Prognostic value of markers in associative genetics

The article presents an algorithm for genetic-statistical analysis of a rare chronic disease with a hereditary predisposition. The results of genotyping obtained by one of the authors by the method of associative genetics were used. Multiple sclerosis is a disease with a hereditary predisposition. At present, the association of multiple sclerosis and a large number of genes has already been discovered, but the genetic control is not completely clear. An important step in combating the disease can be preventive measures among groups of people with hereditary predisposition. In this article, using the example of the C677T genetic polymorphism of the MTHFR gene, we show how to determine the effectiveness of identifying individuals at increased risk. Multiple sclerotic patients (n=180) and healthy (n=231) residents of southern Iran were examined by one of the authors previously. Based on these data, population-genetic indicators and statistical characteristics of the test were calculated. The distribution of genotypes in healthy people: CC – 65 %, CT – 29 %, TT – 6 %, in patients with multiple sclerosis CC – 35 %, CT – 46 %, TT – 19 %. The major allele in the population of southern Iran is C (pC = 0.797; qT = 0.203). The frequency of the minor T allele is doubled in the group of patients compared with healthy ones (qT = 0.419). The T allele is considered to be provocative; the allele C is protective. The CC genotype reduces the likelihood of multiple sclerosis by almost half compared with the empirical risk. In heterozygotes of CT, the risk is increased by more than one and half times, in homozygotes of TT more than three times. 95 % CI confidence intervals for the OR odds ratio indicator are: CC (0.19–0.44), CT (1.36–3.10), TT (1.99–7.61), CT + TT (2.29–5.21). The statistical characteristics of the test indicate its low power when used in screening programs. The sensitivity when testing carriers of the T allele (CT + TT genotypes) is 65 %. The very low prognostic value of a positive test makes it inappropriate to use for screening, but this test may be useful in individual genetic counselling for patients with multiple sclerosis, as well as their relatives. The analysis scheme can be used in other studies of traits with a genetic component.


Introduction
Multiple sclerosis is a complex neurological disease that affects the central nervous system resulting in debilitating neuropathology. Pathogenesis is primarily defined by inflammation and demyelination of nerve axons. Although multiple sclerosis was first described in patients over 150 years ago, the exact etiology and pathogenesis of the disease remain unclear. There are certainly immunological factors, which are involved in the disease pathogenesis. However, epidemiological studies suggest that still unknown genetic factors also can contribute to the etiology of this disease. The precise etiology of multiple sclerosis remains elusive with a complex interplay between environmental factors, genetic susceptibility, and agedependant exposure to viral infection (Levin et al., 2005).
Genetic predisposition has long been suspected in the etiology of this disease. At present, its associations with a large number of genes have already been discovered, but the genetic control is not completely clear. An important step in combating the disease can be preventive measures among groups of people with hereditary predisposition. The association between MTHFR polymorphisms and multiple sclerosis has been investigated in different ethnic groups (Alatab et al., 2011;De Marco et al., 2002;Jonasdottir et al., 2003;Fekih Mrissa et al., 2013;Klotz et al., 2010). The association between MTHFR C677T variants and multiple sclerosis has been revealed in the southern Iranian population (Naghibalhossaini et al., 2015). We used these results to continue the analysis in terms of population genetics and examined whether this polymorphism can be used in a screening programs and in the genetic counseling.

Materials and methods
The distributions of genotypes in the groups of patients and healthy people were compared using the criterion χ 2 : χ ; df = k -1 n1 and n2 -the number of observations in the compared groups (N = n1 + n2), f1 and f2 -the number of genotypes in the compared groups, df -the degree of freedom, k -the number of classes.
The coefficient of association r between allele and disease was calculated as: a -the number of T alleles in the group of patients, b -the number of C alleles in the group of patients, c -the number of T alleles in the group of healthy people, d -the number of C alleles in the group of healthy people, n -the total number of alleles in two groups, df -the degree of freedom.
Allele frequencies pC and qT were calculated as: n -the number of the genotype.
The odds ratio (OR) was calculated: bc ad OR = a -the number of patients with the predisposition genotype, b -the number of patients without the predisposition genotype, c -the number of healthy people with the predisposition genotype, d -the number of healthy people without the predisposition genotype.
The confidence interval (CI) of OR was carried out in the logarithmic scale.
The statistical error lnOR was calculated as: The slnOR was used to obtain the 95 % confidence interval of lnOR. The limit values of the confidence interval lnOR were converted to the boundary values of the confidence interval OR by a back procedure: exp lnOR (Armitage, Berry, 1994).
The index of sensitivity (Sen) and specificity (Spe) of the tests was calculated using the formulas: a -the number of patients with the test genotype, b -the number of healthy people with the test genotype, с -the number of patients without the test genotype, d -the number of healthy people without the test genotype.

Results and discussion
The group of healthy people due to the negligible prevalence of multiple sclerosis (Izadi et al., 2015) can be considered as a sample from the whole population. In the group of patients, the proportion of CC genotype is almost two times less than in the group of healthy people. The number of patients with CT genotype is one and a half times more than in the control group, the number of people with TT genotype is three times more than in control group, so the presence of the T allele in the genotype is considered as an indicator of increased risk for multiple sclerosis in the population of southern Iran.
The major allele in the Iranian population is C (pC = 0.797). The frequency of the minor allele T in patients (qT = 0.419) is two times higher than in the group of healthy people (qT = 0.203; table 1). The distribution of genotypes in this group corresponds to the Hardy -Weinberg equilibrium. The prevalence of multiple sclerosis can be considered as an empirical risk for this disease if there is no other information. The risk changes if new information appears. CC genotype reduces the likelihood of the disease in comparison with the empirical risk almost twice (0.54, table 1). TT genotype increases the likelihood 3.38 times compared with empirical risk. In heterozygotes (CT) the probability of the disease is increased by 1.56 times. Thus, the T allele is considered to be provocative for multiple sclerosis, and the C allele is protective.  Given that the probability of disease in heterozygotes CT is half that the probability of homozygotes TT it can be argued that the interaction of these alleles exhibits a half-dominant effect or incomplete dominance with regard to multiple sclerosis. Since the presence in the genotype T allele increases the risk of multiple sclerosis, we combined these two genotypes (CT + TT) and designated this common group as a group of increased risk for multiple sclerosis. The individuals with the CC genotype are designated as the anti-risk group.
Confidence intervals of OR do not include 1 (table 2), so all indices are statistically significant (p<0.05). Therefore, MTHFR C677T polymorphism can be used as a marker of genetic predisposition to multiple sclerosis. The statistical characteristics of the test were calculated in order to estimate the predictive value of this polymorphism. The prevalence of multiple sclerosis in this population (π = 0.000721, Izadi et al., 2015) was used for further calculation.
The sensitivity of this test is rather low. The test for homozygote genotype TT reveals only 19 % individuals, which have the hereditary predispositions to multiple sclerosis, and the test for heterozygosity reveals 46 %. The T-allele in whole without taking into account the genotype identifies 65 % of potential patients (table 3). The prognostic value of the positive result is negligible (less than 1 %) and is of no practical value. High prognostic value of the negative result (> 99.9 %) is of no practical significance as well (table 3). Thus, the using of this polymorphism for screening has no practical value. Nevertheless, this polymorphism can be useful in the individual genetic counseling of the multiple sclerotic patients and their relatives. The analysis algorithm described in the article can be used to study a variety of traits with a genetic component and diseases with a hereditary predisposition.