Biomedical subjects
Publications and source records attributed to A Cao.
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Two brothers are reported who share mental retardation, conjunctival teleangectasias (mainly equatorial) and characteristic flat face with small mouth and thin prolabia. At the neuropsychological examination, the older brother at 14 years showed a full scale IQ of 40 (WISC), with verbal IQ 45 and performance IQ 44. The younger brother at 7 years showed a full scale IQ of 58 (WPPSI), with verbal IQ 67 and performance IQ 55. Chromosome studies showed a duplication Xp22-Xpter in both brothers and in the inactivated X of their mother. The anomaly was not present in a 3rd healthy brother and in other healthy relatives. The mother has normal intelligence and did not present any of the physical features of her affected sons.
The presence of serological markers of hepatitis B virus (HBV) infection and of hepatocellular HBV DNA were investigated in 19 HBsAg-negative patients with clinically and histologically significant chronic liver disease. Four cases negative for antibodies to HBsAg (anti-HBs), to the core antigen (anti-HBc), and to the e antigen (anti-HBe) were classified as non-A, non-B hepatitis. The remainder, positive for one or more of the three antibodies, were classified as hepatitis B. Histologic diagnosis was chronic active hepatitis in five, chronic persistent hepatitis in 11, micronodular cirrhosis in two, and fatty liver in one patient. The DNA extracted from limited amounts of liver biopsies, without cleavage by restriction endonucleases, was analyzed by the Southern blot technique for the presence of episomal HBV DNA. Autoradiographs showed a single band of less than 4.0 kilobase (kb) corresponding to the monomeric form of HBV DNA in five patients, several bands of larger forms (4.0 to 18.0 kb) in three patients, both the monomeric and the larger forms in eight patients, and no HBV DNA in three patients. While HBV DNA was detected in the hepatocellular DNA of six patients who underwent splenectomy, hybridization was negative with the DNA extracted from their spleens. The episomal viral DNA larger than 4.0 kb may represent concatemeric forms or free oligomers which could not be distinguished from rearranged and/or integrated viral DNA in the limited analyses of the hepatocellular DNA hydrolyzed with HindIII or EcoRI. Our observations suggest the presence of HBV-like agents in the liver of serologically HBsAg-negative patients with chronic liver disease.
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This paper reports the results of first trimester prenatal diagnosis in a twin pregnancy at risk for homozygous beta 0-thalassaemia (beta 0-39 mutant). Trophoblast samples from both twins were obtained at 10 weeks gestation with a forceps guided by ultrasound. Trophoblast DNA analysis, carried out with the oligonucleotide technique, revealed that one fetus was homozygous and the other heterozygous for the beta-39 mutant. This diagnosis was confirmed at 17 weeks gestation by amniocyte DNA analysis. DNA polymorphism analysis within the alpha-globin gene provided useful genetic markers for twin differentiation.
In this report we have summarized our experience with the prenatal diagnosis of beta-thalassemia in 1000 pregnancies followed at least until 12 months after birth. In the majority of these cases, the thalassemia lesion was the nonsense mutation at the codon corresponding to amino acid 39, which produces the hematological phenotype of beta o-thalassemia. Fetal blood sampling was carried out by placental aspiration, by which a sufficient amount of fetal blood for analysis was obtained in the majority of cases (99 per cent). The fetal mortality associated with fetal blood sampling was 6.3 per cent. Those placental samples contaminated by maternal cells were successfully purified by Orskov lysis. Fetal blood was analysed by globin chain synthesis on CM-52 columns, which gave reliable results. Two misdiagnoses (0.2 per cent) have been made of which one was due to a non-globin protein co-migrating with the beta-chains while the other resulted from a misclassification of the type of thalassemia segregating in the family.
The promoter region of adult beta globin genes in humans and other mammals contains conserved regions of pivotal importance for their regulated tissue specific expression. These include the CACCC and CAAT motifs. The CACCC motif is duplicated in humans and other mammals. The human delta-globin gene lacks these conserved regions and its expression in normal individuals is about 3% that of the beta globin gene. Previous studies have shown that the introduction of the beta-globin CACCC or CAAT can activate the delta-globin gene promoter, but the effect of the distal CACCC element has not yet been tested. In the present study, using site-specific mutagenesis, we have introduced the consensus sequence for the distal and proximal CACCC motif and the CAAT box alone or in combination in the wild-type delta-globin gene promoter. The resulting mutants, as well as the wild type (wt) delta- and beta-globin gene promoters, have been analyzed in a transient expression assay in Cos7, K562, and MEL cell lines. The results show that the CACCC boxes can increase the transcription efficiency of the delta-globin gene promoter in both erythroid and non-erythroid cell systems. The contribution of the two CACCC elements is almost equal in the non-erythroid (Cos7) and erythroid embryonic-fetal cell lines (K562), while the proximal CACCC element is more active in adult erythroid cells (MEL). Nonetheless, duplication of this element does not appear to affect the efficiency of the promoter synergistically. Furthermore, to assess the competitive ability of the delta globin promoter containing the proximal or distal CACCC consensus sequences over the wt beta globin gene promoter, we have carried out transient expression experiments using DNA constructs in which the delta and beta globin gene promoters are linked in cis and are sharing a single enhancer (competitive transient expression). The results show that both CACCC elements are able to activate the delta globin gene promoter in Cos7 and K562 cells, although to a different extent, whereas only the proximal CACCC element is effective in increasing the transcription efficiency in MEL cells. These findings are in agreement with the more severe clinical phenotype produced by the beta-thalassemia mutations affecting the proximal CACCC box as compared with those within the distal CACCC box. The Erythroid Kruppel Like Factor (EKLF) is a nuclear protein restricted to erythroid cells which specifically bind the CACCC box sequence and activate the beta-globin gene. In the present study we carried out transactivation experiments of the mutagenized delta-globin gene promoter by introducing an EKLF expressing construct in erythroid cells. Constructs containing the proximal but not those bearing the distal CACCC element are transactivated. Our results indicate that the proximal CACCC box and, to a lesser extent, also the distal box have a role in the regulated stage specific expression of a beta-like globin gene, and show that the insertion of a single CACCC motif in the delta-globin gene promoter is sufficient to increase its activity. Nevertheless only the delta globin gene promoter containing the proximal CACCC element is able to compete with the wt beta globin gene promoter in the adult erythroid environment. These findings have potential relevance for the future prospective treatment of inherited hemoglobinopathies based on the conversion of the low functioning delta-globin gene into a high functioning beta-like globin gene.
Primary open-angle glaucoma (POAG) can be subdivided into two groups according to age of onset: (1) the more prevalent middle to late-age-onset chronic open-angle glaucoma (COAG) diagnosed after age 40, and (2) the less common form, juvenile open-angle glaucoma (JOAG), which occurs between 3 years of age and early adulthood. Susceptibility to either COAG or JOAG has been found to be inherited. The discovery of several genetic markers spanning the region 1q21-q24 in genetic linkage with autosomal dominant juvenile open-angle glaucoma (adJOAG) represents a major breakthrough towards the localisation of gene(s) responsible for the disease. Linkage analysis is a powerful means of distinguishing disease loci in large families with dominant disease. However the size of the group of families may represent a crucial factor for the linkage analysis. Sardinia is an island with a relatively isolated ethnic group showing a relatively high frequency of ad JOAG and COAG (Fossarello et al, 1994) and it is genetically more homogeneous than most Western populations. Therefore it represents an ideal ethnic group to search for linkage. We identified 18 families affected by POAG in which the disease appears to be inherited as autosomic dominant trait. In all families but two, occurrence of both JOAG and COAG in the same kindred was observed. Identification of adPOAG locus was performed by linkage analysis using 9 microsatellite markers spanning the region 1q21-q24. No significant linkage was observed. Our findings provide further evidence for genetic heterogeneity in autosomal dominant primary open angle glaucoma, even in a geographic area where a relatively homogeneous genetic background exists.
This paper reviews the methodology available to make prenatal diagnosis of inherited hemoglobinopathies by DNA analysis and the strategy to be used for the large scale application of this procedure to high-risk populations. The most straightforward approach for prenatal diagnosis is nowadays based on the analysis of DNA enzymatically amplified by the polymerase chain reaction (PCR). The mutations, produced by gross structural rearrangement of the DNA and those affecting a restriction recognition site, are directly detected by visualization following ethidium bromide staining of the electrophoretic pattern resulting from enzymatic digestion of amplified DNA. The remaining ones are detected by dot blot analysis with allelic specific oligonucleotide probes. Because in each population a limited number of specific beta-thalassemia mutations are prevalent, prenatal diagnosis by DNA analysis may be carried out by a population-specific strategy based on the amplification of those regions of the beta-globin genes containing the mutations most frequently occurring in each population followed by dot blot analysis with allelic specific oligonucleotide probes. This approach has the great advantage of being very simple, because radioactive probes are not necessary, very rapid, the results being obtained within 24 hours from sampling and very sensitive, only a limited amount of DNA in the order of 50 ng being necessary.
The diffusion translational coefficient Dt of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10(-4) to 2M Na+ or K+. The observed values of DT are independent of particle concentration in the range 0.1-2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40 degrees -120 degrees. When the salinity is progressively raised an increase of DT from 1.9.10(-7) cm2s-1 to 3.2.10(-7) cm2s-1 was observed at about 2.10(-3) M NaCl followed by a decrease of DT beyond 0.6 M NaCl.
In this study, we report the results of haplotype and mutation analysis of the ATP7B gene in Wilson disease (WD) patients of Greek origin. We have analysed 25 WD families and two single patients and characterised 94% of the WD chromosomes investigated. We have found 12 different molecular defects (three frameshifts, two splice site, two nonsense, five missense mutations), four of which are novel. Five of the mutations are widely prevalent accounting for 74% of the WD chromosomes analysed. These results may enable preclinical diagnosis in the large majority of WD patients of Greek descent, thereby improving genetic counselling and disease management.
BACKGROUND: Hemoglobin (Hb) Sabine (beta 91 Leu-->Pro) is an unstable variant detected for the first time in a 16-year-old Scottish-English-German girl affected by moderately severe hemolytic anemia. A second case was described in a patient of Yugoslavian descent. We report another case of this Hb variant arising as a de novo mutation in a Sardinian patient. METHODS: Definition of the mutation was obtained by DNA direct sequencing on amplified beta-globin gene, as well as by structural analysis of the hemoglobin variant. RESULTS AND CONCLUSION: The patient presented a moderately severe hemolytic anemia with red blood cell inclusion bodies. Hemoglobin electrophoresis showed that quantitatively the abnormal fraction represented 9% of the total Hb amount. beta globin gene analysis revealed a single nucleotide substitution, T-->C, at codon 91, which gives rise to a leucine-->proline substitution. Structural analysis of the variant confirmed the amino acid substitution (Leu-->Pro) predicted by DNA sequencing.
BACKGROUND: Patients with aplastic anemia show to a variable degree an increase of the red blood cell volume and percentage of HbF. The extent of HbF reactivation in sickle cell anemia and thalassemia major is related to the presence of XmnI polymorphism at -158 G (gamma). In this study, we have investigated whether in Fanconi's anemia the increase of the HbF is also related to the XmnI polymorphism. METHODS: Restriction site polymorphisms in the beta-globin gene cluster were analyzed to define the beta-globin haplotype. The presence of a C --> T substitution at position -158 G (gamma) was investigated by XmnI digestion. RESULTS: We found that patients with the XmnI site at -158 G (gamma), which was contained either in the 5' -+-++ or in the rare -+--- sub-haplotype, tend to have higher HbF and MCV values. The differences between XmnI positive and XmnI negative patients were highly significative (p less than 0.0025) for the MCV values, but barely significant for HbF levels (p less than 0.05). CONCLUSIONS: Our results suggest that in Fanconi's anemia both the extent of HbF reactivation and the fetal-like erythropoiesis, which is responsible for high MCV, are at least partially related to the beta-globin haplotype.
Short tandem repeats (STRs) are highly informative loci within the human genome, consisting of short nucleotide sequences tandemly repeated in variable numbers. This results in different alleles of variable length. Herein we describe two STRS located 5' to the beta-globin gene. They can be detected by non radioactive methods and may be used to make prenatal diagnosis of beta-thalassemia.
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This paper describes the status of iron stores, the incidence and the hematological characteristics of iron deficiency anemia in children heterozygous for beta-thalassemia. In beta-thalassemia heterozygotes, iron stores were similar to the controls in infancy and tended to increase with age, reaching levels of moderate iron overload solely in adult males. Iron deficiency anemia occurred less frequently in children heterozygous for beta-thalassemia as compared to normal controls, while no difference between the two groups was observed in the incidence of iron deficiency. Ineffective erythropoiesis, typical of heterozygous beta-thalassemia, by causing an increase of iron absorption may limit the effect of iron shortage. At similar levels of iron depletion, however, children heterozygous for beta-thalassemia develop a more severe anemia as compared to non beta-thalassemic children. With the exception of two children, HbA2 levels in the presence of iron deficiency anemia remain in the range of heterozygous beta-thalassemia. In conclusion, our results indicate that children heterozygous for beta-thalassemia have normal iron stores but are relatively protected against the development of iron deficiency. When iron deficiency anemia develops, its clinical expression is usually more severe than in non beta-thalassemic children.
A new differential pH technique for glucose-6-phosphate dehydrogenase quantitative determination in whole blood has been evaluated. It is a rapid (90 s/analysis), reproducible (C.V. within-run 3.7%; between-run: 2.8%) and accurate method (in comparison with WHO method: r = 0.970). Reference intervals in non-deficient males were evaluated in 167 non-thalassaemics and in 60 beta-thal heterozygotes. The G6PD activity in beta-thalassaemia carriers is higher than in normals; this is particularly true if the activity is expressed in terms of U/g Hb. The phenotypic distribution measured in females is in agreement with that calculated by the Hardy-Weinberg law based on the incidence of the Gd(-) gene in males.