Gino Schilirò

βs haplotypes in various world populations

SummaryWe have determined the βs haplotypes in 709 patients with sickle cell anemia, 30 with SC disease, 91 with S-β-thalassemia, and in 322 Hb S heterozygotes from different countries. The methodology concerned the detection of mutations in the promoter sequences of the Gγ- and Aγ-globin genes through dot blot analysis of amplified DNA with 32P-labeled probes, and an analysis of isolated Hb F by reversed phase high performance liquid chromatography to detect the presence of the AγT chain [Aγ75 (E19) Ile→Thr] that is characteristic for haplotype 17 (Cameroon). The results support previously published data obtained with conventional methodology that indicates that the βs gene arose separately in different locations. The present methodology has the advantage of being relatively inexpensive and fast, allowing the collection of a vast body of data in a short period of time. It also offers the opportunity of identifying unusual βs haplotypes that may be associated with a milder expression of the disease. The numerous blood samples obtained from many SS patients living in different countries made it possible to compare their hematological data. Such information is included (as average values) for 395 SS patients with haplotype 19/19, for 2 with haplotype 17/17, for 50 with haplotype 20/20, for 2 with haplotype 3/3, and for 37 with haplotype 31/31. Some information on haplotype characteristics of normal βA chromosomes is also presented.

Structural and ultrastructural study of the ovary in childhood leukemia after successful treatment

Ovarian biopsy specimens from ten girls (three postmenarcheal) who had undergone antiblastic treatment for acute lymphoblastic leukemia (ALL) and were in complete remission were examined by light microscope. the biopsy specimens from four of these patients (three postmenarcheal) were also observed by electron microscope. the structural and ultrastructural analysis showed a reduction in the number of follicles which were otherwise normal. No follicles were found in the thin sections from two of the three postmenarcheal girls, whereas normal follicles were observed in the third. the cortical stroma showed moderate to severe signs of fibrosis and changes of capillaries. All of these alterations were more evident in patients where ALL was diagnosed at an older age and this finding suggests that they are at a higher risk for low fertility or early menopause.

Clonal stability in children with acute lymphoblastic leukemia (ALL) who relapsed five or more years after diagnosis

Although most relapses of childhood acute lymphoblastic leukemia (ALL) occur 24–36 months after first CR has been achieved, few patients relapse 5 or more years after CR achievement. The assessment of clonality has proved to be useful in determining whether even those very late events represent the reoccurrence of the original clone or alternatively a secondary leukemia. To gain further information on clonal stability in such late relapse, we performed detailed comparative Southern blotting and PCR analyses of TcRδ and TcRγ gene rearrangements in five ALL at presentation and subsequent relapse which occurred more than 5 years after diagnosis. At least one stable rearranged allele of the TcRδ and TcRγ loci was traced in all cases at presentation and clinical relapse despite a wide heterogeneity of the pattern of rearrangements. Our study extends to a larger series of patients previous findings which have sought to analyze the phenomenon of clonal evolution in children relapsed after more than 5 years of CCR. With respect to the potential pitfalls in monitoring minimal residual disease in childhood ALL for the presence of clonal evolution, our results highlight the combination of two target genes (such as TcRγ and TcRδ) as a tool to reduce false negative MRD results.

A Search for Anomalies in the ζ,α,β, and γ Globin Gene Arrangements in Normal Black, Italian, Turkish, and Spanish Newborns

Globin gene mapping analyses of DNA from numerous Black babies, and from newborns from Sardinia, Sicily, Turkey, and Spain have identified the followingA high incidence of α-thalassemia-2 heterozygotes among Black babies with less than 1% Hb Barts at birth and a high incidence of α-thalassemia-2 among Sardinians, but not among Sicilian, Turkish, and Spanish babies. A relatively high incidence of ζ-thalassemia was present among Black babies only, while triplicated ζ was seen in four of the five populations. Two Black babies were each found to have a different θl deletion; two Sardinian babies had a newly discovered β 2.5 kb deletion between ζ and ψζ; four babies had the rare Bgl II polymorphism between ψζ and ψα; and one Black baby lacked the Eco RI site 3’ to ζ Quantitation of the ζ chain by reversed phase high performance liquid chromatography showed that two-thirds of the babies with four α genes (αα/αα) had levels between 0.1 and 1.0%, while nearly 90% of the babies with -α/αα had similar levels (aver...

Growth and growth hormone in children during and after therapy for acute lymphoblastic leukaemia

Growth impairment and growth hormone (GH) deficiency have been reported in children treated for acute lymphoblastic leukaemia (ALL). We have studied growth and GH secretion in a group of 50 patients, affected by ALL, during a 2- to 5-year period after diagnosis, and in 12 “long-term-survivors”. We observed a significant decrease in growth velocity during the 1st year (in particular during the first 6 months) of therapy and a catch-up growth after the end of therapy. “Longterm survivors” did not exhibit a significant reduction of height standard deviation score (SDS), as compared to height SDS at diagnosis. None of the patients showed GH deficiency. Our data indicate that chemotherapy significantly affects growth of patients treated for ALL, whereas radiotherapy-at the doses used in this study-does not induce GH deficiency, at least not within 9 years after diagnosis.

Molecular basis of α-thalassemia in Sicily

Abstract To evaluate the allelic frequency and genetic diversity of α-thalassemia defects in Sicily, both epidemiological and patient-oriented studies were carried out. For the epidemiological study, phenotypic data were collected on more than 1000 Sicilian individuals. Among them, 427 were explored at the molecular level for nine α-thalassemic variants known to be common in the Mediterranean region. Our data reveal an allele frequency of 4.1% for α+-thalassemia matching that of β-thalassemia in this region. The presence of α°-thalassemia (––MEDI and ––CAL) was observed only in the group of referred patients. Newly acquired nucleotide sequence data on the deletional breakpoint of ––CAL allowed us to design a simple PCR-based procedure for exploring this allele. The data also provide additional information concerning the genetic mechanisms involved in such large deletions.

Possible factors influencing the haemoglobin and fetal haemoglobin levels in patients with β‐thalassaemia due to a homozygosity for the IVS‐I‐6 (T→C) mutation

Summary. We have collected haematological, haemoglobin (Hb) and DNA sequence data for 29 patients with a homozygosity for the IVS‐I‐6 (TC) mutation with the intention of identifying factors contributing to the observed variability in the severity of the disease. None of the patients had received blood transfusion therapy for at least 6 months prior to the study. Hb levels varied from 5·0 to 9·9 g/dl. Patients with high Hb F (more than 1·5 g/dl or <20%) had high total Hb levels (7·5–9·7 g/dl) but some with low Hb F also had high total Hb levels; two had a concomitant α‐thalassaemia‐2 (α‐thal‐2) heterozygosity. An inverse correlation between the Hb F and Hb A2 levels was observed. The majority of the patients were homozygous for haplotype VI (49/58 chromosomes) but haplotypes IV (2/58) and VII (7/58) were also present. The only haplotype IV homozygote had high Hb F levels with high Gγ values and the CT mutation at position – 158 in the Gγ promoter, while both high and low Hb F levels were observed among patients with haplotypes VI and VII. Analysis of sequence variations in regulatory regions included the 5 hypersensitive sites (HS) 4, 3 and 2 of the locus control region (LCR), the Gγ and Aγ 5 flanking regions, the second intervening sequence (IVS‐II), and the 5 β‐globin gene region in two patients with high Hb F (one homozygote each for haplotypes VI and IV), and in two patients with low Hb F levels (one homozygote each for haplotypes VI and VII). Haplotype specific differences were observed in the LCR 5 HS‐2 and in the Gγ and Aγ flanking and IVS‐II regions; however, no differences were present between the low and high Hb F‐producing haplotype VI chromosomes, suggesting a major role for factors which are not linked to the β‐globin gene cluster in mediating γ‐globin gene expression in patients with this type of β‐thal.

Allergy to desferrioxamine

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γ‐mRNA and Hb F levels in β‐thalassaemia

Summary. The Hb F levles in β‐thalassaemia can be affected by factors both linked and unlinked to the β‐globin gene cluster. We have recently analysed a group of patients with a homozygosity for the IVS‐1–6 (T → C) mutation, showing a wide variation in Hb F levels (2–47%) which could not be accounted for by any sequence variation within regulatory elements of the β‐globin gene cluster. In order to further investigate factors underlying this phenotypic difference we have developed a competitive reverse trascription/polymerase chain reaction procedure and used this method to determine the relative amounts of γ‐and β‐mRNAs in 10 patients with the IVS‐1–6 homozygosity and 15 heterozygous parents, two IVS‐I‐6δβ‐thalassaemia compound heterozygotes, five homozygotes for the βd́; IVS‐I‐110 (G → A) mutation, and in two with a homozygosity for the β† codon 39 (C → T) mutation. Three heterozygotes were also included. The percentages of γ/(γ+β) mRNA were 10–73% in the IVS‐I‐6 homozygotes and <2% to 10% in their heterozygous parents. A direct relationship existed between the level of mRNA and the % Hb F. However, the relative γ‐mRNA levels in the IVS‐I‐6 homozygotes were higher than their Hb F levels, indicating a possible competition between the γ and β transcripts for translational factors with a less effcient initiation of protein synthesis on the γ‐mRNA or a preferential survival of cells with mainly β‐globi gene expression at the post‐reticulocye stage. The γ‐mRNA levels in the two IVS‐I‐6/δβ‐thalassaemia compound heterozygotes were 71% and 62%, similar to their Hb F levels (63% and 59%), and averaged 82% (range 65–91%) in the five IVS‐I‐110 homozygotes and 97.5% in the two codon 39 homozygotes. The correlation between these values and the % Hb F could not be evaluated because of the transfusion regimens; however, the levels, of γ‐mRNA were as expected for patients with these β‐thalassaemia alleles.

Clinical, Hematological, and Molecular Features in Sicilians with Sickle Cell Disease

We report the clinical, hematological, and molecular findings observed in 32 Sicilian patients with sickle cell disease. None of our patients received regular blood transfusions and careful infectious disease prophylaxis was carried out for all. Haplotyping of beta S chromosomes was performed in all patients; all were homozygous for haplotype #19 (Benin). Gene mapping excluded the presence of an alpha-thalassemia in 13 of our patients; none of the relatives showed any evidence of the presence of alpha-thalassemia. Hb F levels were 11.8 +/- 5.9% with G gamma representing 39.6 +/- 3.6% of total gamma chain. Hb F levels were higher in females than in males (12.5 +/- 5.9% versus 9.7 +/- 6.5%) but the difference was not statistically significant. All patients, regardless of age and sex, were anemic with normal mean corpuscular hemoglobin concentration, high mean corpuscular volume and mean corpuscular hemoglobin, and mild reticulocytosis. Analysis of clinical manifestations suggests that our patients have a disease of moderate severity.