Screening for mutations in exons encoding the ligand-binding domain of the LDL receptor gene using PCR-CFLP and PCR-SSCP.

Primary hypercholesterolemia includes both monogenic disorders and polygenic conditions. Two well defined monogenic disorders are familial hypercholesterolemia (FH) and familial defective apolipoprotein (apo) B-100 (FDB). Both disorders convey high risk of premature coronary artery disease. FH and FDB are caused by mutations in LDL receptor and apo B-100 genes, respectively. In the present study, mutations in both genes in Thai subjects with primary hypercholesterolemia were screened. For apo B-100 gene, a common mutation R3500Q was screened. This mutation was not observed in the patients (n = 45). For LDL receptor gene, mutations in the exons encoding the ligand-binding domain were screened. By PCR-CFLP analysis, 18 abnormal CFLP patterns in exon 4, the hot spot for mutations, were found in patients (n=45). One of the DNA samples with abnormal CFLP patterns was previously identified and reported as a possible disease-causing mutation, namely D151Y. For the other exons, the screening technique was PCR-SSCP. Abnormal SSCP patterns in DNA samples from patients (n=20) were found as follows, two in exon 3, one in exon 5 and another one in exon 6. Further characterization by DNA sequencing and family studies for these abnormal patterns are underway.

Inexpensive, rapid and convenient PCR-minigel SSCP protocol for polymorphisms and mutations analyses of LDL receptor gene.

Hypercholesterolemia has been recognized as a major risk factor of atherosclerosis and coronary artery disease. The elevation in plasma low density lipoprotein (LDL) cholesterol is frequently due to genetic alteration at the genetic locus specifying the LDL receptors, leading to defective catabolism of LDL. In order to facilitate the molecular diagnosis of LDL receptor disorder, single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) amplified genomic DNA fragments has become a simple and sensitive screening method for identification of DNA polymorphisms and mutations in LDL receptor gene prior to DNA sequencing. In addition, SSCP patterns can be detected by silver staining to avoid hazardous radioactive material or other costly nonradioactive detection techniques. However, the original SSCP protocol is generally large-formatted, which is both time and reagents consuming as well as cumbersome. Minigel SSCP protocols have thus been devised but they involve, although commercially available, costly precast gels. We describe here a nonradioactive PCR-minigel SSCP protocol which is sensitive, inexpensive, rapid, reproducible and manually convenient. The results in this study demonstrate that minigel-SSCP (gel size: 10 cm x 7.3 cm x 0.075 cm) can detect conformation polymorphisms in PCR-fragments with a comparative sensitivity to large gel SSCP (gel size: 30 cm x 40 cm x 0.04 cm) as exemplified by the SSCP analyses of exon 13 of the LDL receptor gene. For minigel SSCP, the reagents for gel components and silver staining are reduced approximately 9 times and 10 times, respectively. For electrophoresis, electrical power is also reduced 10 times. This improved technique can become routinely used for molecular diagnosis of LDL receptor defect as well as for other genetic disorders.

Homozygous DNA variants in exon 9 of the LDL receptor gene in a Thai patient with primary hypercholesterolemia phenotype.

Mutation in low density lipoprotein (LDL) receptor gene causes an inherited primary hypercholesterolemia namely familial hypercholesterolemia (FH). In this study, 46 Thai patients with primary hypercholesterolemia were screened for mutations in exon 9 of the LDL receptor gene by polymerase chain reaction-restriction fragment length polymorphism (PCR - RFLP). The analysed fragment was 224 bp in length. According to the published cDNA sequence, exon 9 of the LDL receptor gene contains several hypermutable CpG dinucleotides. Three of these sites are Hpa II recognition sites. PCR product of exon 9 obtained from amplification of wild-type DNA sample would yield four fragments after Hpa II digestion. The expected sizes of these restriction fragments were 15, 30, 40 and 139 bp. All normocholesterolemic subjects (n = 33) showed normal RFLP. However, in one patient (72 year old female), abnormal RFLP from Hpa II digestion of the amplified exon 9 was observed, i.e., a fragment of 70 bp and another one smaller than 139 bp. Such RFLP reflects that exon 9 of both alleles of the LDL receptor gene in this patient lost one and gained one Hpa II site. It is interesting that this patient, eventhough harbouring two mutations on both alleles of the LDL receptor gene (presumably homozygous genotype of FH), apparently revealed lipid levels of heterozygous phenotype of FH without symptoms of coronary artery disease. It has yet to be proved whether these genetic variations are disease-related mutations or presumably common DNA polymorphisms.

Screening for mutations in exon 4 of the LDL receptor gene in Thai subjects with primary hypercholesterolemia: detection of a novel mutation D151Y by PCR-CFLP.

A mutation in low density lipoprotein (LDL) receptor gene causes an autosomal codominant disorder namely familial hypercholesterolemia (FH). Mutations in the LDL receptor gene are very heterogeneous at the DNA levels, occurring in all 18 exons of the gene. However, exon 4 has been found to be the hot spot for mutational events. In this study DNA from 45 Thai subjects with primary hypercholesterolemia was screened for mutations in the hot spot exon 4. The DNA samples were amplified by Polymerase Chain Reaction (PCR) and screened for mutation by Cleavase Fragment Length Polymorphism (CFLP) technique. Identification of mutation was performed by direct sequencing of PCR product. From this screening, one female patient was found to be heterozygous for a novel mutation which was due to a G to T transversion at nucleotide 514. This transversion would change the species-conserved amino acid at codon 151 from charged R group aspatic (GAC) to uncharged R group tyrosine (TAC), termed D151Y. From the same screening strategy, we found that this mutation was absent in 33 healthy normolipidemic subjects. In this index subject, Arg 3500 Gln mutation in apo B-100 gene, causing hypercholesterolemia namely familial defective apo B-100 (FDB), was not found. Therefore, hypercholesterolemia in this index subject was possibly caused by the D151Y mutation in the LDL receptor gene.

Effect of Ava II and NcoI polymorphisms at the low density lipoprotein receptor gene on plasma lipid levels in a group of Thai subjects.

The contribution of common genetic variations at the LDL receptor gene in determining interindividual differences in plasma lipid levels in the general population has been observed in several studies. In this study, we employed the PCR-RFLP method to investigate such an effect of the common Ava II (exon 13) and Nco I (exon 18) polymorphisms at the low density lipoprotein (LDL) receptor gene locus in 54 normolipidemic Thai subjects. The mean LDL-C level was slightly higher in the Ava II (+/+) genotype than the other Ava II genotypes. This difference was significant at the 5 per cent level although there were only three homozygotes with Ava II (+/+) genotype. The average effect of Ava II (+) allele was to increase LDL-C level by 6.75 mg/dl. A gene-dosage effect was not observed for this polymorphism. In addition, the subjects with Ava II (+/+) genotype also tended to have high serum total cholesterol and triglyceride and low HDL-C levels. Nco I polymorphism revealed no statistically significant effect on lipid levels in these subjects. However, the subjects with (+) allele tended to have high levels of LDL-C, serum total cholesterol and triglyceride.

Screening for a D9N common mutation in exon 2 of the LPL gene in Thai normolipidemic and hyperlipidemic subjects.

Lipoprotein lipase (LPL) is a multifunctional protein, playing a major role in the hydrolysis of triglyceride-rich lipoproteins. It also affects the maturation of high density lipoprotein (HDL) and low density lipoprotein (LDL). A D9N substitution is a frequent mutation found in exon 2 of the LPL gene. It is due to a G --> A transition causing a substitution of Asp by Asn at amino acid residue 9 of the protein. This mutation was screened for in 94 Thai primary dyslipidemic (46 hypercholesterolemic and 48 combined hyperlipidemic) subjects compared to 32 normal healthy subjects using PCR-RFLP. Such a mutation has not, yet, been detected in any of these Thai subjects.

The renin--angiotensin system gene polymorphisms and clinicopathological correlations in IgA nephropathy.

Genetic variability in the renin-angiotensin system (RAS) may modify renal responses to injury and disease progression. We examined whether RAS alleles affect severity of IgA nephropathy. These genetic variants include angiotensin I converting enzyme deletion polymorphism in intron 16 (ACE I/D), a point mutation in the angiotensinogen (AGT) gene resulting in a methionine to threonine substitution at residue 235 (M235T) and an angiotensin receptor type I (ATR) A to C transition at bp 1166 (A 1166 C). A total of 53 patients with biopsy-proven IgA nephropathy and 80 normal control subjects were recruited for study. These patients were classified into two groups according to serum creatinine at renal biopsy. Group 1 patients (n = 40) had normal renal function, serum creatinine < or = 1.5 mg/dl and group 2 patients (n = 13) had renal insufficiency with serum creatinine > 1.5 mg/dl. The blood pressure and urinary protein of group 2 patients were higher than group 1 (p < 0.01). The mean scores of histological parameters including mesangial proliferation, glomerular sclerosis (global and segmental), the interstitial fibrosis and crescent formation in group 2 patients were significantly higher than in group 1 patients (p < 0.05). The most frequent genotype in IgA patients was ID (47%) genotype, followed by II (45%) and DD (8%) genotype of ACE gene. The mean serum ACE activity in the DD group was significantly higher than in the II group (p < 0.05) but was not significantly different from that of the ID group. No statistically significant differences were found with respect to allele frequencies between IgA group 1, group 2, or between controls and all IgA patients. Furthermore, no significant difference in AGT alleles, ATR alleles frequencies was detected between groups of IgA patients, although a trend for a higher frequency of DD genotype and AGT-TT genotype were noted in IgA group 2. The combined analysis of the ACE-DD and AGT-TT genotypes did not show any genetic influence on the risk of the disease susceptibility. To resolve the true role of ACE genotype and any dependent effect on progression, larger collaborative studies are required.

Lipoprotein(a) in nephrotic syndrome.

Serum lipoprotein(a) levels were measured in 27 patients with idiopathic nephrotic syndrome (NS), 14 patients with systemic lupus erythematosus (SLE) and 30 healthy controls. Lp(a) levels were significantly elevated in both idiopathic NS (53.4 + 36.2) and SLE (49.3+ 41.9) compared with controls (9.5+ 5.7) (p < 0.001). Fifty nine percent of idiopathic NS and 50 percent of SLE had Lp(a) more than 30 mg/dl. In 19 patients with idiopathic NS, serum Lp(a) levels fell markedly in 12 patients who responded to prednisolone therapy while, 7 patients with partial and no response had serum Lp(a), cholesterol, triglycerides and albumin levels not different from pretreatment period vs 6 months therapy. Lp(a) levels correlated significantly with proteinuria, serum cholesterol and triglycerides (r = 0.8, 0.6, 0.6) in idiopathic nephrotic syndrome and correlated inversely with serum albumin (r = -0.9). The SLE patients had the same pattern of correlation among these parameters and Lp(a) levels except for triglycerides. The high levels of Lp(a) in the NS could be one of the risk factors for atherosclerosis and thrombotic events associated with this disorder. In conclusion, the present study confirmed that patients with idiopathic NS and SLE had markedly increased serum level of Lp(a), in conjunction with other lipid abnormalities. The serum Lp(a) levels decreased substantially in all NS patients who experienced remission. In addition, the study also demonstrated a relationship between serum Lp(a) levels and serum albumin, cholesterol and triglycerides. An elevated Lp(a) level may be useful in guiding the physician towards more aggressive care to detect coronary artery disease early in patients at risk.

Reduced free radical scavengers and chronic renal failure.

Endogenous oxygen radical scavengers such as glutathione peroxidase (GSH-Px), catalase (CAT), glutathione and vitamin E are powerful regulatory systems against free radical toxicity. These oxidative injuries are increased in patients with chronic renal failure leading to various abnormalities including anemia. In this study, activities of GSH-Px, CAT, glutathione and vitamin E were measured in the erythrocytes of 54 chronic renal failure patients compared with 32 healthy controls. GSH-Px activities were lower significantly from controls (20.5 +/- 6.79 vs 28.3 +/- 9.0 u/gHb, p < 0.001). Erythrocytes CAT (6.52 +/- 2.3 vs 7.54 +/- 1.9 u/gHb, p < 0.05), glutathione (63.59 +/- 20.2 vs 75.1 +/- 6.3 mg/dl, p < 0.05) vit. E (2.23 +/- 0.53 vs 3.38 +/- 0.44 g/ml RBC, p < 0.001) were also lower in the patients group. Plasma malondialdehyde (MDA) known as lipid peroxidation product was higher significantly than controls (p < 0.001). Abnormal erythrocyte osmotic fragility test, expressed by glycerol lysis time (GLT50) was found in the patients group (p < 0.001) and correlated significantly with RBC vitamin E. Results demonstrated defects in erythrocytes enzymatic antioxidant defense mechanism in chronic renal failure patients. To improve antioxidant systems seems to be promising in preventing hemolysis and anemia in these patients.

Lipoproteins and lipid peroxidation abnormalities in patients with chronic renal disease.

Increasing experimental and clinical evidence suggests that lipoproteins and lipid peroxidation can be important modulators in progressive kidney disease. A group of 54 patients with varying degrees of kidney impairment was studied to find the abnormalities in lipoproteins and lipid peroxidation. Lipoproteins and lipid peroxidation products, malondialdehyde (MDA) were measured in the plasma of 54 chronic renal disease patients CGN 33, nephrosclerosis 11, 7CTIN, 1PCKD, unknown 2 and compared with values obtained from 32 healthy controls. The patients were divided into 5 groups according to serum creatinine levels: Group 1 (serum creatinine of 2 mg/dl), group 2 (S. creatinine > 2-4 mg/dl), group 3 (S. creatinine > 4-8 mg/ dl), group 4 (S. creatinine > 8-12 mg/dl), group 5 (S. creatinine > 12 mg/dl). Plasma cholesterol was higher significantly than controls in patients with group 1, 2 and 3 (p < 0.01, < 0.001, < 0.05) respectively while plasma LDL-chol was statistically significantly different from controls only in group 2 patients (p < 0.001). Plasma VLDL-chol, beta-VLDL-chol, triglycerides, ratio of chol/HDL and LDL/ HDL showed high levels in all groups compared with controls but more evident in patients of group 2. Plasma HDL-chol decreased during the progression of renal failure. All groups had significantly elevated plasma malonyldialdehyde (MDA) vs controls (p < 0.001), especially highest value was found in group 2. Triglycerides, beta-VLDL chol, VLDL-chol LDL/HDL, chol/HDL correlated very closely with plasma MDA levels and also with serum creatinine. Patients with chronic renal disease showed lipoprotein abnormalities and accelerated lipid peroxidation. The evidence was more marked in patients with normal to mild renal insufficiency which suggested the role of oxidative stress early in the course of nephron injury.

Significance of ascitic fluid white blood cells, pH, lactate, and other chemistry in immediate diagnosis of spontaneous bacterial peritonitis.

From 1989 to 1991, 68 cirrhotic patients, 47 with uninfected ascites and 21 with SBP were studied for the significance of ascitic fluid pH, lactate, PMN count and other chemistry for immediate diagnosis of SBP. It was revealed that ascitic fluid PMN count if over 500 per mm3, the increased lactate, or decreased glucose level, strongly supported the diagnosis of SBP. In cases of suspecting SBP but with low PMN count the ascitic values of lactate, glucose and pH will guide the diagnosis. If the ascitic lactate plus glucose, or lactate plus pH are above the cut off levels (lactate > 25 mg/dl; glucose < 60 mg/dl and pH < 7.35) the diagnosis is strongly suggestive. The ascitic fluid pH and A-AF pH gradient were not of diagnostic value due to instability of pH after tapping. For other chemistry in the ascitic fluid, there was a slight increase in ADA level in SBP, but for glucose, protein and glutamine levels, there was no difference among the groups with and without SBP.

Analysis of serum urinary proteins in normal and renal diseases by cellulose acetate electrophoresis.

A method using dried polyacrylamide gel to concentrate urine samples has been described, tested and used for the purpose of urine protein analysis. Concentrated urine samples from 10 normals and 100 patients with IgM nephropathy and systemic lupus erythematosus (SLE) were analysed by cellulose acetate electrophoresis (CAE). The results demonstrated that the patterns of proteins in the electrophoresis could be used to discriminate the two diseases. The best discriminating power was found in the logarithm of gamma globulin to albumin ratio. In IgM nephropathy the ratio of gamma globulin to albumin is much smaller than the ratio in SLE, indicating that relatively larger gamma globulins were excreted in SLE. In addition, the ratio can be used to discriminate subgroups of patients with IgM nephropathy. Urine from patients with IgM nephropathy with focal and segmental changes showed a significantly higher ratio. The study indicated the usefulness of the technique in discriminating the two common glomerular diseases.

Red cell and plasma calcium, copper and zinc in beta-thalassemia/hemoglobin E.

Beta-thalassemia/Hb E is a genetic disease prevalent in Thailand. This study has used atomic absorption spectroscopy to evaluate red cell and plasma calcium, copper and zinc in patients with beta-thalassemia/Hb E, both splenectomized and non-splenectomized. The levels of these trace elements in both red cells and plasma were different between the non-thalassemic controls and the disease patients. The most prominent result was that calcium concentration in red cells increased significantly in thalassemia subjects, particularly in splenectomized cases. These results might reflect the abnormal trace element metabolism and defects in the calcium transport system of the red cell membrane in thalassemia.

The association of adenosine deaminase activity with T-lymphocytes and subsets in pulmonary tuberculosis and bronchogenic carcinoma.

Simultaneous determination of blood/lung ADA activity and T-lymphocyte subsets was conducted in 12 patients with active pulmonary tuberculosis, 12 patients with bronchogenic carcinoma and 11 healthy volunteers. Differences were significant only in the tuberculosis patients, namely, increased mean enzyme values in both the peripheral blood (36.68 +/- 10.90 U/L) and in the BALF (4.25 +/- 2.19 U/L), and correlation of ADA activity between the blood and the diseased lung only; the difference in elevated enzymatic activity between the tuberculosis group and the cancer group was of no statistical significance. We conclude that simultaneous ADA analysis of the blood and the BALF may be of diagnostic value in cases suspected of having tuberculosis as yet undiagnosed by other means. Based on the lowest value of enzymatic activity in the blood of patients with tuberculosis (28 U/L), the test has a sensitivity of 75 per cent and a specificity of 100 per cent; whereas the lowest value in the BALF of tuberculosis patients (2.9 U/L), the test has a sensitivity of 77 per cent and a specificity of 82 per cent. Findings that there was a blood-lung correlation of elevated ADA activity and a correlation of enzymatic elevation with increased numbers of T-cells bearing IL-2 receptor in cases of pulmonary tuberculosis only provide evidence in support of T-lymphocytes actively participating in the ongoing immune process.