Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
From www.bloodjournal.org by guest on June 17, 2017. For personal use only. Phenotypic Effect By Maria In this study. mapping 10 normal carriers one with with ( the -a), ( a I and aa) - a) type. -THALASSEMIA genotype we basis of these heterogeneity, found populations where incorporate the the defect show in found remarkable with hematologic mani- ranging from an isolated a condition of moderately increase severe of HbA2 thalasse- manifestations.2’4 This confirmed by cr-globin restriction thalassemia endonuclease of different Another coinheritance nase (G6PD) suggestion has gene analysis mapping in both racial backgrounds.5 already known ameliorating of the glucose-6-phosphate defect of the Mediterranean has also been seen to produce larger and more completely cells than seen in the majority #{176} analysis ping we report carried in a group zygotes, the results out by restriction endonuclease of 1 0 Sardinian fl#{176}-thalassemia maphetero- showed acteristics and whose an increase of HbA2 normal hematologic only distinguishing levels. In 8 of these the Institute Cagliari Medical University. Institute University Supported in part (HL-241 (Rome, Italy) of San Biology Francisco, della study 73-03), Project Program MEE Thalassemias and the of Preventive 81.361.83. and ratios Mediterrean Anemias, Address reprint Biologia del/Eta (Case/la Postale © 1983 by Grune 27. 1982; requests Evolutiva, 251) 09100 & Stratton, 0006-4971/83/6106-0018$O1.OO/O 226 accepted to December Professor Cagliari. Inc. Sardinia, Cao, Cagliari, Italy. in the first set of (-a/-a) a-globin Mediterranean type. we may conclude that may be the cause of that have been seen in From factor our data, contributing effect could defect of the MATERIALS AND METHODS ji#{176}-thalasscmia on the basis balanced I Seven ). it to the be the Mediterranean or heterozygotes of normal can Sardinian All synthesis of a Ii#{176}-thalassemia children remainder, be f-thaIassemia chain major/intermedia The also in this cell indices. globin carriers of thalassemia values, since cc/si are obligatory included red blood reduced 3#{176}-thaIassemia. Southern S regularly Counter was quantitated assumed is almost detected to be by carriers exclusively of of the population.6 according grams e to WHO.9 of DNA II and Bam hr under restricted from a-globin G6PD white and with in cells West the electrophoresed in 0.8% It Vol. was prepared 62. No. blood by phenolchloroform by Blood, peripheral Ten 30 U of restriction probe F was determined precipitation.” Mannheim, filters.’2 (4C, Hemoglobin-A2 et al.’#{176} recommended gene Counter and hemoglobin to Kan blood Coulter standard activity synthesis ethanol (Boehringer, was to nitrocellulose 32P-labeled Diagnostic). chain was digested DNA Coulter according extraction HI the a commercial microchromatography7 Globin conditions with with method.8 was prepared alcohol measured Control, was performed Bgl ferred Cell denaturation reticulocytes DNA were calibrated by DE-52 by an alkali Sar- C/mica the genes a-globin the (-a/aa) genotype we or the hemizygous state of phenotypic Red blood cell indices the Via Jenner, structural of a single G6PD HbA2 in the model 13. 1982. Antonio Universit#{224} Studi this homozygous isoamyl April HbA2 should Methods CNR Cagliari, of fl#{176}-thalassemia, dinia. Submitted in prevalent. heterozygotes. additional showed increased of Institutefor of the an selected (Table with e Sanit#{224} Medicine CNR with gene, as they are parents Hughes Medicine, Institute In three 10 Sardinian of them Age. Igiene National therefore. $-thalassemia ct-globin deletion of the were Coulter the Assessorato the may Subjects CA. Sardegna, two f3-thalassemia association type. was we of Developmental Italy, and the Howard and the Department of by grantsfrom Autonoma Health and Sardinia, Laboratory of california. Regione Study of Clinic of defect seems that production type From of in 2, the (-a/aa). several char- feature carriers, suggest, is also On the basis of these findings, the association of a-thalassemia the normal red blood cell indices picture of red blood heterogene deletion the G6PD f3- ofct-globin all of whom We of complex heterozygous determination of red association gene effect. for one production The a-thalassemia and The article, to this genotype and in one with also found the homozygous been with and the gene zygotes.3 In this the thalassemia programs (-a/-a), factor is the dehydrogetype, which a hematologic hemoglobinized offl-thalassemia to of either may mia.’4 Genetic studies and globin chain synthesis analysis have provided evidence that the coinheritance of a-thalassemia may be a cause of the less severe phenotypic recently lead fl#{176}-thalas- deletion tests. of the we heterozygous to the indices. this Cao Antonio of may contribute screening and due normal with that and findings. heterozygotes phenotypic also (G6PD) with of Interaction interaction genes. defect eventually two In three cells G6PD of the TuVeri, a-thalassemia. a-globin blood increased Teresa that with or two whom remaining a-globin Furbetta, semia of deletion (-a/act). genotype. On the the in the gene of and found dehydrogenase Mediterranean festations levels to I a - all Mario $#{176}-Thalassemia conclude genotype indices we Galanello, endonuclease a-globin cell a-globin the - blood ( -al glucose-6-phosphate tJ the a and Renzo restriction subjects, of a single Pirastu, heterozygotes, red genes deletion these out In 8 of these a-globin the carried to characterize with levels. two Mario $#{176}-thalassemia presented HbA2 A. Melis, we in order Sardinian of Heterozygous micro- endonucleases Germany) for 24 manufacturers. agarose hybridized by 1 (July), The and with trans- a specific nick-translation, 1983: pp. as 226-229 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. fl’-THALASSEMIA a AND Table INTERACTION 1 . Hematologic 227 Fee tures and Gb bin Chain RBC Sex Sub,ects (x 1O’2/L,tec) Sy nthesis Anal ysis in Alpha Hb MCH MCV HbA, (g/dl) (pa) fl) (%) and Beta Th alassemia HbF (%) Do uble Heterozygotes a/ G6PD (Ratio) a-Glob,n (Ul/gHb( Genes Arrangement L.G. M 5.67 17.1 30.3 88 5.24 0.88 0 -cs/-n S.D. M 4.92 14.4 29.4 90 5.00 - 0.86 0 -a/-sn PT. F 5.25 13.6 26.0 79 5.51 0.73 - 0 -a/-a cL. M 5.70 14.9 26.2 79 5.15 0.62 0.73 8.4 -a/-a P.S. M 5.50 15.0 27.2 80 5.21 0.50 0.83 7.2 -a/-a R.G. M 4.90 13.2 27.2 83 5.13 0.73 0.98 9.0 -a/-a S.M.B. F 4.63 12.1 26.9 77 5.17 0.51 - 6.1 -a/-a GS. M 5.86 15.7 27.5 80 4.44 0.55 0.89 5.8 -a/-a V.A. F 5.05 13.7 27.1 84 5.66 - - 0 -a/aa A.F. M 5.31 14.9 28.2 83 4.76 2.7 1.30 7.2 -a/aa M 5.18 Normal 080’ ± 15.9 (3.65-6.69) F 14.0 (3.88-5.81) Obligate M 6.12 f(-ThaI. F 5.44 + 2 SD described plasmid by JW probe and ranges prepared 30.2 16.3) ± 21.9 + 4.3 87.6 (9. 1- ± 4.6 66.5 21.9 14) et al.,’3 The filters from were 66.2 (18.5-25.8) a-globin gene also hybridized by nick-translation ± ofa In normal DNA digested ized with an a-globin-speciflc Hinfl cloned with the in a c-specific fragment with ofthe pBR HI and hybridthe duplicated Barn probe, loci reside in a DNA fragment (Kb) in length.’5 Restriction maps of /3#{176}-thalassemia carriers showed that they shortened 10.5-Kb fragment, characteristic zygous a-thalassemia-2 (-a/-a). The a-globin both the 14.5- and 10.5-Kb the silent carrier state semia-2 enzyme probe, the by Bgl II digestion. and hybridized DNA that contain respectively.’6 can be 14.5 kilobase 8 of the 10 had only the of homoremaining 2 fragments, which or heterozygous ( -a/aa). characterized normal ± 0.44 (1.86-2.98) 11.0 2.52 ± 4.99 ± 0.52 0.36 1.02 0.98 4.98 ± 0.56 ± ± 1.02 0.54 ± 0.12 (0.92-1.14) (0.14-0.98) (4.09-6.57) 8.7 0.37 (0.11-1.13) (1.53-3.09) 11.5 (55-78) RESULTS a-thalassemia-2 We further 2.55 100) ± plasmid. showed indicates 10.2 (59-85) 3.1 ± ± (77- (19.5-25.1) 1.8 ± (79-102) (25.4-34.6) 1.6 11.8 1) 89.1 #{247} 13.3 0.90 3.9 ± ± 1.12 (0.16-2.5) 1.04 (4. 19-6.29) 0.94 ± 1.83 1.18 ± 0.52 (1.40-2.32) (0.12-3.35) in parentheses. Maniatis l0I.’ 1.8 (11.5-15.3) (4.02-6.7 ‘Means 30.9 (26.9-35.0) ± (10.51.06 ± (4.99-7.38) Heteroz. 2.1 ± (12.5-19.1) 4.69+0.86 ‘ 080 yields nature When digested with with an a-globin-specific 1 2.5- and 7.0-Kb the a, and a, globin The a-thalassemia-2 produced by two of a-thalas- chromosome the rearranged and unequal I 6.0-Kb fragment from while in the homozy- an additional chromosome, gous state for this lesion, only the 1 1 .0-Kb and 16.0-Kb fragments can be seen. Mediterranean a-thalassemia-l heterozygotes (--/aa) gave a threefragment pattern with the normal bands, 1 1 .0 and I 2.0 Kb in length, and an additional 14.0-Kb deleted chromosome. specific probe, we have the rightward With confirmed deletion in the homozygous hetenozygous state. state and Moreover, did we detect band from the Bgl II and the -globinthat 8 of our subjects a-thalassemia-2 genotype in the cases 2 the same lesion in none of these the a-thalassemia- I haplotype. DISCUSSION fragments structural haplotype different this taming the 2 and a 12.0-Kb fragment, containing the -,, and a, genes’7 (Fig. I). DNA from the nightwa rd heterozygous a-thalassemia-2 genotype yields the two normal fragments from the normal loci, (-a) crossover the In this study, restriction endonuclease analysis of a-globin structural genes in 10 /3#{176}-thalassemia heterozygotes selected on the basis of normal red blood mechanisms, indicated as the nightward or leftward deletion.’6 Digestion of the DNA from the leftward deletion a-thalassemia-2 haplotype produces only the cell characteristics more frequently, This association 7.0-Kb the two study,’8 the frequency of the single a-gene deletion ( -a) in a random group of /3-thalassemia heterozygotes from the same racial background was found to be 0.18. The coinheritance of heterozygous /3-thalassemia with the deletion of two a-globin loci reduces both a and /3 globin chain production to half that observed in fragment, normal while fragments a 15.8-Kb in DNA fragment from the replaces rightward deletion.’6 We found the nightward deletion in all the carriers analyzed, both in the homozygous and heterozygous We state. also analyzed haplotype hybridizing by (--) with our samples for a-thalassemia-l digesting DNA a nick-translated with Bgl II -globin-speciflc probe. When normal DNA is cleaved with hybridized with a -globin-specific probe, ments are produced, i.e., an 1 1.0-Kb and Bgl II and two frag- fragment, con- showed either the deletion ofone or, of two a-structural globin gene loci. is highly significant as, in a previous the unaffected subject. This results in an almost balanced globin chain synthesis, which could, in turn, explain the quasi-normal volume and hemoglobinization of the red blood cells. However, the same amelio- From www.bloodjournal.org by guest on June 17, 2017. For personal use only. MELIS 228 I 2 ET AL. 3 i6.okb 12.0 11.0 S -a/-a -a/aa aa/aa Fig. 1. Autoradiogram of Bgl-ll-digested DNA hybridized with a ‘-gIobin-specific probe. Lane 1: a-thalassemia-2 heterozygote; lane 2: a-thalassemia2 homozygote; lane 3: normal a-globin gene genotype. A map of the Bgl II restriction sites (B) in the ‘-a globin gene cluster is shown at the bottom. The ‘. ?.i B (L2 tpal (Li B B 11.0-Kb fragment Kb fragment i’,, the gene ‘2 and the 12.0- a2 genes. The 16.0-Kb fragment containing the i’,. ‘,l’a, and only one a-globin locus, is the product of an unequal crossing over. deleting a 3.7-Kb fragment between the two a-genes. 3k 5’ contains 5K;-’ the and a,. rating effect was also found in two /3#{176}-thalassemia heterozygotes with only one a-structural gene deleted. A possible explanation for one of these cases might be previous study,4 the finding of normal red blood cell indices in several /3-thalassemia heterozygotes seems to represent the maximum expression of the ameliorating the association of the G6PD defect of the Mediterranean type. This defect, when associated with heterozygous /3-thalassemia, has already been found to lead to tendency due to the coinheritance This extreme effect is relatively of a-thalassemia. frequent, since in the Southern Sardinian semia heterozygotes 3.5% to have greater volume was never seen indices. mean This red chronic ever, may nondeletion and increased to produce red effect blood slight has cell Hb blood content/cell,3 cells with but normal been attributed to a younger population, resulting from hemolysis.’9 Another be the undetected a-thalassemia presence resulting possibility, tologic a how- may light (aat/_a) genotype. In our population, such nondeletion defects have been observed with a frequency of 25% of all the a-thalassemia-2 haplotypes.’8 We conclude, therefore, that the interaction of heterozygous /3#{176}-thalassemia with the deletion of either one or two a-globin genes blood cell indices. Moreover, that the contribute Based association of to this effect. on the evidence the may produce normal red from our data it seems G6PD defect may also in this and carrier screening due based miss a significant of these results, incorporate to the coinheri- testing on MCV/MCH proportion it would for HbA2 in popula/3-thalasvalues of carriers. In the seem advisable to in the first set of tests for heterozygous a-thalassemia in /3-thalasis also prey- alent. ACKNOWLEDGME We Rita Forget a probably of /3#{176}-thalasnormal hema- ofa-thalassemia gene(s).2G Therefore, where a-thalassemia is also prevalent, genetic screening programs semia in populations where plasmid produced characteristics, tance tions semia of an associated in an effective population, were found are grateful Loi to Dr. for editorial for the pBR, to us before plasmid and Y. W. assistance. Dr. publication. JW 101, E. Kanavakis Kan We Drs. NT for his continuing would also like J. Lauer for making and advice to thank T. and Dr. Maniatis his results available B. for From www.bloodjournal.org by guest on June 17, 2017. For personal use only. a AND f3’-THALASSEMIA INTERACTION 229 REFERE NCES 1. Weatherall 3). Oxford, 2. Di, Blackwell Bianco Thalassaemia Syndromes (ed 1981 for I, Salvini $ a and P. Silvestroni microcytemia. Hum 3. Sanna ciency F, Melis between MA, Galanello R, De Virgiliis the glucose-6-phosphate thalassemia genes at 5, Cao dehydrogenase 4. R, Melis E, Cao #{176}-thalassemia A2 levels. 5. Rev Kanavakis Furbetta phenotype Furbetta Globin chain level. Galanello with M, Br isolated 23:193, JS, R, Angius synthesis with Fr Hematol E, Wainscoat M, a-thalassaemia 6. A: MA, heterozygotes Nouv J Haematol Cao MT. A, Scalas analysis types increase in obligate Huisman of hemoglobin Weatherall T: The AM, THJ, A, Angius Maccioni DJ, for 86:700, 1975 9. A, Rosatelli MA, J Med of 10. WA, Brodie of hemoglobin ME, small J Clin Scientific McWade Galanello AN, R: HE, Lie-Injo LE, a-thalassemia 1974 sequences electrophoresis. among J Mol DNA Biol 98:503, A, Kleid DG: Proc Nucleotide NatI sequence Acad Sci USA of the 72:1 184, SM, Clin Embury Pathol of 25:738, Group: DJ: foetal Reliable hemoglobin routine by gene deletions. 1972 of procedures dehydrogenase. WHO Tech for the trait: Schwartz thalassemia F, Nathan syndromes. DG: J Clin Cell Haematol Globin Invest chain synthesis in Maccioni 47:25 1 2, 1968 JA, Dozy AM, L, of synthetic Nucleic a-globin genes Sci USA 75:5950, Kan organizations YW, Acid lie close Chang account a-thalassemia-2 Ci, J 1978 V. Todd for genotype. C, Cao the single Clin Invest Galanello L, trait chromosomal Sequence Dozy AM, Kan Zachariades Alpha 60:509, 5, T: The genes: homology arrangeand cs-globin 20: 1 I 9, I 980 A: Blood Z, thalassemia YW, Stamatoyannopou- Angius in A, two Furbetta M, Mediterranean 1982 Siniscalco M: The dehydrogenase between 16:537, thalassemia Maniatis MG, interaction Ser Villa-Komaroff Insertion plasmids. human Acad M, Lee KY, Piomelli Rep bacterial Nail like globin a Hadjiminas 20. YW, the glucose-6-phosphate Standardization JK, SM: Proc molecular of Pirastu 19. Miller J, Shen of human populations. alkali Rid 1980 Lauer G, into duplicated DNA. different ment los de Weissman genes The SH, gene 18. Med SH: in cellular Two LB. BG, 1978 Orkin 17. 1967 the alpha gel Wilson globin 66:1319, A simplified J Lab JT, of human a-globin 18:196, Mayson Ill. A2. P. Weatherall percentages of glucose-6-phosphate Kan Varmus in homozygous of specific of phage. Forget Rosatelli of N 366, by A, together 16. C, Tuveri Genet of haemoglobins. the determination Pembrey WHO YW, lesion 25 1:392, T, Jeffrey Wilson 15. Cao Br J Haematol L, Melis Sardinia. Schroeder method study 14. copies interaction f-thalassaemia. in Southern J: Microchromatography denaturation. Kan Detection operation Res 5:563, WG, Wood M, Ximenes Jakway 8. Maniatis Efstratiadis 1981 estimation Nature EM: separated I 3. MT. 1981 Sophocleous heterozygous Falchi fJ-thalassemia 7. AM, 1975 D: A, Furbetta Scalas fetalis). 1 2. Southern rightward 1982 52:465, (hydrops Dozy D: Genetic I975 Galanello Paglietti JM, J, Todd defi- 1980 44:555, Taylor fragments G, Frau and I I. Ganesan Hered 1973 A: Interaction T, The B, Mastromonaco heterozygosity 23:454, JB: Scientific, I, Graziani E: Double A, Clegg haematological deficiency the two genes MA, Ruggeri at the effects and phenotype of thalassemia level. Br J 1969 R, Melis Furbetta M, in Sardinia. Angius Hemoglobin R, Addis A, Tuveri 3:33, M, T, 1979 Scalas Cao MT. A: 3#{176}- From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 1983 62: 226-229 Phenotypic effect of heterozygous alpha and beta 0-thalassemia interaction MA Melis, M Pirastu, R Galanello, M Furbetta, T Tuveri and A Cao Updated information and services can be found at: http://www.bloodjournal.org/content/62/1/226.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.