A Biochemical Genetic Investigation of Transketolase in the Wernicke-Korsakoff Syndrome

The Wernicke-Korsakoff syndrome (WKS) is a severe neuropsychiatric disorder most commonly seen as a result of malnutrition associated with chronic alcohol abuse. In its acute form (Wemicke's encephalopathy, WE) it is characterised by ophthalmoparesis, nystagmus, ataxia and an apathetic confusional state. In those that survive this initial phase, over 70% will be left with a specific form of memory impairment with a varying degree of severity. In those most severely affected (~50% Korsakoff s psychosis, KP), the impairment will be such that they are incapable of independent living and many will require long-term institutional care. In Scotland there is good evidence of an increase, over the past two decades, in the number of people suffering from alcohol-related brain damage, of which KP is the major form (Smith & McColl, 1992). As a result the absolute number of patients in long term psychiatric care and suffering from alcoholic KP has grown. They form a small but significant proportion of beds in the refractory wards of mental hospitals. One general argument that has fuelled interest in the possibility of genetic factors in alcoholic KP is the fact that only a small percentage of those who chronically abuse alcohol and become malnourished ever develop the characteristic neuropathology. In 1930 Wagner and Weir reported on the association of the WKS with thiamine depletion. Thiamine exists in three forms, thiamine monophosphate, triphosphate and pyrophosphate (TPP): the last making up 80-90% of total thiamine in cells. TPP is a cofactor for many enzymes of carbohydrate metabolism including pyruvate dehydrogenases, alpha-ketoglutarate dehydrogenase and transketolase (TK). Several studies have implicated TK in the pathogenesis of the WKS. The aim of this study was to investigate the role of TK in the WKS using quantitative and qualitative methods of analysis in patient and control groups. Blood from 30 hospitalized patients with the WKS and 25 normal controls was analysed for erythrocyte transketolase (ETK) activity and stimulated erythrocyte transketolase (ETKs) activity by the addition of TPP using an end point assay. By employing a modified TK assay (Smeets et al., 1971; Bayoumi & Rosalki, 1976), mean ETK activity in 10 normal controls was found to be 0.418 +/- 0.193 lU/mg protein which was not significantly different (p>0.1) from the 20 patients (mean ETK activity 0.406 +/- 0.155 lU/mg protein). The same was true for the ETKs activity in which normal controls had a mean of 0.738 +/- 0.208 IU/mg protein and patients 0.680 +/- 0.190 IU/mg protein. This difference was not statistically significant (p>0.1). A report (Kaczmarek & Nixon, 1983) suggesting the existence of several species of TK was investigated by polyacrylamide gel electrophoresis (PAGE). TK activity was detected on PAG by specific TK enzyme stain. All normal controls and patient samples were run on PAG and then stained both specifically and generally (Coomassie-Brilliant Blue (CBB) R-250 and silver stain). They yielded two ETK bands which varied in intensity and were cathodal to haemoglobin. Although qualitative analysis strongly suggested ETK to be heterogeneous, it did not provide means of investigating WKS by biochemical analysis. Because the analysis of ETK (quantitatively and qualitatively) showed no apparent differences between the two groups, it was further investigated following the purification of the enzyme. PAGE crosslinked with DHEBA, a form of crosslinker which allows gels to be solubilized under alkaline conditions, provided means of isolating pure fractions of TK from red blood cells of normal controls and patients. These extracted fractions were subjected to analysis using high performance liquid chromatography (HPLC), isoelectric focusing (lEF) and endpoint assay. Despite confirmation of TK heterogeneity (with respect to ionic strength, isoelectric point (pi) and ETK activity), no differences in electrophoretic profile between patients and controls were revealed. In all samples two distinct bands of varying intensity appeared at pi 7.35 and 6.55 on BEF gels. Further studies on native TK were carried out after purification from white blood cells which had been collected as buffy coats from normal individuals (Mocali & Paoletti, 1989). Enzyme recovery declined as the steps of purification progressed, therefore another purification method was developed from the method of Takeuchi et. al.(1986) (where enzyme recovery was not sufficient for amino acid analysis). The fractions isolated by this method were subjected to fast protein liquid chromatography (FPLC) on Mono Q column which yielded two peaks containing TK activity. The fractions were also loaded on an SDS-PAGE where the protein band had a molecular weight (MW) of 68kD (kiloDaltons). These results suggested that TK exists in at least two forms varying in ionic strength as revealed by the chromatographic profile from FPLC

Re-sequencing of the APOAI promoter region and the genetic association of the -75G > A polymorphism with increased cholesterol and low density lipoprotein levels among a sample of the Kuwaiti population

BACKGROUND: APOAI, a member of the APOAI/CIII/IV/V gene cluster on chromosome 11q23-24, encodes a major protein component of HDL that has been associated with serum lipid levels. The aim of this study was to determine the genetic association of polymorphisms in the APOAI promoter region with plasma lipid levels in a cohort of healthy Kuwaiti volunteers. METHODS: A 435 bp region of the APOAI promoter was analyzed by re-sequencing in 549 Kuwaiti samples. DNA was extracted from blood taken from 549 healthy Kuwaiti volunteers who had fasted for the previous 12 h. Univariate and multivariate analysis was used to determine allele association with serum lipid levels. RESULTS: The target sequence included a partial segment of the promoter region, 5’UTR and exon 1 located between nucleotides −141 to +294 upstream of the APOAI gene on chromosome 11. No novel single nucleotide polymorphisms (SNPs) were observed. The sequences obtained were deposited with the NCBI GenBank with accession number [GenBank: JX438706]. The allelic frequencies for the three SNPs were as follows: APOAI rs670G = 0.807; rs5069C = 0.964; rs1799837G = 0.997 and found to be in HWE. A significant association (p < 0.05) was observed for the APOAI rs670 polymorphism with increased serum LDL-C. Multivariate analysis showed that APOAI rs670 was an independent predictive factor when controlling for age, sex and BMI for both LDL-C (OR: 1.66, p = 0.014) and TC (OR: 1.77, p = 0.006) levels. CONCLUSION: This study is the first to report sequence analysis of the APOAI promoter in an Arab population. The unexpected positive association found between the APOAI rs670 polymorphism and increased levels of LDL-C and TC may be due to linkage disequilibrium with other polymorphisms in candidate and neighboring genes known to be associated with lipid metabolism and transport

The Genetic Structure of the Kuwaiti Population: mtDNA Inter- and Intra-population Variation

This is the publisher's version, also available electronically from http://digitalcommons.wayne.edu/humbiol/vol84/iss4/7/.This study investigated: (1) the mitochondrial DNA (mtDNA) genetic variation in 116 unrelated individuals who originated from the Arabian Peninsula, Iran, or were of Bedouin ethnicity and (2) the genetic structure of Kuwaiti populations and compared it to their neighboring populations. These subpopulations were tested for genetic homogeneity and shown to be heterogeneous. Restriction fragment length polymorphism (RFLP) and mtDNA sequencing analyses of HVRI were used to reconstruct the genetic structure of Kuwait. The results indicated that the combined Kuwaiti population has a high frequency of haplogroup R0 (17%), J (12%), and U (12%) similar to other Arabian populations. In addition, contemporary African gene flow was detected through the presence of sub-haplogroup L (L1 and L2) (2%) and the absence of L3 which is reflective of an earlier migration. Furthermore, the multidimensional scaling (MDS) plot showed that the Kuwaiti population clusters with neighboring populations, including Iran and Saudi Arabia indicating gene flow into Kuwait. According to this study, the Kuwaiti population may be undergoing an expansion in a relatively short period of time, and the maternal genetic structure of Kuwait resembles both Saudi Arabia and Iran

The Genetic Structure of the Kuwaiti Population: mtDNA Inter- and Intra-population Variation

This study investigated: (1) the mitochondrial DNA (mtDNA) genetic variation in 116 unrelated individuals who originated from the Arabian Peninsula, Iran, or were of Bedouin ethnicity and (2) the genetic structure of Kuwaiti populations and compared it to their neighboring populations. These subpopulations were tested for genetic homogeneity and shown to be heterogeneous. Restriction fragment length polymorphism (RFLP) and mtDNA sequencing analyses of HVRI were used to reconstruct the genetic structure of Kuwait. The results indicated that the combined Kuwaiti population has a high frequency of haplogroup R0 (17%), J (12%), and U (12%) similar to other Arabian populations. In addition, contemporary African gene flow was detected through the presence of sub-haplogroup L (L1 and L2) (2%) and the absence of L3 which is reflective of an earlier migration. Furthermore, the multidimensional scaling (MDS) plot showed that the Kuwaiti population clusters with neighboring populations, including Iran and Saudi Arabia indicating gene flow into Kuwait. According to this study, the Kuwaiti population may be undergoing an expansion in a relatively short period of time, and the maternal genetic structure of Kuwait resembles both Saudi Arabia and Iran

Increased Risk of the APOB rs11279109 Polymorphism for CHD among the Kuwaiti Population

Background. Coronary heart disease (CHD) is among the leading causes of death in Kuwait. This case-control study investigated the genetic association of APOB rs11279109 with CHD in Kuwaitis. Methods. The polymorphism was genotyped in 734 Kuwaiti samples by direct amplification. Statistical analysis with genetic modeling was used to assess its association with CHD. Results. A statistically significant association (P<0.001) between the rs11279109 DD genotype (OR: 2.43, CI: 1.34–4.41) with CHD was observed. A codominant genetic model revealed a 2.69 risk increase (CI: 1.57–4.61) for the DD genotype (P=0.009) independent of age, sex, BMI, smoking, hypercholesterolemia, and ethnicity suggesting APOB rs11279109 as an indicator for the increased risk of CHD. Conclusion. The DD genotype may explain molecular mechanisms that underline increased LDL oxidation leading to arthrosclerosis. The findings emphasize the need to identify genetic markers specific to the CHD patient ethnic group in order to improve prognosis and help in early diagnosis and prevention

Sequence Analysis of APOA5 Among the Kuwaiti Population Identifies Association of rs2072560, rs2266788, and rs662799 With TG and VLDL Levels

Common variants of Apolipoprotein A5 (APOA5) have been associated with lipid levels yet very few studies have reported full sequence data from various ethnic groups. The purpose of this study was to analyse the full APOA5 gene sequence to identify variants in 100 healthy Kuwaitis of Arab ethnicities and assess their association with variation in lipid levels in a cohort of 733 samples. Sanger method was used in the direct sequencing of the full 3.7 Kb APOA5 and multiple sequence alignment was used to identify variants. The complete APOA5 sequence in Kuwaiti Arabs has been deposited in GenBank (KJ401315). A total of 20 reported single nucleotide polymorphisms (SNPs) were identified. Two novel SNPs were also identified: a synonymous 2197G&gt;A polymorphism at genomic position 116661525 and a 3′ UTR 3222 C&gt;T polymorphism at genomic position 116660500 based on human genome assembly GRCh37/hg:19. Five SNPs along with the two novel SNPs were selected for validation in the cohort. Association of those SNPs with lipid levels was tested and minor alleles of three SNPs (rs2072560, rs2266788, and rs662799) were found significantly associated with TG and VLDL levels. This is the first study to report the full APOA5 sequence and SNPs in an Arab ethnic group. Analysis of the variants identified and comparison to other populations suggests a distinctive genetic component in Arabs. The positive association observed for rs2072560 and rs2266788 with TG and VLDL levels confirms their role in lipid metabolism

Identification and Characterization of Variants in Intron 6 of the LPL Gene Locus among a Sample of the Kuwaiti Population

Lipoprotein lipase (LPL) is responsible for the hydrolysis of lipoproteins; hence defective LPL is associated with metabolic disorders. Here, we identify certain intronic insertions and deletions (InDels) and single nucleotide polymorphisms (SNPs) in intron 6 of the LPL gene and investigate their associations with different phenotypic characteristics in a cohort of the general Kuwaiti population. Two specific regions of intron 6 of the LPL gene, which contain InDels, were amplified via Sanger sequencing in 729 subjects. Genotypic and allelic frequencies were estimated, and genetic modeling was used to investigate genetic associations of the identified variants with lipid profile, body mass index (BMI), and risk of coronary heart disease (CHD). A total of 16 variants were identified, including 2 InDels, 2 novel SNPs, and 12 known SNPs. The most common variants observed among the population were rs293, rs274, rs295, and rs294. The rs293 &ldquo;A&rdquo; insertion showed a significant positive correlation with elevated LDL levels, while rs295 was significantly associated with increased BMI. The rs274 and rs294 variants showed a protective effect of the minor allele with decreased CHD prevalence. These findings shed light on the possible role of LPL intronic variants on metabolic disorders

Identification and Characterization of Variants in Intron 6 of the <i>LPL</i> Gene Locus among a Sample of the Kuwaiti Population

Lipoprotein lipase (LPL) is responsible for the hydrolysis of lipoproteins; hence defective LPL is associated with metabolic disorders. Here, we identify certain intronic insertions and deletions (InDels) and single nucleotide polymorphisms (SNPs) in intron 6 of the LPL gene and investigate their associations with different phenotypic characteristics in a cohort of the general Kuwaiti population. Two specific regions of intron 6 of the LPL gene, which contain InDels, were amplified via Sanger sequencing in 729 subjects. Genotypic and allelic frequencies were estimated, and genetic modeling was used to investigate genetic associations of the identified variants with lipid profile, body mass index (BMI), and risk of coronary heart disease (CHD). A total of 16 variants were identified, including 2 InDels, 2 novel SNPs, and 12 known SNPs. The most common variants observed among the population were rs293, rs274, rs295, and rs294. The rs293 “A” insertion showed a significant positive correlation with elevated LDL levels, while rs295 was significantly associated with increased BMI. The rs274 and rs294 variants showed a protective effect of the minor allele with decreased CHD prevalence. These findings shed light on the possible role of LPL intronic variants on metabolic disorders

Apolipoprotein E Genotyping Among the Healthy Kuwaiti Population

Apolipoproteins (lipid-free) are lipid-binding proteins that circulate in the plasma of human blood and are responsible for the clearance of lipoproteins. Apolipoprotein E (ApoE) is one of the several classes of this protein family. It acts as a ligand for the low-density lipid (LDL) receptors and is important for the clearance of very low-density lipid (VLDL) and chylomicron remnants. The APOE gene locus is polymorphic, with three major known alleles, APOE*3, *4, and *2. We investigated the distribution of the allele frequency of the APOE gene locus and describe here the genetic variation in four Kuwaiti subpopulations: Arab origin (Arabian peninsula), Arab Bedouin tribes, Iranian origin, and the heterogeneous population. We also describe the use of Spreadex gels in resolving the amplified and digested products of the APOE gene locus. DNA was extracted from whole blood and subjected to PCR and then to RFLP analysis. Allele and genotype frequencies were estimated for the total population and for each subpopulation. Statistical analysis showed no difference in the allele frequencies between the four groups. The frequency of APOE*3 in the Kuwaiti population was highest (88.4%) followed by the frequency of APOE*4 (6.5%) and APOE*2 (5.1%). The genotype and allele frequencies obtained for the Kuwaiti population fell within the reported worldwide distribution for the APOE gene locus. Moreover, the results obtained in this study showed no statistical difference ( p \u3e 0.05) between the APOE allele and genotype frequencies between the subgroups for all six genotypes and three alleles, supporting the assumption of admixture in the Kuwaiti population and that the obtained frequencies were in Hardy-Weinberg equilibrium. Finally, we found that the distribution of the APOE alleles in Kuwait differs somewhat from those reported in other Arab populations, suggesting that the Arabs originating from the Arabian peninsula are different from those of Lebanon, Morocco, and Sudan