Association of the leukemia inhibitory factor gene polymorphism rs 929271 with idiopathic mild intellectual disability

Aim. To investigate the possible association of LIF gene polymorphism rs929271 with mild intellectual disability (ID). Methods. The group of patients with mild (IQ score between 50 and 70) idiopathic intellectual disability consisted of 64 individuals including 40 (62.5 %) males and 24 (47.5 %) females. The control group consisted of 238 healthy volunteers from different regions of Ukraine. Polymorphic variants of LIF gene rs929271 were detected using PCR followed by Hinf1 RFLP analysis. Results. The data concerning LIF genotypes and allelic variants distribution in ID patients and control group were obtained. Statistical analysis shows signifi cant differences at rs929271 for both genotype and allele frequency when comparing ID cases and controls (p = 0.01 and 0.02, respectively). Conclusions. Our results suggest that LIF gene polymorphism rs929271 is associated with idiopathic mild intellectual disability. Therefore, we propose LIF as a new marker of genetic susceptibility for intellectual disability.


Introduction
Intellectual disability is a neurodevelopmental disorder, affecting about 3 % of the population, and is associated with a series of social and medical handicaps [1].The causes of intellectual disability vary with the severity of the condition: moderate-to-severe intellectual disability (IQ less than 50) is much more likely to be due to a single pathological cause (genetic or environmental) whereas mild ID (defi ned as an IQ score between 50 and 70) is rather due to the complex condition in origin [2].
Leukemia inhibitory factor (LIF) is a member of the neuropoietic family of neurotrophins and was found to regulate the neuronal phenotype and coordinates astrocyte, oligodendrocyte, microglia, and infl ammatory cell responses [3][4][5].Furthermore, LIF is shown to act as a survival factor for neurons and oligodendrocytes [6,7].
Aim of this study is to evaluate the possible association of the LIF gene polymorphism rs929271 with mild intellectual disability.

Materials and Methods
DNA-samples were extracted from peripheral blood leucocytes of unrelated volunteers from different regions of Ukraine and ID patients by the standard phenol-chloroform method.Informed consents we re obtained from all the individuals participating in our study.
The group of patients with mild (IQ score between 50 and 70) idiopathic intellectual disability consisted of 64 individuals including 40 (62.5 %) males and 24 (47.5 %) females, where previous extensive genetic investigations have revealed no abnormalities.All patients underwent physical and neurological examination (test used for IQ: WISC III, WISC-R, WISC) and standard G-banding ka ryotype analysis.DNA tests to determine Fragile X status (FRAXA, FRAXE, FRAXF loci) and Prader Willi/Angelman syndromes (PW/ AS) were perfor med to rule out the known genetic causes of ID prior to further investigation.Array-CGH analysis (400K resolution) revealed no any pathological rearrangements in all patients.
The control group consisted of 238 individuals including 128 (53.8 %) males and 110 (46.2 %) females.This group may be considered representative for the estimation of DNA polymorphism frequency in autosomal genes [21,22].
The presence of LIF polymorphism rs929271 was examined by PCR-RFLP (restriction fragment length polymorphism) analysis.Specifi c oligonucleotides, designed and synthesized in accordance to corresponding sequences of LIF ge ne, were used as primers: forward: 5'-GGGGACACAGAAACAAG GACAGGG -3' and reverse: 5'-AAGGGTCGGAT CTGAGAGAATGGG-3'.Pri mers were desig ned us-  ing a web-based PRIMER 3.0 program (http://workbench.sdsc.edu).We used the «BLAST» program at http://www.ncbi.nlm.nih.gov/blast to check for the specifi city of the primers.Hypothetical RFLP results R. V. Gulkovskyi, L. S. Volkova, L. A. Livshits were tested using NEB cutter V2.0 (http://tools.neb.com/NEBcutter2).The PCR amplifi cation was performed in a fi nal volume of 25 μl containing 1 × PCR buffer, 1.5 mM MgCl2, 200 μM of each dNTP, 1 μM of each pri mer, 0.2 units of Taq-DNA polymerase and 200 ng of the DNA template.The cycling conditions were as follows: initial denaturation at 95 C for 5 min, 30 cycles consisting of denaturation at 94 C for 30 s, annealing at 63 C for 30 s, extension at 72 C for 30 s and a fi nal elongation step at 72 C for 3 min.The amplifi ed fragments were digested with HinfI.Digestion was performed in 15 μl reaction volume containing 1 X reaction buffer, 0.5 units of the restriction enzyme and 10 μl of purifi ed PCR product, incubated at 37 C overnight and analyzed in 2 % standard agarose electrophoresis.
The results were statistically assessed using Ope-nEpi software and Fisher's 2 by 2 exact test, as well as odd ratio (OR) calculation; p < 0.05 was considered to be statistically signifi cant test [23].

Results and Discussion
The designed primers successfully amplifi ed the cor responding fragment (298 bp) of the LIF gene.The T to G transition in rs929271 variant removes a restriction site for endonuclease HinfI.Thereby three different patterns could be observed for the rs929271 variant after the restriction digestion: a 298 bp band (for rs929271 T/T); a 298 bp, a 159 bp and a 139 bp bands (for rs929271 T/G); a 159 bp and a 139 bp bands (rs929271 G/G) (Fig. 1).
Based on the RFLP analysis of the rs929271 variant, the individuals were classifi ed into three groups: TT, TG and GG.Genotypes and allele frequencies of the rs929271 polymorphism are presented in Table 1.The observed genotype distributions showed no deviations from Hardy-Weinberg expectations in general population of Ukraine and in ID patients group.
It was determined that total frequency of heteroand homozygous carriers of the LIF gene rs929271 minor allele, in this case that of guanine (G), is reliably higher (p = 0.01) in the ID patients group (71.9 %) compared to the control group (55 %).The minor G-allele occurred less frequently in the control gro-up -0.328 than in the ID patients gro up -0.445 (p = = 0.02).It was shown that the risk of mild ID development increased for both hetero-and homozygous carriers of minor rs929271 G-allele and the odd ratio was 2.09 (95 % CI: 1.14-3.81).
Our results suggest that the LIF gene polymorphism rs929271 is associated with idiopathic mild intellectual disability.These data can be explained by a decrease of LIF levels in the LIF gene rs929271 minor allele G carriers that in turn, may affect the regulation of neurogenesis and neuroprotection at least via LIF/ERK/MAPK signaling.
Therefore, we propose LIF as a new marker of genetic susceptibility for intellectual disability.Future investigations are necessary to explain the molecular mechanisms of the LIF involvement in ID pathogenesis.