Mutations in KERA, encoding keratocan, cause cornea plana.

Specialized collagens and small leucine-rich proteoglycans (SLRPs) interact to produce the transparent corneal structure. In cornea plana, the forward convex curvature is flattened, leading to a decrease in refraction. A more severe, recessively inherited form (CNA2; MIM 217300) and a milder, dominantly inherited form (CNA1; MIM 121400) exist. CNA2 is a rare disorder with a worldwide distribution, but a high prevalence in the Finnish population. The gene mutated in CNA2 was assigned by linkage analysis to 12q (refs 4, 5), where there is a cluster of several SLRP genes. We cloned two additional SLRP genes highly expressed in cornea: KERA (encoding keratocan) in 12q and OGN (encoding osteoglycin) in 9q. Here we report mutations in KERA in 47 CNA2 patients: 46 Finnish patients are homozygous for a founder missense mutation, leading to the substitution of a highly conserved amino acid; and one American patient is homozygous for a mutation leading to a premature stop codon that truncates the KERA protein. Our data establish that mutations in KERA cause CNA2. CNA1 patients had no mutations in these proteoglycan genes.

Unstable minisatellite expansion causing recessively inherited myoclonus epilepsy, EPM1.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1; MIM 254800) is an autosomal recessive disorder that occurs with a low frequency in many populations but is more common in Finland and the Mediterranean region. It is characterized by stimulus-sensitive myoclonus and tonic-clonic seizures with onset at age 6-15 years, typical electroencephalographic abnormalities and a variable rate of progression between and within families. Following the initial mapping of the EPM1 gene to chromosome 21 (ref. 6) and the refinement of the critical region to a small interval, positional cloning identified the gene encoding cystatin B (CST6), a cysteine protease inhibitor, as the gene underlying EPM1 (ref. 10). Levels of messenger RNA encoded by CST6 were dramatically decreased in patients. A 3' splice site and a stop codon mutation were identified in three families, leaving most mutations uncharacterized. In this study, we report a novel type of disease-causing mutation, an unstable 15- to 18-mer minisatellite repeat expansion in the putative promoter region of the CST6 gene. The mutation accounts for the majority of EPM1 patients worldwide. Haplotype data are compatible with a single ancestral founder mutation. The length of the repeat array differs between chromosomes and families, but changes in repeat number seem to be comparatively rare events.

Increased expression of scavenger receptor type I gene in human peripheral blood from hyperlipidemic patients determined by quantitative additive RT-PCR.

The scavenger receptors type I and II are mediators for the binding and uptake of chemically modified lipoproteins and are restricted to cells of monocyte origin. These receptors are highly expressed during the process of monocyte to macrophage differentiation. Quantitative mRNA levels of scavenger receptors from peripheral blood mononuclear cells have been analyzed in 29 hyperlipidemic patients and 15 healthy controls. Macrophage scavenger receptor isoforms transcripts were studied in circulating peripheral blood mononuclear cells with a modified RT-PCR method based on the use of a non-modified internal standard and a mathematical logistic adjustment of the standard curve. This method makes it feasible to study the variation in the expression of the scavenger receptors gene in peripheral blood during different physiopathological conditions. We studied the expression of the scavenger receptors gene in different blood cell lines and was present in only those of monocytic origin. The results have shown evidence that levels of scavenger receptor type I transcripts were proportional to apoB/cholesterol levels whereas type II receptors did not show any transcriptional variability. These findings suggest that the cholesterol level exerts a selective up-regulation of the scavenger receptor type I which is detectable by the induced increment of circulating monocytes in the blood of hyperlipidemic patients.

Angiotensin I-converting enzyme genotypes and angiotensin II receptors. Response to therapy.

In the present study, we studied angiotensin II type 1 (AT1) and type 2 (AT2) receptor messengers by quantitative reverse transcriptase-polymerase chain reaction. We examined peripheral blood mononuclear cells from 30 healthy subjects and 50 subjects with primary hypertension, in whom angiotensin I-converting enzyme genotype was determined, before and after 15 days of treatment with different antihypertensive drugs. The medication included a calcium channel antagonist, an angiotensin I-converting enzyme inhibitor, and a beta 1-blocker. We also studied the relationship between AT1 receptor gene expression and biochemical parameters of the renin-angiotensin system. AT1 receptor messenger levels were positively correlated with plasma renin activity in both normotensive and untreated hypertensive subjects. Increases of this messenger and plasma angiotensin II levels were correlated with the D allele in the same individuals. AT1 receptor messenger levels decreased significantly with angiotensin I-converting enzyme inhibitor treatment in subjects with the DD genotype, and a significant decrease was observed in subjects with the II and ID genotypes treated with a calcium antagonist. No changes were observed in mRNA with the beta 1-blocker. We conclude that the AT2 receptor is not expressed in peripheral leukocytes and that AT1 receptor messenger levels vary in relation to angiotensin I-converting enzyme genotype and pharmacological treatment. These results suggest that angiotensin I-converting enzyme genotype may be an important factor when deciding on antihypertensive therapy in individuals with primary hypertension.

The genotype interactions of methylenetetrahydrofolate reductase and renin-angiotensin system genes are associated with myocardial infarction.

We analyzed the evolution with age of the frequencies of the I/D polymorphism of the angiotensin I-converting enzyme (ACE), a1166c of the angiotensin II AT1 receptor (AT1R), M235T of the angiotensinogen (AGT) and A225V of their methylenetetrahydrofolate reductase (MTHFR) gene in a healthy (H) population and the subsequent comparison to age- and sex-matched groups of myocardial infarction (MI) subjects. A total of 472 H subjects were divided into three groups < 30, 30-55 and > 55 years old and 277 individuals with MI into two groups 30-55 and > 55 years old. The evolution with age showed that the AGT M allele (P < 0.001) and the MTHFR V allele (P < 0.05) frequency decreased with age in H men. The comparison between healthy and MI groups showed that the MM genotype frequency increased in MI men > 55 years (OR =4.16; 95% CI; 1.72-10.1) The cc genotype showed a similar behaviour (OR = 3.96; 95% CI; 1.21-12.9). In men, all the combinations with MM genotype presented a high risk, with OR values between 1.10 and 7.22. In women, the cc genotype increased in the MI > 55 group (OR = 6.66; 95% CI; 2.02-21.9). All the combinations with the cc genotype showed OR values between 1.71 and 13.3. The MM genotype in men and cc genotype in men and women, are independent risk factors for MI. We propose that the study of the allele frequency evolution in an H population at different ages is essential to determine risk factors for MI in case-control studies, since data from isolated age-matched groups can be misinterpreted.

[Myocardiopathies (II). Genetic changes in the etiopathogenesis of hypertrophic myocardiopathy. The therapeutic prospects]. / Miocardiopatías (II). Alteraciones genéticas en la etiopatogenia de la miocardiopatía hipertrófica. Perspectivas terapéuticas.

The genetical characterization of any disease implies its immediate theoretic and practical reorganization since all the basic clinical aspects such as ethology, diagnosis, prognosis, prevention and finally treatment are affected. This can be the fact for hypertrophic myocardiopathy in the near future. Recently, mutations in some new genes causing this alteration, apart from those found in the beta-myosin heavy chain gene, have been identified. Hypertrophic cardiomyopathy could be classified etiopathogenetically as primary, if it is due to a genetic alteration in any of the components of the sarcomere, and secondary when the initial factor is external, although it will be eventually reflected in a malfunction of the sarcomere. Therefore hypertrophic cardiomyopathy could be defined as a myocardial disorder with and autosomic hereditary pattern which is characterized by a ventricular hypertrophy due to alterations in the cardiac sarcomere. Detected mutations so far, which have been admitted to be the primary alteration in this disease, are localized in the beta-myosin heavy chain gene (14q1), in the alpha-tropomyosin gene (15q2), in the cardiac troponin T gene (1q3), as well as in the chromosome 11 p13-q13 loci. Different authors have pointed out a possible epigenetic mechanism produced by endogenous or environmental secondary factors as responsible for hypertrophic myocardiopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

[P53 protein gene. Review of the literature and assessment of the prognostic impact of its mutations in bladder tumors]. / Gen de la proteína P53. Revisión de la literatura y valoración de las implicaciones pronósticas de sus mutaciones en el tumor vesical.

The p53 protein, of the core protein group, was initially considered an oncogene but it was later noted to be included in the group of tumour-suppressive genes, the function of which seems to be focused in the control of cell growth, regulation of DNA transcription, and inhibition of certain oncogenes. A mutant protein variety has been observed with longer than normal mean life and altered function, so that it is not effective for the inhibitory control of cell growth. Mutations of that protein's gene, located in the short arm of chromosome 17, have been discovered in a large variety of tumours in humans, and in the urogenital region they have been consistently seen in both vesical and prostate tumours. The objective of the study was to confirm the need of this gene mutation, so that the vesical transitional cell tumour develops an infiltrant nature. The presence of mutations in this gene's exons 5 and 8 in infiltrant transitional cell tumours (28.5% cases) was demonstrated using as controls either surface transitional cell tumours, non-transitional tumours and healthy vesical tissue obtained together with the tumour specimen in each surgical procedure. The methods used were PCR (polymerase chain reaction) and the identification of the mutated specimen through TGGE (temperature gradient electrophoresis). The absence of mutations in the control strips together with the observation of mutations in the specimens from infiltrant tumours confirms the above hypothesis.

Direct quantification of HIV-1 RNA in human plasma by free solution capillary electrophoresis (FSCE).

SUMMARY: The levels of human immunodeficiency virus type 1 (HIV-1) RNA have been directly quantitated, after an isolation step, in plasma from patients with primary HIV-1 infection by free solution capillary electrophoresis (FSCE) with ultraviolet detection. HIV-1 RNA was detected and quantified at physiological levels by measuring the absorbance by FSCE. All the patients with primary infection showed concentrations in a range of 1.08-1.71 x 10(8) virions/ml of plasma. No signals were observed in seronegative donors. This procedure represents a practical alternative to other methods to quantify HIV-1 RNA and may be useful in assessing the efficiency of antiretroviral agents, especially during the early stage when other conventional viral markers are often negative.

Quantification by additive RT-PCR of HIV-1 RNA plasma levels in different stages of HIV-1 infection.

In this study, virion-associated RNA was measured in plasma from twenty six patients in various stages of HIV-1 disease by the additive RT-PCR method. Plasma viral RNA levels were inversely correlated (r = -0.72894) with total CD4+ cell counts and directly (r = 0.86964) with serum titre beta 2-microglobulin in chronically infected patients. This additive RT-PCR is based on a mathematical logistic adjustment of the standard curve and the use of an internal standard identical to the target molecule, which represents a control system for the efficiency of RT-PCR and allows a continuous assessment of the accuracy based on the recovery.

Both alleles of the M235T polymorphism of the angiotensinogen gene can be a risk factor for myocardial infarction.

We have studied the role of three polymorphic genes of the renin-angiotensin system (RAS) as independent risk factors for myocardial infarction (MI) and their correlation with three of the major coronary risk factors: serum cholesterol (CH), hypertension (HT) and smoking (SM). A population of 392 men was genotyped for the M235T polymorphism of the angiotensinogen (AGT) gene, the insertion/deletion of the angiotensin-converting enzyme (ACE) and the all66c of the angiotensin-II type 1 receptor (AT1R), by means of polymerase chain reaction (PCR) and restriction enzyme analysis. It was observed that the T allele frequency increased significantly in the MI with HT, CH, and SM subgroup (0.58 vs 0.31) (p<0.01). In contrast, the M allele frequency was higher in the MI without HT, CH, and SM (0.69 vs 0.42) (p<0.01). A strong association between the MM genotype and MI (p<0.001, odds ratio=4.29, confidence interval=1.95-9.42) was found when age-matched MM control subjects were compared to MI individuals with none of the other known major coronary risk factors. Futhermore, subjects with the MM genotype showed a significantly higher plasma renin activity (PRA) profile than those with the TT genotype (p<0.001). It can be concluded that the M allele is an independent risk factor for MI and the T allele modified the risk when other major risk factors are present.