.
Mutations in the gene encoding FVIII cause haemophilia A. Loss-of-function mutations in the protein C or protein S genes result in severe thrombotic disease, while different mutations in the gene for factor V might result in severe bleeding as well as thrombotic tendency. Inherited factor V deficiency, which is a result of loss-of-function mutation in the factor V gene show autosomal recessive inheritance. Prevalence of factor V deficiency is 1:1,000,000. Contrary to this, when a specific mutation affects the primary cleavage/inactivation site for APC, the arginine at position 506, will result in the opposite effect, that is, increased risk of thrombosis (thrombophilia). The consequence of this mutation (the so-called factor V Leiden mutation) is that the mutant active, procoagulant factor V will stay longer in the circulation leading to increased risk of thrombosis. Heterozygous genotype increases the risk by 5-10-fold compared to wild type individuals, while homozygous mutant genotype increases the risk of venous thrombosis by 50-100-fold. The mutation being highly prevalent, it is also important at the level of public health. In Hungary, 1 out of 10 is heterozygous. Molecular testing of Factor V Leiden is one of the most commonly performed diagnostic assays in the developed world.
For the final proof of a given inherited disease, molecular genetic assays are frequently complemented with protein-based tests, as it has been demonstrated in the case of Duchenne/Becker muscular dystrophy. Such an analysis is shown in Figure 10.5. The figure shows the result of antigen measurement in the case of a family with a factor V deficient member (generation IV, arrow). As has been shown, the autosomal recessive factor V deficiency is a rare disease with a prevalence of 1:1,000,000. Two mutations are needed to develop the symptoms, inherited in trans. According to the situation shown in the picture, on the father’s side, the mutation can be traced back to the paternal grandmother (II. 2). Persons with heterozygous genotype will not have symptoms, as factor V levels around 50% are enough to maintain coagulation. The other disease-causing mutation (or more precisely, its consequence, the decreased factor V amount) can only be seen in the proband's mother (III. 2), which indicates its de novo generation. Antigen levels show that factor V deficiency in this family is a result of the decreased amount of factor V protein (CRM-, cross reactive material - ), meaning that the mutations interfere with the expression or stability of the mutant factor V.
Figure 10.5. Figure 10.5. Complementer tests for molecular genetic analysis: quantifcation of proteins by ELISA