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Sequence Analysis of the y-Globin Gene Locus from a Patient with the Deletion Form of Hereditary Persistence of Fetal Hemoglobin By Catherine A. Stolle, Laura A. Penny, Sharlene Ivory, Bernard G. Forget, and Edward J. Benz, Jr. The 7-globin genes from a patient homozygous for a deletion form of hereditary persistence of fetal hemoglobin (HPFH-1) have been cloned and sequenced. The DNA sequence of the patient‘s 7-globin genes corresponds to a previously identified sequence framework (chromosomeA) with the exception of 10 base changes. Seven of these base changes can be attributed to normal allelic variation generated by small gene conversion events. The remaining three base changes are present in a 0.76 kb Hindlll fragment containing a putative enhancer located 3’ to the “7-globin gene. The same three base changes have also been described in the Seattle variant of nondeletion HPFH. We have analyzed 16 alleles from non-HPFH individuals and five alleles from individuals with nondeletion or deletion HPFH for the presence of these base changes by polymerase chain reaction amplification of cloned or chromosomal DNA and hybridization to allele-specific oligonucleotide probes. Although these base changes were found in an individualwith HPFH-2, they were not found in the DNA from two patients with nondeletion HPFH. More importantly, all three base changes were detected in DNA from five non-HPFH individuals and appear to be common in blacks. We conclude that these base changes do not correlate with an HPFH phenotype and that the significant mutation in HPFH-1 is the deletion of over 100 kb of genomic DNA. 0 1990 by The American Society of Hematology. H from a patient homozygous for HPFH-1. Of the 10 base changes identified, 7 correspond to known sequence polymorphisms, and 3 base changes located in the *y gene 3‘ enhancer” have been described previously in the Seattle variant of nondeletion HPFH.26Further analysis of D N A from non-HPFH individuals indicates that these three base changes are present in normal y-globin gene sequences and are unlikely to contribute to the increase in y-globin gene expression observed in HPFH-1. EREDITARY PERSISTENCE of fetal hemoglobin (HPFH) is a benign condition in which fetal hemoglobin expression persists into adulthood at levels greater than 1% in the absence of erythropietic stress or thala~semia.’-~ Because this condition can be viewed as a failure to switch from fetal (HbF, a2y2)to adult (HbA, ad2)hemoglobin synthesis, it has been studied as a model for the developmental control of gene expression. Two major categories of HPFH have been delineated based on mutations associated with these disorders. Nondeletion HPFH is commonly associated with single base changes in the 5’ flanking region of the y-globin genes. These mutations may alter promoter sequences or binding sites for trans-acting factors, resulting in a corresponding over expression of either the ‘7-globin or *y-globin gene.5-’2Deletion HPFH is characterized by large (= 105 kb) deletions in the &globin gene cluster that remove the adult (6 and @)globin genes and result in increased expression of both Gy- and *y-gl~bin.’~-’’ One hypothesis to explain the increase in y-globin gene expression in the deletion form of HPFH described in American blacks (HPFH-1) is that the sequences juxtaposed to the y-globin genes as a result of the deletion contain an enhancer-like element that serves to maintain a transcriptionally active domain. A small fragment of D N A from the region immediately 3’ to the breakpoint in HPFH-1 has been found to have enhancer-like activity when transfected into erythroid cells.20.2’This region of D N A is also hypomethylated and DNAse I hypersensitive in erythroid tissues and contains a large open-reading frame (ref 20-22; Elder and Groudine, personal communication, March, 1989). Furthermore, deletions that extend beyond the enhancer-like element result in SPo thalassemia instead of HPFH.2’323However, no single model has been proposed that successfully explains the mechanism underlying all forms of deletion HPFH.24 The sequences of the y-globin genes from patients with deletion HPFH have never been directly examined for mutations analogous to those present in nondeletion HPFH. Such mutations might provide an additional explanation for the persistence in expression of the y-globin genes in deletion HPFH. We have cloned and sequenced the y-globin genes Blood, Vol 75, No 2 (January 15). 1990: pp 499-504 METHODS Cloning and sequencing of HPFH-I y-globin genes. Genomic DNA from a lymphoblastoid cell line, LAZ-149;’ obtained from a black patient homozygous for HPFH-1,I5was digested to completion with BglII (New England Biolabs, Beverly, MA). EMBL 3 phage arms (Promega Biotec, Madison, WI) were prepared by digestion of the vector with BamHl and EcoR1, followed by precipitation with isopropanol, as described in the manufacturer’s protocol. Phage arms and digested genomic DNA were ligated in a 10 pL reaction containing 66 mmol/L Tris-HC1 (pH 7.5), 5 mmol/L MgCl,, 5 mmol/L DTT, 1 mmol/L adenosinetriphosphate (ATP), and 3 units of T4 DNA ligase (Pharmacia, Piscataway, NJ). Ligation reactions were incubated at 16OC for 16 hours. A portion of the ligation mixture was then packaged using Gigapack packaging extracts (Stratagene, San Diego, CA) according to the manufacturer’s ~ From the Departments of Internal Medicine and Human Genetics, Yale University School of Medicine. New Haven, CT. Submitted June 9,1989; accepted September 8. 1989. Supported by a Research Career Development Award to E.J.B. and grants from the NIH to E.J.B. and B.G.F. C.A.S. was supported by a National Research Service Award. Presented in part at the annual meeting of the American Society for Clinical Research in Washington, DC, May, 1988, and has been published in abstract form (Clin Res 36:569,1988). Address reprint requests to Edward J. Benz, Jr, MD, Department of Internal Medicine, Yale University School of Medicine. 333 Cedar St, New Haven, CT 0651 0. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 6 1990 by The American Society of Hematology. 0006-4971/90/7502-0017$3.00/0 499 STOLLE ET AL 500 NaOH and 25 mmol/L EDTA and blotted onto a Nytran membrane (Schleicher and Schuell, Keene, NH) using a slot blot manifold. Oligonucleotide probes corresponding to the *y-globin gene published sequence (TTGGAAGGTTTACTTAA; AGGTTTAGC TGAGCTCA; AGCACCTTACACTGAGT)32.33 or the Seattle variant (TTGGAAGGCTTACTTAA; AGGTTTAGATGAGCTCA; AGCACCTTGCACTGAGT)26 were 5' end-labelled to a specific activity of 1 x lo7 cpmlpg using T4 polynucleotide kinase (Boehringer, Mannheim, W Germany) and y- [32P]dATP(Amersham, Chicago, IL). Each probe was hybridized to the membrane-bound amplified DNA at 5OC below its calculated melting temperature. The membrane was then washed at 2OC below the probe melting temperature to remove nonspecifically bound probe.34 The filters were exposed to X-ray film (Kodak, Rochester, NY) at -7OOC with an intensifying screen. suggestions. VCS 257 bacteria were infected with the packaged phage DNA and plated using standard methods." Approximately lo5recombinant phage plaques were screened by hybridization with a nick translated 2.6 kb EcoRI fragment from the 5' end of the Ay-globingene. Potential positive clones were counter screened with a 1.6 kb EcoRI fragment from the 3' end of the Gy-globin gene. Positive identification was achieved by restriction enzyme digestion, and Southern blot analysis of insert DNA was compared with a 13 kb BglII fragment of normal DNA containing the Gy-and *y-globin genes. Insert DNA containing the y-globin genes from one of the patient's chromosomes was excised from the vector by digestion with SalI. A Gy-globinfragment (2.6 kb StuIISphI) digested with AluI and an Ay-globinfragment (2.2 kb RsaI) digested with HaeIII were blunt end ligated into M13mpl0, which had been linearized with SmaI. In addition, a 0.76 kb Hind111 fragment from the region 3' to the patient's Ay-globingene was similarly subcloned. Single stranded or double stranded M13 templates were sequenced using the dideoxy sequencing method and Sequenase (US Biochemicals, Cleveland, Each fragment was sequenced at least twice using several independent isolates of M13 subclones (Fig 1). Amplification of genomic DNA and hybridization with allelespecijc oligonucleotide probes. Genomic DNA (1 p g ) was amplified by the polymerase chain reaction technique for 30 cycles with 1.5 minute denaturations at 94OC, primer annealing for 2 minutes at 5S0C, and extensions for 3 minutes at 720C3' Amplified DNA (1% to 2% of reaction product) was denatured in a solution of 0.4 N GY - 4 Sal I/ Bgl II n. I nm II -, -AY RESULTS The LAZ-149 cells used in this study were derived from a patient who was the subject of previous report^.'^^'"^*^ The patient is homozygous for a form of HPFH common among American blacks (HPFH-1) as determined by Southern blot analy~is.'~-'~ The two y-globin gene loci were indistinguishable by restriction enzyme mapping and presumed to contribute identically to the HPFH phenotype. The '7- and *yglobin genes from one of the patient's two chromosomes e+ n n II / s a I I stu I stu-TGi?l I SI" I 50kb Rsa I R s I~ --ET--- Hind 111 Hind 111 b 1 2 . 1 L Sph I I SI" I AA C St" I L.-. Sph I A A A A A A A A AAAA AA A Fig 1. Sequencing strategy for HPFH-1 7-globin gene clones. (a) Diagramatic representation of '7- and 'y-globin gene fragments isolated from a 13 kb Sa/I/Bg/ll cloned DNA insert from a patient with HPFH-1. The solid bar indicates the portions of the gene fragments sequenced. lb) Sequencing strategy for the '7-globin gene and (c) sequencing strategy for the '7-globin gene and putative 3' enhancer. The location of the A M (A) or Haelll (HI restriction enzyme sites in the y ' or ' 7 globin genes (respectively) used to prepare subclones for sequencing in M13 are illustrated below the diagram of each gene. Sequencing was obtained using singlestranded clones and universal primer ( 4 1 , double-stranded clones and reverse primer (h), or synthetic oligonucleotide primers (I+). Differences in the nucleotide sequence compared w i t h published sequence are indicated by a numbered arrow above the diagram of each gene. The numbers refer to sequence differences listed in Table 1. 501 y-GLOBULIN GENE SEQUENCE IN HPFH 'y-globin gene and the 'yAy inter-genic region derived from the patient's cloned D N A was subcloned into M13 and sequenced as described above using a universal primer. Three Sequence Change Position Normal Sequence Fw additional base changes were detected: a deletion of one A in Gy-Globin a string of four at -601 to -603; an insertion of an A at -T +1,611 to +1.618 Gr(B) -605; and a deletion of a T at -61 1 (Fig 1C and Table 1). CG+T + 1,800 and + 1,801 G7(B) The three base changes support the notion that a gene "7-Globin -T -61 1 *Y(B) conversion event has occurred in which the 5' end of the +A - 605 "y(B) Ay(A) gene has been converted to an Ay(B) gene. However, -A -603 to -600 "y(B) the predicted base change at position -588 (A-G), which C+G -369 "-AB) would have been consistent with a gene conversion event "7 3' Enhancer involving the entire sequence from -369 to -61 1, was not c T-C +2,276 observed. GG+M +2,357 and +2,358 "y(B) Small regions of converted gene sequence interspaced by C-+A +2,451 c nonconverted regions have been identified previously in the A+G +2,667 I y-globin genes and have been called "patchy" gene conThe position of each base change is relative to the cap site for the "7version~.~'.~* Such gene conversions are relatively common in or Ay-globingenes, respectively. y-globin gene sequences and frequently result in an Ay-globin *Base changes not identifiedin normal y-globin gene sequence. gene converted to a 'y-globin gene sequence. The base changes described here clearly suggest conversion of an Ay bearing the deletion form of HPFH-1 were sequenced (Fig 1) (A)-type chromosome to an Ay (B)-type sequence, and not and compared with the sequence of previously reported '7conversion by a 'y-globin gene (Fig 2). In any event, the and Ay-globin conversion of the sequences of a normal y-globin gene to that The sequence of the '7-globin gene from -383 to +2,209 of another normal y-globin gene cannot explain the phenobp (relative to the cap site) was identical to 'y-globin from the previously reported chromosome A sequence f r a m e ~ o r k , ' ~ type of y-globin gene expression in this patient. Since no base changes similar to the mutations that give except for a deletion of one of eight T's (+ 1,611 to + 1,618) rise to nondeletion HPFH were detected, the sequence of the and a CG-T substitution (+ 1,800 and + 1,801; Fig 1B and putative enhancer located 3' to the Ay-globin gene was Table 1). Both of these nucleotide base changes are present examined. A 0.76 kb HindIII fragment from the 3' end of the in the normal '7-globin gene of the B chromosome frameAy-globingene (Fig 1) was isolated from a digest of the 13 kb work (Slightom, personal communication, February 1988) Sal1HPFH- 1 fragment by gel electrophoresis and electroeluand, as such, are not likely to be responsible for the increase tion. The HindIII ends were filled using a mixture of all four in expression of both '7- and Ay-globin genes characteristic dNTPs and the large fragment of D N A polymerase. The of this condition. A T at - 158 (which creates a restriction fragment was then blunt end ligated into M13mplO and enzyme cleavage site for XmnI) is normal for a 'y-globin D N A templates were sequenced as described above. Four gene from chromosome A and is present in the patient's sequence differences were detected:( 1) T-C at +2,276; (2) D N A sequence. The presence of a T a t -158 has been GG-AA a t +2,357 and +2358; (3) C-A at +2,451; and associated with a 'yAy ratio of 60:40 in nonanemic adults (4) A-G a t +2,667 relative to the Ay-globin gene cap site and a small increase in H b F (of the Gy variety) in patients is (Fig 1C and Table 1). One of these changes (GG-AA) with sickle cell anemia or B thala~semia,~' but cannot explain characteristic of a normal Ay-globin gene of the B chromothe large increase in both '7- and Ay-globin observed in this patient. The presence of a T at - 158 of the 'y gene has also been demonstrated in other individuals with HPFH-1 .36 -611 -605 -602 -588 -369 +25 +65 The sequence of the Ay-globin gene from -383 bp to ___ + 1,760 bp (relative to the cap site) was also identical to the Ay(A) T (-) A A C published sequence for a chromosome A y-globin gene, ___ except for a single base change (C-G) at position -369 (-) A (-) G Ay (B) -__ __ (Fig 1C and Table 1). A G a t position -369 is characteristic C A C c (4 A A Gy (A) of a normal Ay-globin gene from chromosome B33 and is therefore not a candidate for the mutation underlying HPFH. Ar (HPFH-I) (-) A (-) A G A C The C to G base change a t -369 could be the result of a Fig 2. Sequence variation in the 6' flanking region of the random mutation at a single base or a gene conversion event "y-globin gene from a patient with HPFH-1. The sequence obinvolving the 5' end of the Ay-globingene. If indeed a gene tained from the 5' flanking region of the "7-globin gene from a conversion had occurred, one might expect to see additional patient with HPFH-1 ("y[HPFH-l]) is compared with the normal base changes a t positions where the Ay(A) and Ay(B) genes "'y-globin gene sequence of the chromosome A ("y[A]) or chromosome B("'Y[B]) frameworks and the "y-globin gene sequence differ (ie, -588, -603, -605, and -61 l ) , depending on the ("y[A]). The "y(HPFH-1 1 gene sequence can be accounted for by a size of the gene segment involved in the conversion (ref 33; "patchy" gene conversion event(s) involving the "y(A) and *y(B) Slightom, personal communication, February 1988). genes, most simply illustrated as the sequence between the solid To obtain sequences from the area further 5' of the bars above. The exact boundaries of the gene conversion event(s) Ay-globin gene, a 5.0 kb StuI fragment containing the are unknown (dashed lines). Table 1. Summary of Sequence Differences in the "7- and "7Globin Genes from a Patient With HPFH-1 l--T--u-- I F - STOLLE ET AL 502 some framework. The remaining three base changes have been identified in a variant of nondeletion HPFH in Seattle.26 To determine whether these three base changes represent mutations that underlie HPFH, cloned or genomic DNAs from patients with deletion or nondeletion HPFH. other unrelated disorders (HbS, @ thalassemia and HbE), and without hemoglobinopathies were amplified using the polymerase chain reaction, applied to a nylon membrane, and hybridized to allele-specificoligonucleotideprobes. Representative results for the sequence variation found at position +2,667 are shown in Fig 3. The results of this analysis (Table 2) indicate that the three base changes are ( I ) usually inherited as a cluster; (2) not found in two nondeletion HPFH alleles; (3) present in a patient homozygous for another major form of deletion HPFH (HPFH-2); but (4) also present in patients with hemoglobinopathiesunrelated to HPFH (ie, HbS) as well as in four individuals without hemoglobinopathies, all of whom had normal HbF levels. Interestingly, sequences corresponding to the Seattle variant were detected in the genomic DNA from 9 of 1 I black individuals, suggesting that these sequences may represent a cluster of polymorphisms common among blacks. However, the presence of the variant sequence in individuals with normal HbF levels indicates that these base changes alone are insufficient to increase Gy- and *y-globin levels and are not likely to contribute to the phenotype of HPFH. DISCUSSION The phenotypic expression of HPFH is variable.'" The distribution of HbF may be heterocellular or pancellular in erythrocytes. The composition of HbF may reflect an increase in predominantly Gy-globin, 'y-globin, or both. The increase in HbF may be associated with large deletions in the @-globin gene cluster, point mutations in the promoter regions of the Gy- or '?-globin genes.' or as yet undefined mutations either linked to the @-globingene cluster or at some distant l o c u ~ . ~ ~ ~ " O A common form of HPFH in American blacks (HPFH-I) is associated with a deletion of approximately 105 kb in the @-globingene cluster, which removes the 6- and @-globin genes and extends over 80 kb 3' to the @-globingene."." HPFH-I is characterized by an increase in both Gy-and "7- Table 2. Hybridization of Allele-Specific Oligonucleotide Probes Corresponding t o the Normal or Variant "7-Globin Gene Sequence t o Clones or Genomic DNA 3 4 5 C A C A G 2276 2451 2667 2276 2451 2667 Single Allele HPFH-1 - - - Nondeletion HPFH ( 202 Oy) + + + HbS GreekBthalassemia + + + Greek HPFH (- 117 A ~ + + - - - ) Genomic DNA 66 thalassemia heterozygote HbS heterozygote HbE heterozygote HPFH-2 homozygote normal normal normal normal + + - - + + + + - - + - + - + - - - - + + + + + + + + + + + + + + + + + + + + + + + + + + - + + Cloned DNA corresponding to a single allele and genomic DNA was polymerase chain reaction-amplifiedfor the region corresponding to the enhancer 3' to the Ay-globin gene and bound to a nylon filter for hybridization to allele-specificoligonucleotide probes. A ( 1 represents hybridizationto an oligonucleotide probe containing the normal or variant base: a ( - 1 represents lack of hybridizationto the probe. The position of the normal or variant base relative to the 'y-globin gene cap site is indicated. With the exception of Greek &thalassemia and Greek HPFH, the DNA examined was that of black individuals. + globin expression (Gy, 50.7% f 4.3%)," an HbF level of approximately 30% in heterozygotes, and pancellular distribution. Feingold and Forget" have identified an enhancerlike sequence in the region 3' to the deletion breakpoint in HPFH-I, which is brought to within 8 to 10 kb of the y-globin genes as a result of the deletion. The enhancer-like region increased the activity of a linked CAT gene when plasmid constructs were transfected into K562 cells. The DNA from this region was also hypomethylated and DNase I hypersensitiveonly in erythroid tissues and contained a large VA R IA NT 2 T Base Number NORMAL 1 Variant SeqUaKa Namal Sequencs DNA Fig 3. Hybridizationof allde-mpdfic ofie0nUd.otides t o polymerasechain raaction amplifik DNA. Cloned DNAs were amplified using primers flanking the 0.75 kb Hindlll fragment located 3' t o the "y-globin gene. The top row was hybridized t o the normal oligonuceotide spanning the +2,667 region, and the bottom row was hybridized t o the variant oligonuceotide spanning the 2,667 region. DNA samples are as follows: Lane 1, HPFH-1; lane 2, nondeletion HPFH (-202 'y1: lane 3, HbS lane 4, Greek &thalassemia; lane 6, Greek nondeletion HPFH ( - 117 "y). + 503 -/-GLOBULIN GENE SEQUENCE IN HPFH open reading frame. The enhancer-like region may serve to maintain the y-globin genes in a transcriptionally active configuration, and, as a result, contribute to the phenotype of HPFH. However, it is also possible that as yet undetected mutations linked to the @-globin gene cluster or at some distant locus may be responsible (in part or in whole) for the increase in y-globin gene expression in HPFH-1. Unlike the mutations identified in nondeletion HPFH, such a mutation(s) would have to account for the increase in both ‘y- and Ay-globin gene expression observed in the deletion form of HPFH. We have examined the sequence of the ‘7- and Ay-globingenes from a patient homozygous for HPFH-I for mutations that might contribute to the phenotype. The ‘7- and Ay-globin genes from an HPFH-1 patient revealed a total of 10 sequence differences compared with published sequences.’,32333 Two of these base changes are located within 230 bp of the ‘7-globin gene poly A signal, and both are present in normal allelic variants of ‘y-globin. Four base changes are located 5’ of the Ay-globin gene and appear to be vestiges of a “patchy” gene conversion event in which an Ay (A)-chromosome was converted to an *y (B)-type gene sequence. Four sequence differences were identified in the putative enhancer located 3‘ to the Ay-globin gene. One sequence change (G-AA) is found in normal Ay-globin genes of a B chromosome framework (Fig 1 and Table 1). The remaining three base changes have also been reported by Gelinas et a126in the 3’ enhancer region in cis to the y-globin genes of a patient heterozygous for nondeletion HPFH (Seattle variant). The presence of an identical cassette of base changes in both deletion and nondeletion forms of HPFH raised the possibility that one or all of these base changes may contribute to the increase in y-globin gene expression observed in these conditions. However, analysis of amplified genomic D N A from a variety of patients with normal or elevated H b F by hybridization to allele-specific oligonucleotide probes failed to detect a correlation between elevated y-globin gene expression and the presence of the three base changes. Furthermore, the presence of these base changes in patients unaffected by HPFH argues against a role for these base changes in increasing y-globin gene expression. While this manuscript was in preparation, two other groups reported similar findings. Han et a142sequenced the 3‘ Ay-globingene enhancer from four individuals with various levels of H b F expression (including one patient with nondeletion HPFH) and found that the presence of the Seattle variant sequence did not necessarily correlate with high H b F levels. Bouhassira et a143analyzed 196 chromosomes from normal individuals of various ethnic groups for the presence or absence of the variant sequence by polymerase amplification of the 3’ Ay-globin gene region followed by allele-specific oligonucleotide hybridization or restriction enzyme digestion. They observed the three base changes in every ethnic group studied. In particular, the changes were present in 100% of native Africans homozygous for HbS, and 93% of American blacks with HbA. I t is of interest to note that our black patient with ‘y -202 nondeletion HPFH does not have these variant sequences. Thus, our data and that of other^^^,^^ suggest that these base changes are not causally related to HPFH, but instead represent clustered polymorphic sites common in the black population. Such a clustering of polymorphic sites has been described previously in the 5’ flanking region of the &globin gene.44 In summary, sequence analysis of the promoter regions of the ‘7- and Ay-globin genes and the 3’ Ay-globin enhancer element from a patient with deletion HPFH failed to reveal a mutation(s) likely to increase both ‘7- and Ay-globin expression. It is possible that as yet unidentified mutations within the /3-globin gene cluster or at some distant site (such as in the gene for a trans-acting factor) contribute to the HPFH phenotype. However, in the absence of other detectable mutations that correlate with increased ‘7- and Ay-globin levels, we conclude that the significant mutation in HPFH-1 is the deletion of over 100 kb of genomic DNA. ACKNOWLEDGMENT The authors gratefully acknowledge Dr Jerry L. Slightom for comparison of the sequence data with chromosome A and chromosome B sequence frameworks, Sina Nasri-Chenijani for assistance with the EMBL-3 genomic cloning, and Nicole E. Sykes for preparation of the manuscript. REFERENCES 1. Collins FS, Weissman SM: The molecular genetics of human hemoglobin. Prog Nucleic Acid Res Mol Biol31:315, 1984 2. Weatherall DJ, Clegg JB: The Thalassemia Syndromes (ed 3). Oxford, UK, Blackwell, 1981 3. Bunn HF, Forget BG: Hemoglobin: Molecular, Genetic and Clinical Aspects. Philadelphia, PA, Saunders, 1986 4. Stamatoyannopoulos G, Nienhuis AW: Hemoglobin switching, in The Molecular Basis of Blood Diseases. Stamatoyannopoulos G, Nienhuis AW, Leder P, Majerus PW (eds): The Molecular Basis of Blood Diseases. Philadelphia, PA, Saunders, pp 66-105, 1987 5. Collins FS, Stoeckert DJ, Serjeant GF, Forget BG, Weissman SM: ‘-/pi hereditary persistence of fetal hemoglobin: Cosmid cloning and identification of a specific mutation 5‘ to the G~ gene. Proc Natl Acad Sci USA 81:4894,1984 6. Collins FS, Meatherall JE, Yamakawa M, Pan J, Weissman SM, Forget BG: A point mutation in the A-/-globingene promoter in Greek hereditary persistence of fetal hemoglobin. Nature 313:325, 1985 7. Gelinas R, Endlich B, Pfeiffer C, Yagi M, Stamatoyannopou10s G: G to A substitution in the distal CCAAT box of the A~ globin gene in Greek hereditary persistence of fetal hemoglobin. Nature 313:323,1985 8. Gelinas R, Bender M, Lotshaw C, Waber P, Kazazian H Jr, Stamatoyannopoulos G: Chinese *Y fetal hemoglobin: C to T substitution at position -196 of the A-/ gene promoter. Blood 67:1777,1986 9. 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