¶ … 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...

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…