Link Between CD24 Gene and Multiple Sclerosis

¶ … risk of development and progression of Multiple Sclerosis with the different CD 24 polymorphisms: V/V, a/a and a/V.

To assess the expressivity of various gene polymorphism on T cells.

To check any tendency in the development of Multiple Sclerosis within family members and different ethnic groups.

Materials and Methods:

The study checked the single nucleotide polymorphism in a population of Multiple Sclerosis patients and controls. These patients were diagnosed with Multiple Sclerosis, using the Mc Donald criteria. The short nucleotide polypeptide was recognized by using PCR-RFLP, using primers, and tests were applied to check for differences in observations between the groups.

The studies established a link between CD 24 and the development of Multiple Sclerosis and its progression.

INTRODUCTION:

The etiology of multiple sclerosis involves interplay between genetic and environmental factors. It is a disease of northern Europeans and occurs less frequently in other racial groups. The lifetime risk of multiple sclerosis in northern Europe is 1:800, increasing to 1:50, 1:20 and 1:3 for the offspring, siblings and monozygotic twin partners of affected individuals, respectively. (D.A, 1996)

In Multiple Sclerosis, the myelin producing oligodendrocytes of the central nervous system are the target of recurrent cell mediated autoimmune attack, which starts with the entry of activated T-cells through the blood-brain barrier. These recognize myelin derived antigens on the surface of the nervous system's antigen presenting cells, the microglia, and undergo clonal proliferation. The resulting inflammatory cascade releases cytokines and initiates destruction of the oligodendrocyte myelin unit by macrophages. (D.A, 1996)

Other than the already established candidate genes (DRB, 1.1501, DRB 5.0101, DQA1.0102 AND DQV2.0602) preliminary evidence also associates CD 24 to multiple sclerosis, as a factor, modifying disease progression. These genes are not the causative factors but play a role in modifying the susceptibility of an individual towards multiple sclerosis. (D.A, 1996)

CD 24 is a cell surface glycoprotein present on many cells, including activated T cells, B cells, macrophages, dendritic cells and local antigen presenting cells in the CNS. Many of these CD24 expressing cells are involved in the pathogenesis of multiple sclerosis. (Zhou, Rammohan, Lin, Robinson, & Li, 2005)

This review has been extracted from two studies; 'CD 24 V/V is an allele associated with the risk of developing multiple Sclerosis in the Spanish population' . (D Otaegui1, A Sa'enz1, P Caman "o1, 2006) and 'CD24 is a genetic modifier for risk and progression of Multiple Sclerosis' (Zhou, Rammohan, Lin, Robinson, & Li, 2005)

MATERIAL AND METHOD:

In the above mentioned studies of CD24 and the role of genetic polymorphism in the risk and progression of Multiple Sclerosis, the following method was applied.

The samples were approved by the institutional review board and informed consent was taken from all the subjects. These patients were diagnosed with Multiple Sclerosis using the Mc Donald criteria. According to the Mc Donald Criteria, diagnosis is made on clinical attack and lesions observed on MRI as follows:

Patient presenting with two or more lesions on MRI along with two or more clinical attacks provides no additional need for further investigations. This clinical evidence, on its own, forms the basis of diagnosis.

For diagnosing Multiple Sclerosis in patients who present with two or more clinical attacks and one lesion, there needs to be dissemination in space or two or more lesions on MRI consistent with MS plus positive CSF.

In patients who present with one clinical attack and two or more objective lesions, there needs to be dissemination in time, for diagnosis.

If there has been one clinical attack and one lesion, there needs to be dissemination in space by two or more MRI lesions, consistent with MS plus positive CSF, and dissemination in time by MRI.

To make a diagnosis, the above mentioned criteria must be present. If there isn't sufficient evidence, one must wait for another clinical attack.

Patients with no clinical attacks but one or more lesions must be followed for a year, to evaluate disease progression, and at least two out of the following three must be checked:

Positive brain MRI ( 9 T2 lesions and 4 or more T2 lesions with positive VEP)

Positive spinal cord MRI (2 or more focal T2 lesions)

Positive CSF (Boon, 2006)

The degree of disability was assessed with the Expanded Disability Status Scale (EDDS), which is scored using the following basic rules.

The EDSS provides a total score on a scale that ranges from 0 to 10. Levels, 1.0 to 4.5, refer to patients with a high degree of ambulatory ability, whereas, the subsequent levels, 5.0 to 9.5, refer to its loss. Category 0 is with a normal neurological exam. Patients who are disabled to an extent that they cannot perform full daily activities, but are still ambulatory without aid or rest for 200 meters are classified as category 5. In category 10, death occurs as a result of Multiple Sclerosis. (Tarver, PhD, 2009)

The obtained samples were compared with a control group.

In the first study, the focus group was recruited from the neurological departments in Gipzukoa, Northern Spain, and were grouped as Basque Natives and Caucasians. These patients were mostly women, with an average age of 42.9+_ 12.7 years. A control group of healthy donors, from the Gipzukoa blood bank, with similar age and sex ratios, were chosen, and similarly divided into Caucasians and Basque natives, using their surnames. This was to check any propensity in the occurrence of Multiple Sclerosis in between the different ethnic groups. An average age of onset and evolution time was calculated, along with the average degree of disability, which was found to be 30.8 +_ 9.5 years, 13.1 +_ 8.9 years and 4 +_ 2.5 respectively. (Zhou, Rammohan, Lin, Robinson, & Li, 2005)

However, the second article chose MS patients and non-MS relatives as their subject population, in order to test evidence of familial aggregation of the 'V' allele in families. This was carried out through the transmission disequilibrium test using family data. Families were divided into two groups:

1) Trios: these were the type 1 families in which there was only one child, who was an MS patient. Both the parental genotypes were available, with at least one being heterozygous.

2) Sibships: these were the type 2 families, with more than one child. The genotypes of the affected and unaffected siblings were available with at least two different genotypes in the sibships.

3) Families that could be either type 1 or 2, were classified as the type 1 family, according to the recommendation of Spielman and Ewens.

Left over blood samples from the American Red Cross was used as a population control. Most of these subjects were from Central Ohio, so it was assumed that the genetic distribution of this sample would reflect that of the Central Ohio population. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)

The samples from both studies were checked for CD 24 polymorphism. The genotype of every individual was calculated. The reported Single Nucleotide Polymorphism, characterized by PCR RFLP, for CD 24 was a replacement of T. with C, at the 226 nucleotide position, in the coding region of exon 2, resulting in the substitution of the amino acid Alanine, 'A', at position 57, by Valine, 'V', near the GPI anchorage site of the mature protein. The results from PCR were the following:

T/T (homozygous for alanine) genotype was visualized as a single, undigested 453- bp fragment; the C/C (homozygous for valine) genotype was digested at a single site yielding two fragments of 317 and 136 bp; finally, the T/C genotype generated two restriction sites, yielding three bands o f 453, 317 and 136 bp. The T/C genotypic subjects reflected heterzygosity.

In the first study, after the genotypes of the subjects were concluded, the chi square Fisher and Man Whitney test was applied. (Zhou, Rammohan, Lin, Robinson, & Li, 2005)

The EDSS was then translated to the Multiple Sclerosis Severity Score (MSSS). The MSSS is a 10-point scale that is based on EDSS but also takes into account disease duration. If two people with MS have the same EDSS but different disease durations, the one who has had the disease longer will have a lower MSSS, and vice versa. The scoring was based on a reference population of Europeans with MS. An individual with an MSSS of 1 or less would have a disease severity equivalent to the least severely affected 10% of that reference population. These scores can also be divided into 6 different severity grades (1 = least severe, 6 = most severe). (RL, & J, 2008)

The MSSS test performs the Kruskal wallis test using asymptomatic approximation or an exact permutation method. (Zhou, Rammohan, Lin, Robinson, & Li, 2005)

In the second study, Chinese hamster ovary cells were tranfected with the two allele types, to test their expression efficiency. The anti-human CD 24 produced was used to check the expression of human and mouse CD 24, using flow cytometry. This was done by isolating peripheral blood leukocytes from fresh blood samples and was stained with saturating amounts of anti-CD 24 antibodies in conjunction with anti-CD 3 antibodies to mark the T cells among the Peripheral blood leukocytes. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)

Pearson's chi square test was used to perform the homogeneity test between the two distributions of the genotypes. Yate's chi square test was utilized to perform tests to compare frequencies of the CD 24 V/V genotype between cases and controls. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)

Patients with an EDDS classification of 6.0 were classified into three groups according to their genotypes: V/V, V/A, A/A. To assess differences in MS progression, the Kaplan Meier method was used to estimate the survival curve. This was compared with the log- rank test. Here survival was taken to mean that a patient had not reached EDDS 6.0 yet, and the time span was measured by the number of years that had lapsed since the first symptom. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)

RESULTS:

In the first study, the CD 24 V/V SNP was observed to occur more in the MS group compared to the control group. There was no significant difference between these groups in the other two variations, the CD 24 A/V and the CD 24 A/A SNP. In addition, there was also no relationship between age, gender and ethnic group (Basque and Caucasians) regarding the presence of the CD 24 V/V SNP. The average MSSS in patients with CD 24 V/V was 6.1613 compared to CD 24 V/V non-carriers. The MSSS test program yielded a P. value of 0.123791. (Zhou, Rammohan, Lin, Robinson, & Li, 2005)

The second paper was a much more extensive study, which tested the effects of the CD24 genotypes in relation to the progression of the disease and the efficiency of the three CD 24 genotypes that were expressed on the cell surface. In their population study, the samples were obtained from central Ohio. Most of the patients with MS were Caucasians indicating the high incidence of MS in this particular ethnic group. After PCR was conducted, it was observed that the CD 24 A/A and A/V genotypes in both the normal and effected population were nearly the same. However 6.3% of the normal individuals and 13.2% of the effected individuals had the CD 24 V/V genotype indicating a double risk of acquiring multiples sclerosis with this genotype. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)

A family study was carried out using TDT analysis to check the association of MS with CD 24 'V' allele. In the type 1 families the Xtdt value was 13 compared to the expected 7.5. In the type 2 families the TDT value was 20 compared to the expected 18.57. The total observed TDT values for both family types was 33 which yielded a P. value of 0.017 which indicates significant results. Thus the population and family study both suggest that there is a link between allele CD 24 V/V and its preferential transmission to MS patients as compared to non-MS individuals. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)

The effect of CD 24 genotype and disease progression was also evaluated with the help of the EDSS score. All patients with an EDDS score of 6 (indicating a loss of ability to walk without aid) were interviewed or followed up. The patients with each genotype were asked how many years it took to reach an EDDS value of 6. 50% of patients with CD24 V/V reached EDSS in 5 years, compared to 13 and 16 years in CD 24 A/A and CD24 A/V respectively. This indicates a relationship between CD24 V/V and progression of MS, concluding that homozygous patients have an increased risk of a more rapid disease progression. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)

The expression of the three alleles and how efficiently they are expressed on the cell surface was also determined. The expression of CD24 on peripheral blood leukocytes of age, sex and disease status matched CD24 A/A and V/V by two color flow cytometry. CD24 is expressed in both T cells and non- T cells of all the genotypes, however, there is a greater expressivity, on T cells, in patients with CD 24 V/V genotype. Also, the CD 3+ cells expressed 6 times more cell surface receptors in CD 24 V/V patients compared to AA patients. The intensity of expression was higher amongst the peripheral blood leukocytes of CD 24 V/V patient. CD 24 A/A and A/V were also compared for difference in expressivity. CD 24 A/V shows less than two fold increase in expressivity compared to CD A/A which shows that it does not have a significant effect on the progression of MS. To directly check for expressivity CD 24 'A' and 'V', cDNA were cloned and transfected in Chinese Hamster Ovary cells with different concentrations of plasmids. Three days later, the expression of the CD 24 genes were analyzed with flow cytometry revealing that CD 'V' showed 30-40% more expressivity than CD 'A' c DNA. The increased expressivity of the homozygous Valine gene could be a reason for its more rapid disease progression. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)

DISCUSSION:

Existing evidence associates CD 24 to multiple sclerosis, as a factor, modifying disease progression. The lesions of ms are mimicked by those of EAE, an autoimmune disease induced in animals by immunization with myelin. This happens when t lymphocytes are sensitized to myelin antigens. However, in MS, sensitivity to myelin has not been demonstrated. The interest of this research was based on establishing an association with the CD 24 receptor polymorphism with MS susceptibility. (D.A, 1996)

In both studies, the size of the proteins containing alanine or valine had been clearly demonstrated, indicating that these amino acids could influence the cleavage efficiency and anchor attachment of this region of CD 24 peptide, which is a co-stumulatory molecule that functions independently of CD 28 and that may regulate the recruitment of autoreactive T cells to the CNS. (Zhou, Rammohan, Lin, Robinson, & Li, 2005)

The studies also analyzed the difference of the presence of C/C, V/C and V/V in MS and non-MS patients. Since differences were only observed with the homozygous valine allele, occurrence of which was considerably high amongst MS patients, it is likely that the association is based on the absence of alanine rather than with the presence of valine. (D Otaegui1, A Sa'enz1, P Caman "o1, 2006)