Dissertation committee:
Λεωνίδας Στεφανής, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ελευθέριος Σταμπουλής, Ομότιμος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Παναγιώτα Δαβάκη, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Κωνσταντίνος Κυλιντηρέας, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μαρία Αναγνωστούλη, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ, Επιβλέπουσα
Μαρία Ελευθερία Ευαγγελοπούλου, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γεώργιος Κούτσης, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Summary:
BACKGROUND - AIMS: Multiple Sclerosis (MS), the main Demyelinating Disease of the Central Nervous System (CNS), is considered to have a very complex pathophysiology, which gathers scientific attention during the last fifty years. The disease comes as the result of the interaction of multiple endogenous and exogenous predisposing factors, among which the immunogenetic background holds a leading position. It has been repeatedly observed that the risk of developing MS is correlated with certain class II allelic polymorphisms within the Major Histocompatibility Complex (MHC), mainly the carriage of HLA-DRB1*15:01. Nevertheless, it seems that numerous different Human-Leucocyte-Antigens (HLA) Class I and II, as well as non-HLA genetic loci, and the epistatic mechanisms among them, are also implicated in the overall MS genetic burden. Another fact, that underlines the great significance of the genome in MS, is made obvious by the considerable percentage of familial cases (familial Multiple Sclerosis –fMS), that is estimated around 12.6% of all incidents in the international literature. Hence, the relative lack of scientific information regarding differences between fMS and sporadic Multiple Sclerosis (sMS), concerning their immunogenetic and non-immunogenetic (demographic, clinical, labolatorial and imaging) characteristics, comes as a big surprise. Principal goal of this thesis was the investigation and description of possible immunogenetic (regarding DRB1 locus) and non-immunogenetic differences between fMS and sMS, performed for the first time in a sample of MS patients of Greek origin, from all over Greece. Observation of possible correlations between certain DRB1 polymorphisms and specific demographic, clinical, imaging and laboratorial traits of MS in general, was an additional secondary goal.
MATERIALS - METHODS: 128 fMS and 364 sMS patients of Greek origin, diagnosed with Clinically Definite MS (CDMS) according to 2017 revised McDonald criteria, were included in this study and gave information regarding demographic (age, gender, age at disease onset, other autoimmune disorders in themselves and their 1st degree relatives), clinical [disease type, first symptoms, number of relapses, relapse rate, time of conversion from Clinically Isolated Syndrome (CIS) to CDMS, treatment, degree of disability and disability progression rate], laboratorial [presence of Oligoclonal Bands in the Cerebrospinal Fluid (CSF)], and imaging (lesion distribution in brain and spinal cord, tumefactive lesions) parameters. Among them, 57 non-related fMS and 141 sMS patients gave consent for DRB1 immunogenetic typing, which was performed by a low-resolution SSO technique. Allelic frequencies in the above samples were also compared with the corresponding frequencies of a sample of 246 Greek origin healthy controls, which were described in a previous study, after obtaining the appropriate permissions. Statistical analyses were done with the use of SPSS v.21.0. The level of statistical significance was set at 0.05, while p value was further corrected by the Benjamini–Yekutieli method, in the case of multiple allelic frequency correlations. Hardy-Weinberg proportions (HWP) for DRB1 haplotypes were confirmed by a PyPoP software, while Ewens-Watterson (EW) homozygosity tests for neutrality were also performed.
RESULTS: fMS was found in a frequency of 13.1% among the initially screened sample of candidate participants. 59% of fMS cases were linked to their affected relatives maternally. fMS started less frequently with double vision (3.1% vs 13.7%, p=0.003), and was linked less frequently with brainstem lesions (49.6% vs 64.8%, p=0.006), and subtentorial lesions in general (64.5% vs 74.8%, p=0.048), in comparison to sMS. 1st degree relatives of fMS patients were more frequently diagnosed with Hashimoto’s thyroiditis, with reference to those of sMS patients (9.8% vs 3.3%, p=0.033). fMS displayed internal heterogeneity, with anticipation effects in age at disease onset, between older and younger generation (39.7 vs 24.5 years, p=0.001), and earlier progression from CIS to CDMS in younger generation patients (30.1 vs 83.3 months, p=0.037). Earlier progression from CIS to CDMS was also noted for the fMS subgroup with 2nd or 3rd degree relatives affected (fMS 2nd/3rd) in comparison to the fMS 1st subgroup (1st degree relatives affected, 27.9 vs 43.6 months, p=0.047). HLA-DRB1*15 allelic frequency was significantly higher with reference to normal controls, for both the fMS (31.6% vs 16.7%, p=0.002), and the sMS (31.2% vs 16.7%, p<0.001) study groups, although the statistical power of this finding was degraded in the fMS 1st subgroup (24.1% vs 16.7%, p=0.205), but not in the fMS 2nd/3rd subgroup (39.3% vs 16.7%, p=0.012). On the contrary, HLA-DRB1*11 allelic frequency was found significantly lower, with reference to normal controls, only in the case of the sMS group (31.9% vs 52%, p<0.001), while the fMS patients were carrying the allele in a greater percentage (49.1%), mainly due to the fMS 1st subgroup (58.6%). No significant deviations from the HWPs were detected, while the EW tests for neutrality did not show any preference for homozygosity over heterozygosity, for all the groups and subgroups of patients in the study. In regards to the study’s secondary goal, we found positive correlation of DRB1*03 carriage with younger age at disease onset (26.4 vs 30.3 years, p=0.04), positive correlation of DRB1*04 carriage with presence of 1st degree relatives diagnosed with other autoimmune disorders (61.1% vs 26%, p=0.01), negative correlation of DRB1*13 carriage with optic neuritis as first MS symptom (3.6% vs 24%, p=0.011), negative correlation of DRB1*14 carriage with presence of thoracic demyelinating lesions (28.6% vs 73.3%, p=0.024), and negative correlation of DRB1*16 carriage with presence of thoracic demyelinating lesions (53.6% vs 76.9%, p=0.029), in the total sample of MS patients.
CONCLUSIONS: fMS was found to represent a significant minority in our sample of MS patients of Greek origin (1 out of 8 approximately), and was inherited more frequently through a maternal line. Regarding its phenotype, it was shown to be relatively more isolated in the supratentorial areas in comparison to sMS, as well as more commonly related with familial predisposition for Hashimoto’s thyroiditis. With reference to the immunophenotype, the influence of the DRB1 locus was somewhat degraded in fMS, compared to sMS, with the exception of the DRB1*15 risk allele. HLA genotype – clinical phenotype correlations, which represented the secondary goal of this study, did not reach the level of statistical significance, independently for the fMS group, fact which is futher supportive of the true existence of the above mentioned degradation. Making a first attempt of interpreting this observation, we believe that a predisposing DRB1 immunogenetic background is not sufficient on its own to lead to MS development by multiple close relatives within the same family. Several epistatic mechanisms are also required (probably within the MHC), as well as the concomitant carriage of several other HLA and non-HLA predisposing polymorphisms, additionally to the possible influence of certain superimposed, deleterious, environmental factors.
