Samenvatting

The complement system is a part of the immune system and consists of more than 35 soluble and plasma membrane proteins that interact with each other and other elements of the immune system. Activation of the complement system by the classical, membrane binding lectine (MBL) and alternative pathway cause a cascade reaction, which leads to a large complement response.
For so far known the complement system has three functions: it acts as a chemo-attractant (releasing of complement to attract phagocytes to the site of infection), opsonization of bacteria (complement coats the bacteria, allowing the phagocytes to recognize the bacteria) and killing pathogens by causing pores in the cell membrane. The latter is caused by the assembling of the membrane attack complex (MAC) and consists of the complement components C5b, C6, C7, C8 and C9. Defects in the MAC complement components results in recurrent meningococcal disease.
This study is focused on the complement component 7. It is known in literature that C7 deficiency leads to the loss of lytic function of complement. C7 deficient patients show a higher risk of getting recurrently infected by the Neisseria meningitidis bacteria. Mutations in the C7 gene often results in a stopcodon that leads to a premature truncated C7 protein. Other mutations results in no production or no secretion of the protein or to a nonfunctional protein.

In literature it is known that the C7 protein has different polymorphisms. The correlation between (familial) N. meningitidis infections and C7 polymorphisms is not yet known. 20 patients were analyzed during this study. Iso electric focusing showed that 11 patients have an absence of the C7 protein (C7 deficient patients), 7 patients have a C7 protein with aberrant charge (IEF patients) and 2 patients with a normal charged C7 (C7n), but they are infected with a less virulent serogroup. The hemolytic activity of the latter 2 patient group is normal, giving the information that the complement is working.

The aim of this project is the genetic analysis of the complement C7 gene in the C7 deficient, IEF and C7N patients. The bactericidal activity against the N. meningitidis also needs to be determined. The main question is: what is the cause of the N. meningitidis infection in the IEF and C7n patients.

The genetic analysis was done by PCR and sequencing of the C7 gene in all the patients, to detect polymorphisms and mutations. To confirm the mutations and detect the C7 protein, western blotting was preformed. The bactericidal activity of the patients’ serum was tested in a killing assay with the Neisseria meningitidis.

The C7 deficient patients, all but one, are compound heterozygous. One patient has a novel homozygous mutation. Three other novel mutations were found in the C7 deficient patients and the C7 protein could not be detected on western blot, which confirms that the novel mutations leads to the absence of the protein in serum. The absence of the protein also shows that the C7 patients were not able to kill the N. meningitidis in the killing assay.

The molecular bases of 2 polymorphisms are elucidated by 2 novel polymorphisms found in 2 IEF patients. The polymorphisms cause an amino acid substitution and leads to C7 protein with a different charge then the wild type. This is confirmed with data of previous studies.
No other polymorphisms or combinations of polymorphisms were found what can contribute to the cause of the infection in the IEF patients. The IEF patients were able to kill the N. meningitidis in the killing assay, even better then the controls. For so far the cause of the infection in the IEF patients could not be found.