Luigi F. Bernini

Genetic control of survival in epidemics.

Descendants of Dutch colonists, who emigrated to Surinam in the last century and survived epidemics of typhoid and yellow fever with a total mortality of about 60%, were tested for twenty‐six polymorphisms. The gene frequencies were compared with those of a large Dutch control sample. An analysis of drift indicated that the variations in gene frequencies observed for C3, Gm, HLA‐B, and GLO were unlikely to be due to drift. Therefore these data might indicate selection through genetic control of survival in these epidemics.

Purification and primary structure of ceratotoxin A and B, two antibacterial peptides from the female reproductive accessory glands of the medfly Ceratitis capitata (insecta:Diptera)

In the present article we report the purification and the amino acid sequence of two antibacterial peptides present in the secretion of the female reproductive accessory glands of the dipteran insect Ceratitis capitata. Both peptides consist of 29 amino acid residues, are heat stable, strongly basic and differ from each other for the substitution of two amino acids. Their primary sequence and predicted secondary structure are related to other families of peptides known to have lytic and/or antibacterial activity. We propose the name ceratotoxins (from Ceratitis) for these antibacterial peptides.

Rapid detection of point mutations and polymorphisms of the α-globin genes by DGGE and SSCA

We report the application of DGGE and SSCA for the identification of point mutations causing α‐thalassemia. The α‐globin genes were amplified in three overlapping fragments of 250 bp (I), 540 bp (II), and 600 bp (III), respectively. Fragments II and III were analyzed by DGGE, while fragments I and II were analysed by SSCA. A panel of seven previously identified mutations was employed to test the combined DGGE/SSCA strategy: 5/5 and 6/7 mutations were detected by SSCA and DGGE, respectively. The same approach has also led to the identification of eight disease‐causing mutations in a sample of 18 presumed non‐deletional α‐thalassemia carriers. During this pilot study, two novel mutations as well as three new polymorphisms were found. The combined application of SSCA and DGGE allows the rapid identification of mutations responsable for α‐thalassemia and abnormal globin chain variants. Moreover, it will prove extremely useful for pre‐ and postnatal diagnosis and in screening programs for non‐deletional α‐thalassemias.

The involvement of Alu repeats in recombination events at the α-globin gene cluster: characterization of two α°-thalassaemia deletion breakpoints

Abstract Alu repetitive sequences are frequently involved in homologous and non-homologous recombination events in the α-cluster. Possible mechanisms involved in Alu-mediated recombination events are strand exchange, promoted by DNA pairing between highly homologous Alu repeats, and subsequent strand invasion. Alternatively, Alu sequences might play a more active role in recombinogenic processes in the α-cluster. We describe a novel 33-kb α°-thalassaemia deletion ––DUTCH encompassing the α- and zeta-globin genes and pseudogenes in a kindred of Dutch-Caucasian origin. This deletion appears similar, although not identical, to the previously described ––MEDII deletion. Cloning and sequencing of both the ––DUTCH and ––MEDII deletion breakpoints clearly indicate that the mechanism leading to these α°-thalassaemia deletions involves misalignment between the highly homologous tandemly arranged Alu repeats at both parental sides, which are normally 33 kb apart. Comparison of breakpoint positions along the Alu consensus sequence indicate the involvement of a 26-bp core sequence in two out of five α°-thalassaemia deletions. This sequence has been identified by others as a possible hotspot of recombination. These findings favour the idea that Alu repeats stimulate recombination events not only by homologous pairing, but also by providing binding sites for recombinogenic proteins.

A novel polyadenylation signal mutation in the α2‐globin gene causing α thalassaemia

Summary. In a family of Indian origin we have identified a deletion of two bases at the polyadenylation signal sequence of the α2‐globin gene (AATAAA AATA). Three individuals heterozygous for this mutation display an αo‐thalassaemialike phenotype. Single‐stranded conformation analysis and automatic sequencing showed no additional mutations in either α1‐ or α2‐globin genes. A previously described polyadenylation sequence mutation (AATAAA AATAAG), αTSaudiα, causes HbH disease in homozygotes. In this study the patients heterozygous for the AATA(‐AA) mutation show a similar phenotype observed in the αTSaudiα heterozygotes. This confirms the observation that the inefficient transcriptional termination due to mutations of the polyadenylation sequence of the α2‐gene might interfere with the α1‐gene expression.

Molecular spectrum of beta-thalassemia in the Iranian Province of Hormozgan.

Prevention of β-thalassemia implies knowledge of the molecular spectrum occurring in the population at risk. This knowledge is necessary, especially when a prevention protocol is applied to a multiethnic population. For this purpose, we have recently analyzed a large population of Iranian patients living in the Province of Hormozgan in Iran, and a small group of Iranian patients living in The Netherlands. We have found a different mutation spectrum in both populations as compared to the data obtained by other authors for the Iranian regions of Tehran, Fars, Sistan Balouchestan, Bushehr, and Khouzestan. The IVS-I-5 (G → C) is the most frequent mutant in the province of Hormozgan (69%), followed by the IVS-II-1 (G → A) (9.6%), while the IVS-I-1 (G → A) was the most frequent defect found in the Iranian population sample in The Netherlands. The IVS-II-745 (C →G) mutation in cis with the 5′UTR (untranslated region) +20 (C → T) transition was observed in two unrelated, transfusion-dependent homozygotes, living in the Hormozgan Province where, in contrast with populations living in other provinces of Iran, no IVS-I-110 (G → A) or IVS-I-1 (G → A) mutations were found. We report the molecular spectra of our population samples and compare them with the mutation spectra observed in the Iranian populations by other authors. We discuss the severe phenotype of the patients homozygous for the IVS-II-745 (C → G) mutation, linked in cis to the 5′UTR +20 (C→ T) transition. Molecular analysis using commercial kits is briefly compared with denaturing gradient gel electrophoresis, emphasizing the value of a rapid method of detection for molecular defects in areas where many mutations occur.

Gene Frequencies in a Dutch Population

Gene frequencies at 22 polymorphic loci, including blood groups, serum proteins, and red cell enzymes, were calculated in a Dutch population of 806 individuals. 18 further loci at which little or no variation was found were also studied.

Rapid estimation of hemoglobin A2 by DEAE chromatography

A chromatographic technique for the estimation of Hb A2 in human hemolysates is described. The main advantages of this method are the ease and rapidity of execution, the high reproducibility, the use of very simple equipment, and the possibility of carrying out the whole procedure in the field and at room temperature.

Development of a preparation and staining method for fetal erythroblasts in maternal blood: Simultaneous immunocytochemical staining and FISH analysis†

In order to detect fetal nucleated red blood cells (NRBCs) in maternal blood, a protocol was developed which aimed at producing a reliable staining method for combined immunocytochemical and FISH analysis. The technique had to be suitable for eventual automated screening of slides. Chorionic villi washings, cord blood, and maternal blood samples were used for this study. After a density gradient separation and centrifugal cytology, slides were stained either with 3,3-diaminobenzidin (DAB), a marker for heme, or with antibodies against the gamma-chain of fetal hemoglobin (HbF). FISH analysis for both X- and Y-chromosomes was performed on the same slides. Cytocentrifugation provided a controlled cell density on the slides with good cell morphology. Both the DAB and HbF staining were suitable for manual screening of large numbers of slides. The HbF staining, although supposed to be more specific for fetal NRBCs, appeared to be more sensitive to minor changes in preparation. We were eventually able to combine HbF staining with FISH analysis, and produced a detection efficiency of >85% for both X- and Y-chromosome signals. This preparation protocol simplifies the detection of NRBCs in maternal blood. Immunocytochemical staining and FISH analysis can be performed on the same cell with good image contrast, thus facilitating both manual and automated image analysis. This will facilitate the use of this approach for prenatal diagnosis.

Localization of HLA on the Short Arm of Chromosome 6

SummaryA detailed marker gene study in a large Dutch kindred segregating for a reciprocal translocation between the chromosomes 6 and 20, t(6;20) (p21;p13), revealed a close linkage between the HLA genes and the breakpoint on the short arm of 6. During this study an apparent peak lod score of 2.9 was obtained at a recombination value of 0.05 for a linkage between HLA and the breakpoint, indicating that the chromosomal region, carrying the HLA genes, is situated near the breakpoint in band 6p21 close to the transition to 6p22.