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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Genetic Heterogeneity in Heterocellular Hereditary Persistence of Fetal Hemoglobin By J.E. Craig, J. Rochette, M. Sampietro, A.O.M. Wilkie, R. Barnetson, C.S.R. Hatton, F. Demenais, and S.L. Thein A large English pedigree in which heterocellular hereditary persistence of fetal hemoglobin (HPFH) segregates is described. b-globin cluster deletions and g gene promoter mutations associated with HPFH have been excluded. Of particular importance in this pedigree is the absence of any cosegregating hemoglobinopathy, thus allowing observation of the segregation pattern of this form of HPFH without the complicating effect of a b-globin gene mutation. Information gained in this study confirms that the extent of elevation of hemoglobin (Hb) F and F cells varies between affected individuals. There are one example of incomplete penetrance and three examples of father-to-son transmission, thus excluding X-linked inheritance. Consistent with previous reports, the most likely mode of inheritance is autosomal codominant. Linkage studies using a b-globin cluster microsatellite show no evidence of linkage to this chromosomal region implicating the presence of trans-acting regulatory factor(s). We have recently mapped one such locus to the chromosome 6q region in a very large Asian-Indian pedigree. Linkage to chromosome 6q in the English pedigree was excluded, thus indicating the presence of genetic heterogeneity in heterocellular HPFH. q 1997 by The American Society of Hematology. T is clearly demonstrated by the striking amelioration of the phenotype of individuals homozygous for b-thalassemia or sickle cell disease who also coinherit a HPFH determinant.9,10 Furthermore, the eventual characterization of the genetic basis for this form of heterocellular HPFH will provide important insights into developmentally regulated gene expression, and may lead to new therapeutic strategies for the hemoglobinopathies. Recently, considerable progress has been made in the mapping of heterocellular HPFH determinants by linkage analysis using DNA polymorphisms. One locus has been mapped by our group to an 11-cM interval at the q22.3q23.1 region in chromosome 6 in a very large Asian-Indian family.11 Another locus associated with variation in F-cell levels in sickle cell disease and normal adults has been mapped to the Xp22.2-p23.3 region.12 In the Asian-Indian family, regression analysis indicates that 90% of the variation in F-cell levels is accounted for by three genetic determinants: the 6q linked gene, b-thalassemia, and XmnI polymorphism in the Gg gene promoter.11 We have investigated an extensive English family with heterocellular HPFH in which the HPFH determinant is inherited without a hemoglobinopathy, thus allowing a clearer impression of the segregation pattern for this type of HPFH. The pattern of transmission of the HPFH trait is strongly suggestive of autosomal codominant inheritance; X-linked inheritance is unlikely, considering that father-to-son transmission has occurred. Linkage with the b-globin cluster is excluded (lod score õ 02) at u less than or equal to .01, and multipoint linkage analysis provides no evidence for linkage to the candidate 6q region. The data indicate that there is genetic heterogeneity in the unlinked HPFH phenotype, and that in addition to the 6q gene, there must be at least one other trans-acting autosomal locus that can exert an influence on Hb F levels in adults. HE PHYSIOLOGIC SWITCH from the production of fetal hemoglobin (Hb F) to the adult form of Hb (Hb A) is usually accomplished by 2 years of age. Inherited conditions in which the level of Hb F remains above the normal adult level (õ1%) with normal red blood cell indices and morphology are referred to as hereditary persistence of fetal hemoglobin (HPFH).1 The pancellular forms due to major deletions of the b-globin gene cluster and those associated with promoter mutations in the g-globin genes are characterized by clearly increased levels of Hb F in heterozygotes and demonstrate a mendelian inheritance as alleles of the b-globin complex on chromosome 11p. However, there is another group characterized by modest elevations of Hb F levels (1% to 4%) distributed in an uneven fashion among the F cells (subset of erythrocytes containing Hb F). In this group of HPFH cases (heterocellular HPFH ), no mutations are identifiable within the b-globin cluster, and in many cases the determinant is not linked to the b complex, implicating the presence of trans-acting factor(s).2-5 Surveys show that the distribution of F-cell values is skewed to the right and that approximately 10% of the normal population have at least 4.5% F cells.6-8 The importance of this condition From the MRC Molecular Hematology Unit and the Department of Clinical Genetics, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK; Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Adelaide, South Australia, Pediatrie I et Genetique Moleculaire, Centre Hospitale-Universitaire (CHU) Amiens, Amiens, France; Istituto di Medicina Interna e Fisiopatologia Medica, Università di Milano, IRCCS Ospedale Maggiore, Milano, Italy; Department of Hematology, Wexham Park Hospital, Slough, UK; and Institut National de la Sante et de la Recherhe Medicale (INSERM) U358, Hôpital Saint-Louis, Paris, France. Submitted October 2, 1996; accepted February 21, 1997. Supported by a Nuffield Dominion Fellowship, a European Community (EC) Senior Fellowship, a Wellcome European Fellowship, and a Wellcome Senior Clinical Fellowship. Address reprint requests to S.L. Thein, MD, MRC Molecular Hematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. 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. q 1997 by The American Society of Hematology. 0006-4971/97/9001-0028$3.00/0 SUBJECTS AND METHODS Hematology. Blood samples were collected (with informed consent) in EDTA as anticoagulant, and full blood cell counts were obtained using an automated cell counter. The percentage of Hb A2 was measured by elution and spectrophotometry after cellulose acetate electrophoresis at pH 8.9, and Hb F by alkaline denaturation. F-cell assays were performed in peripheral blood using a monoclonal mouse anti– g-globin chain antibody by microscopy (2 1 Blood, Vol 90, No 1 (July 1), 1997: pp 428-434 428 AID Blood 0019 / 5h38$$$361 08-14-97 10:32:48 bldal WBS: Blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. GENETIC HETEROGENEITY IN HETEROCELLULAR HPFH 103 red blood cells counted per blood smear) and by fluorescenceactivated cell sorting (FACS) (104 cells counted per assay).13 DNA analysis. DNA was extracted from peripheral blood leukocytes and analyzed for seven restriction fragment length polymorphisms in the b-globin gene cluster, and the b-haplotypes were derived.14 The T-C polymorphism at position 0158 of the Gg-globin gene15 was determined by XmnI restriction analysis of the 5* region of the Gg-globin gene amplified by polymerase chain reaction (PCR).16 PCR was used to specifically amplify the Gg and Ag promoter regions from /50 to 0650 relative to the mRNA cap sites, and the regions were directly sequenced as previously described.16 Genotyping. The family was genotyped for 11 microsatellites (D6S408, D6S407, D6S262, D6S435, D6S457, D6S413, D6S472, D6S975, D6S976, D6S270, and D6S292) in the 6q22-q23 region11 and for a microsatellite between the d- and b-globin genes within the b-globin cluster on chromosome 11p.17 Microsatellites were amplified by PCR and analyzed on denaturing 7 mol/L urea 6% polyacrylamide gels. PCR conditions were as follows: 947C (4 minutes) followed by 35 cycles of 947C (1 minute), 557C (45 seconds), and 727C (45 seconds), and a final extension at 727C for 2 minutes. The separated PCR products were transferred onto positively charged nylon membranes (Hybond N/; Amersham, Amersham, UK) and hybridized with either radiolabeled (CA)n or PCR primers. These oligonucleotides were labeled by 3* end–tailing with a32P-dCTP using calf thymus deoxynucleotidyl terminal transferase and the terminal transferase kit from Boehringer Mannheim (Germany). Hybridizations were performed at 427C for 3 hours in 7% polyethylene glycol (PEG 6000 or 8000) and 10% sodium dodecyl sulfate (SDS). Membranes were washed once in 21 SSC and 0.1% SDS for 10 minutes at room temperature. Confirmation of family relationships. The genetic relationships of the kindred were investigated using a panel of probes known to be specific for hypervariable regions of human DNA. Genomic DNA was completely digested with HinfI and Southern blot hybridized with seven minisatellite probes (MS1, MS2, MS29, MS31, MS43, MS51, and plG3) labeled with 32P-CTP by random priming.18 Nonpaternity was excluded in all cases. Linkage analysis. Two-point lod scores were calculated using the MLINK program of the LINKAGE package.19 Linkage calculations were performed under the assumption of autosomal dominant inheritance. Individuals were assumed to be in the genotypic classes AA, Aa, or aa, where the allele A is responsible for high values of F cells (¢8%). A disease gene frequency of 0.01 was used in the analysis, which takes into account the stringent criteria used in assignation of the high-F phenotype. Hardy-Weinberg equilibrium has been assumed. Three liability classes were designated: class 1, individuals with the AA or Aa genotype were assumed to display the phenotype in all cases; class 2, the assumption was made that individuals of the Aa genotype have a probability of .95 of displaying the phenotype; and class 3, individuals were assumed to be affected and to have the AA genotype. Liability class 1 was assigned to all affected individuals (except III-13, III-14, and III-15, described later) and all unaffected marriage partners. Individuals (other than marriage partners) who were coded as unaffected were assigned to liability class 2 to allow for the fact that they may in fact be of the Aa genotype but do not display the phenotype due to incomplete penetrance. The proband (III-13) and her siblings (III-14 and III15), who had an increased level of F cells severalfold that of their parents who were both affected, were assigned to liability class 3. Analyses were made separately in both sides of the extended family, pedigrees A and B, as well as in the combined pedigree (Fig 1). Pedigree A comprises the left side of the kindred, including the proband (III-13), her siblings (III-14 and III-15), their parents, and relatives of their mother (II-6). Pedigree B comprises the right half AID Blood 0019 / 5h38$$$362 429 of the kindred, including the proband and her siblings, their parents, and relatives of their father (II-7). Multipoint analyses were undertaken with two sets of markers, D6S408-(0.023)-D6S407-(0.033)-D6S262 and D6S262-(0.066)D6S976/D6S270-(0.018)-D6S292, using the program LINKMAP and FASTLINK20,21 in the combined pedigree and in both subpedigrees A and B independently. The figures in parentheses refer to the recombination fractions. RESULTS The English Pedigree A 24-year-old white woman (III-13, Fig 1) was found to have an elevated level of Hb F following a Kleihauer test during her second pregnancy. Hb F remained elevated at 3.5% after the pregnancy and delivery of a normal female infant. The F-cell percentage was determined by immunofluorescence on peripheral blood smears and by flow cytometry to be 47% and 44%, respectively. The parents of the proband and her two younger siblings were studied, and it was found that both her brother (III-15) and sister (III-14) had clearly elevated levels of Hb F (7.1% and 3.3%, respectively). Hb F of the proband’s mother (II-6) was also elevated at 1.3%, as was her F-cell percentage (12% and 16%, by peripheral blood smear and FACS, respectively). The father of the proband (II-7) had an apparently normal level of Hb F (0.5%), but the more sensitive immunostaining procedures yielded reproducibly elevated F-cell values (11% and 9% by peripheral blood smear and FACS). The Gg:Ag ratio of the propositus as determined by Triton-urea gel electrophoresis was approximately 0.5. The Gg:Ag ratio of the siblings of the propositus (III-14 and III-15) was determined by reversephase high-performance liquid chromatography. It was found that the common AgT variant was present, as well as A I g . The ratio of Gg:AgT:AgI was 0.28:0.5:0.22 in the proband’s sister (III-14) and 0.1:0.84:0.06 in her brother (III15). The husband of the propositus (III-12) has normal levels of Hb F (0.4%) and F cells (4% by both methods). They have two daughters (IV-7 and IV-8), of whom only the older was available for study. At the age of 5 years, she has a marked elevation of Hb F (10.8%) in a heterocellular distribution (F cells, 80% and 71% by peripheral blood smear and FACS methods, respectively). The pedigree has been extended as far as possible, and relevant hematologic data are displayed in Fig 1 and Table 1. There is no known consanguinity in the family, and false paternity has been excluded. A total of 33 individuals all older than 5 years from three generations have been studied. No members of the pedigree were anemic. In each case, red blood cell indices were normochromic normocytic, and Hb A2 levels were within the normal range. There was no evidence of b- or a-thalassemia. Further individuals on both sides of the extended family have elevated F-cell percentages, although none are as pronounced as those present in the proband, her two siblings, or her daughter. Gene mapping showed the b-globin gene complex to be intact with no evidence of a deletion within the b cluster. A g gene triplication on one allele was revealed from DNA analysis and confirmed by BglII-g restriction mapping as previously described.22 Nine members of the extended family 08-14-97 10:32:48 bldal WBS: Blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 430 CRAIG ET AL Fig 1. Pedigree of the English family with Hb F and F-cell levels by smear and FACS, b haplotype, and XmnI-Gg site. b haplotype is constructed for 7 RFLPs, HindII-e, HindIII-Gg, HindIII-Ag, HindII-Cb, HindII-3*Cb, AvaII-b, and BamHI-b, and denoted by A (Ï""Ï""""), B ("ÏÏÏÏ""), C (Ï""Ï"""), D (Ï"Ï"""Ï), E (Ï"Ï""""), F ("ÏÏÏÏ""), G (Ï""Ï""Ï), H ("ÏÏÏÏ"Ï), I ("ÏÏÏÏÏ"), and J (Ï""""""). The g gene triplication is found on the b chromosome associated with b haplotype A. were heterozygous for the g gene triplication (ggg) as shown in Fig 1 (b haplotype A) and Table 1. Sequence analysis of the Gg and Ag promoter regions of all five members of the proband’s nuclear family did not show any difference from published normal sequences. Two sequence variations were found: the T-C polymorphism at position 0158 of the Gg gene (detected by XmnI restriction analysis)15,16 and a 4-bp deletion at positions 0221 to 0224 of the Ag gene (detected by Fnu4HI restriction analysis)23-26 (Table 1). Considering all the information available, there is evidence of heterocellular HPFH segregating in this family in the absence of any hemoglobinopathy, although the Hb F value of 7.1% in III-15 and 10.8% in IV-7 is higher than the level normally associated with this form of HPFH. Karyotype analysis was performed on the members of the immediate family of the propositus. No abnormality was found. Inheritance Pattern of Heterocellular HPFH Hb F levels in the 33 family members range from 0.1% to 10.8%, which corresponds to F-cell values of 0.5% to 80%. Four individuals have Hb F levels more than 3.0% and F-cell values more than 40% (the propositus, her two siblings, and her daughter). These individuals have been studied on three separate occasions with consistent results. By contrast, the other affected individuals have Hb F levels of less than 1.5% and F-cell levels ranging from 5% to 21%. There are 15 individuals with F-cell values greater than 8% (according to FACS analysis). In these cases, there is a strong likelihood that a HPFH determinant exists. However, four AID Blood 0019 / 5h38$$$362 individuals are reproducibly in the F-cell range of 4% to 6% (II-10, III-3, III-5, and IV-4), which emphasizes the difficulty in drawing an arbitrary line between individuals unaffected by a HPFH determinant (but at the upper end of the ‘‘normal’’ distribution) and those who are affected by the HPFH determinant but show only a modest elevation of F cells. These individuals have also been studied on two separate occasions, and consistent results were obtained. Statistical analysis using the least-squares method in a previous survey8 of 300 healthy adults has suggested that individuals with at least 4.4% F cells may be considered affected with HPFH. However, in view of the difficulty in drawing an arbitrary cutoff point, for linkage purposes in this study, individuals with F cells more than 4% and less than 8% were classified as unaffected but assigned to liability class 2. Incomplete penetrance. Individual II-2 is a maternal aunt of the propositus and has a Hb F level of 0.2% with Fcell values of 1.5% and 3.0% by the peripheral blood smear and FACS methods, respectively. Her two siblings (II-4 and II-6) have F-cell values of 17.5% and 16% (by FACS), respectively. II-2 has three offspring (by two fathers: II-1 and II-3) who were available for study. Of the three children, one is clearly affected (III-1, F cells 21% by FACS) and the other two have F-cell percentages between 4.5% and 6%. The phenotype of the clearly affected daughter (III-1) is very similar to that of her maternal uncle (II-4) and aunt (II-6), and it therefore seems likely that individual II-2 has the HPFH genotype and has passed it to her daughter without expressing the phenotype herself. The phenotypes of the 08-14-97 10:32:48 bldal WBS: Blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. GENETIC HETEROGENEITY IN HETEROCELLULAR HPFH 431 Table 1. Hematologic Data of Family Members F Cells (%) Pedigree No. II-1 II-2 II-3 II-4 II-5 II-6 II-7 II-8 II-9 II-10 II-11 III-1 III-3 III-5 III-6 III-7 III-9 III-10 III-11 III-12 III-13 III-14 III-15 III-16 III-17 III-19 III-20 III-21 IV-2 IV-4 IV-5 IV-6 IV-7 Sex/Age Hb (g/dL) Hb F (%) Hb A2 (%) Smear FACS Xmn I-Gg A g-bp Deletion M/64 F/62 M/70 M/58 F/57 F/48 M/48 F/54 M/51 F/50 M/53 F/40 M/44 F/16 M/36 F/34 F/33 M/32 F/33 M/36 F/24 F/22 M/17 F/18 M/12 F/28 M/27 M/25 M/11 F/16 F/15 M/8 F/5 15.8 14.4 15.4 14.8 14.5 14.5 14.9 13.1 14.9 13.2 17.0 14.5 15.8 14.0 16.3 14.0 12.3 16.3 11.1 15.2 14.1 14.1 14.9 13.8 12.4 12.6 17.1 15.5 13.4 14.3 13.5 12.9 12.6 0.4 0.2 0.3 0.9 0.7 1.3 0.5 0.8 0.3 0.3 0.1 1.0 0.4 0.4 1.4 0.4 0.9 0.3 0.7 0.4 3.5 3.3 7.1 0.2 0.2 0.2 0.2 0.1 0.9 0.6 0.9 1.0 10.8 2.3 2.2 2.2 3.0 2.8 3.0 2.5 2.3 2.5 2.4 2.5 1.9 2.1 2.2 2.5 2.8 3.0 2.2 2.5 2.8 2.8 2.9 2.7 2.5 2.4 2.9 2.4 2.4 2.5 3.0 2.5 2.6 2.9 4.5 1.5 2.0 14 3.5 12 11 12 1.0 4.0 0.5 19 5.0 4.5 17 3.0 6.5 0.5 8.5 4.0 47 40 75 2.0 2.0 3.0 2.5 0.5 8.0 4.0 8.0 6.5 80 2.0 3.0 4.5 17.5 4.0 16 9 12 2.0 4.5 1.5 21 6.0 6.0 20 3.0 10 1.5 9.0 4.0 44 44 57 4.0 1.5 3.5 4.0 0.5 8.0 4.5 8.0 10 71 //0 //0 //0 /// 0/0 //0 //0 //0 //0 //0 0/0 /// //0 /// //0 //0 //0 0/0 //0 /// //0 /// /// /// //0 0/0 //0 0/0 0/0 0/0 /// 0/0 /// 0/0 //0 0/0 0/0 0/0 //0 00*/0 00*/0 0/0 00*// //0 0/0 0/0 0/0 0/0 0/0 0/0 //0 0/0 0/0 00*// 00*/0 00*/0 00*/0 0/0 /// 00*// /// 0/0 //0 0/0 0/0 00*/0 Presence of the 4-bp deletion at positions 0221 to 0224 of the Ag gene is noted by /. * Denotes b cluster allele carrying the g gene triplication (ggg). individuals concerned were reproducible. There are no other examples of incomplete penetrance in this pedigree. There is father-to-son transmission of HPFH. There are three occasions in this pedigree in which an affected male has had children who were available for study (II-4, II-7, and III-6). In each case, there has been an affected son (II4 to III-6, III-6 to IV-2, and II-7 to III-15). II-7 and his son III-15 should probably not be considered as evidence of father-to-son transmission, as the HPFH determinant could have been passed to III-15 by his affected mother (II-6). However, the case of II-4 (F cells 17.5% by FACS) transmitting the trait to his son III-6 (F cells 20% by FACS) is good evidence against X-linkage. The phenotype segregates independently of the b-globin complex but the Xmn I-Gg polymorphism may be involved in expression of the trait. The b haplotypes, Ag4-bp deletion polymorphism, and the g gene triplication served as informative markers for segregation of the b-globin complex, and evidence for independent segregation of the HPFH determinant was provided in several instances. The propositus (III-13), her two siblings (III-14 and III15), her daughter (IV-7), her father (II-7), and her paternal AID Blood 0019 / 5h38$$$362 aunt (II-8) are all clearly affected, and all are carrying the g gene triplication associated with haplotype A (Fig 1). However, individuals III-16, II-10, and III-20 are also carrying the same b allele and are unaffected. Thus, there is good evidence that the phenotype is not linked to the g gene triplication haplotype. It has been shown previously that the g gene triplication arrangement is not associated with increased Hb F levels.22,27 In the other half of the pedigree, inspection of Fig 1 shows that if the HPFH determinant behaves as an allele of the bglobin complex, it must be carried on a chromosome associated with either the C or D haplotype. Of the affected family members, one (III-13) has the C haplotype, eight have the D haplotype, and one has both (II-6). In three instances (III13 to IV-7, III-9 to IV-6, and III-6 to IV-2), transmission of the HPFH phenotype has occurred without the C or D haplotype. XmnI-Gg polymorphism has been shown to influence the level of F cells in normal individuals.6 In this pedigree, while 13 of 15 affected individuals are either XmnI-Gg /// or //0, the other two affected individuals (IV-2 and IV-6) are XmnIG g 0/0, thereby providing evidence that the phenotype in 08-14-97 10:32:48 bldal WBS: Blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 432 CRAIG ET AL Table 2. Two-Point Lod Score for the Microsatellite in the b-Globin Gene Cluster and the Chromosome 6q Markers in the Combined Pedigree and the Two Halves, Pedigrees A and B Lod Score at Recombination Fraction (u) of Marker b cluster Combined Pedigree A Pedigree B D6S408 Combined Pedigree A Pedigree B D6S407 Combined Pedigree A Pedigree B D6S262 Combined Pedigree A Pedigree B D6S435 Combined Pedigree A Pedigree B D6S457 Combined Pedigree A Pedigree B D6S413 Combined Pedigree A Pedigree B D6S472 Combined Pedigree A Pedigree B D6S975 Combined Pedigree A Pedigree B D6S976/D6S270 Combined Pedigree A Pedigree B D6S292 Combined Pedigree A Pedigree B .00 .01 .05 .10 .20 .30 .40 0infini 0infini 0.35 02.17 02.51 0.34 00.88 01.18 0.30 00.42 00.67 0.25 00.11 00.27 0.16 00.03 00.11 0.08 00.02 00.04 0.02 04.24 03.05 03.34 02.40 01.08 01.94 00.76 00.06 00.84 00.15 0.27 00.38 0.21 0.38 00.04 0.22 0.28 0.04 0.12 0.14 0.02 0infini 0infini 03.06 04.77 03.62 01.92 02.93 02.10 01.01 01.94 01.37 00.55 00.83 00.56 00.15 00.28 00.17 00.02 00.05 00.02 0.01 0infini 0infini 05.30 04.41 03.01 02.17 01.93 01.21 00.90 00.89 00.43 00.42 00.06 0.13 00.07 0.15 0.22 0.02 0.11 0.12 0.02 0infini 0infini 07.48 05.31 03.23 02.85 02.53 01.34 01.37 01.33 00.57 00.74 00.37 00.03 00.22 00.07 0.06 00.04 00.00 0.03 0.00 05.89 00.25 07.44 02.33 00.26 02.85 01.39 00.20 01.37 00.82 00.05 00.74 00.18 0.16 00.22 0.05 0.19 00.04 0.07 0.10 0.00 0infini 0infini 02.63 03.06 02.83 01.58 02.12 01.94 00.89 01.49 01.36 00.58 00.71 00.64 00.26 00.28 00.25 00.11 00.07 00.06 00.03 0infini 0infini 02.70 03.35 02.24 01.38 01.79 01.37 00.72 01.11 00.85 00.43 00.44 00.32 00.17 00.12 00.08 00.06 0.00 0.01 00.01 0infini 0infini 05.00 04.29 02.22 01.49 02.01 00.82 00.66 01.02 00.26 00.30 00.23 0.12 00.03 0.02 0.15 0.04 0.03 0.06 0.02 05.69 00.05 05.04 01.35 0.73 01.49 00.00 1.19 00.66 0.49 1.25 00.30 0.72 1.06 00.03 0.58 0.72 0.04 0.30 0.33 0.02 04.14 01.30 02.88 02.46 01.21 01.54 01.38 00.86 00.82 00.76 00.47 00.50 00.12 0.02 00.20 0.12 0.17 00.07 0.13 0.14 00.01 this family is not simply (or completely) related to the presence of the XmnI-Gg site. It is likely that the extent to which the HPFH phenotype is expressed is dependent on the XmnIG g genotype (as we have previously demonstrated in a large Asian-Indian pedigree4). Linkage Analysis With Polymorphic Markers on 6q and the b-Globin Complex Two-point Lod scores were determined between the phenotype and the polymorphic markers in the 6q22-q23 region and in the b-globin complex (Table 2). Since several affected individuals in pedigree A were homozygous for the marker AID Blood 0019 / 5h38$$$362 D6S976 that is tightly linked to marker D6S270, haplotypes were generated for these two markers and analysis was performed using the D6S976/D6S270 haplotype to increase the informativeness for linkage. Individuals with F-cell values of at least 8.0% were considered to be affected and assigned to liability class 1, except for individuals III-13, III-14, and III-15, who have very high F-cell levels and were assumed to have inherited the HPFH determinants from both parents. Individuals III-13, III-14, and III-15 were assigned to liability class 3. Individuals II-10, III-3, III-5, and IV-4 have Fcell values of 4.5% to 6.0% as determined by FACS. These individuals were assigned to unaffected status (liability class 2). The phenotype assigned to the individuals is indicated 08-14-97 10:32:48 bldal WBS: Blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. GENETIC HETEROGENEITY IN HETEROCELLULAR HPFH 433 Fig 2. Multipoint Lod scores for the location of the HPFH locus in relation to 2 sets of markers in the 6q22.3-q23.2 region: (A) D6S408, D6S407, D6S262 and (B) D6S262, D6S976/D6S270, D6S292. Results are plotted as a function of distance from D6S408 (A) or D6S262 (B). Analyses were undertaken in the combined pedigree and independently in each half, pedigree A and pedigree B. Recombination fractions were converted to cM using the Haldane map function. in Fig 1. No adjustment has been made for sex, or the XmnIG g status of individuals in this preliminary study. Linkage with the b-globin cluster is excluded in the combined pedigree and pedigree A (Lod score õ 02) at u less than .01 (Table 2). The data suggest that the genetic determinant causing HPFH is located outside the b-globin gene complex and support previous investigations of the pedigree in which no mutation associated with HPFH has been found in the b-globin cluster. The hypothesis of linkage of the HPFH phenotype to the 6q22.3-q23.2 region was similarly tested. Multipoint analysis provides no evidence for linkage to the 6q candidate region. Multilocus linkage analysis was undertaken in pedigrees A and B both independently and combined (Fig 2). Calculations were confined to four-point analysis using the HPFH locus and two sets of markers in the 6q region—D6S408, D6S407, and D6S262 and D6S262, D6S976/D6S270, and D6S292. The results confirm that there is no evidence for the location of the HPFH gene within the 6q region spanned by D6S408 and D6S292. DISCUSSION The mode of inheritance of unlinked HPFH has been variously described as autosomal dominant, autosomal codominant, X-linked, and polygenic.28,29 An extended English family with heterocellular HPFH has been studied in an effort to gain insight into the phenotype and inheritance pattern of this condition. The family presented is larger than other published pedigrees with heterocellular HPFH segregating in the absence of any hemoglobinopathy despite which a complex situation has emerged. In this English pedigree, Xlinkage is unlikely considering that father-to-son transmission has occurred. The pattern of transmission from II-4 to AID Blood 0019 / 5h38$$$362 III-6 to IV-2 (in each case, a male with an unaffected partner) is suggestive of autosomal dominant inheritance. However, there are complexities that remain unexplained, such as variability in the extent of Hb F and F-cell elevation (variable expressivity) and incomplete penetrance. The tremendous variability in the elevation of Hb F and F cells is not unique to this family, but is also previously observed in other families with HPFH.4 The HPFH is not linked to the b-globin gene complex, although the XmnI-Gg site appears to modify the phenotype; the most markedly affected individuals have at least one XmnI-Gg site. However, the association between affected status and the presence of this polymorphism is not absolute. Autosomal codominant transmission is a strong possibility and would explain the more pronounced elevations of Hb F and F cells observed in individuals III-13, III14, and III-15, who would be considered homozygotes or compound heterozygotes for two HPFH determinants. Their parents (II-6 and II-7) are both affected with modest elevations of F cells. It is not possible in this family to determine if HPFH determinants are allelic or nonallelic. The limited published data regarding heterocellular HPFH in whites in the absence of hemoglobinopathies support the results obtained in this family. The original study by Marti30 and the population survey by Zago et al7 established that the Hb F (and F cell) levels of normal individuals are genetically determined. They traced the parents and siblings of probands with Hb F levels at the upper limits of the population range. In the majority of cases, one of the parents had a similarly increased Hb F (or F cell) level. In both studies, there was at least one example in which neither parent displayed elevated levels of Hb F. Assuming true paternity, this may represent incomplete penetrance of the trait, as found on one occasion in the English pedigree. The hypothesis that heterocellular HPFH in this English 08-14-97 10:32:48 bldal WBS: Blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 434 CRAIG ET AL pedigree is determined by the same gene(s) in 6q responsible for heterocellular HPFH in the large Asian-Indian pedigree11 has been examined. The results obtained strongly suggest that this region is an unlikely candidate for a locus associated with the HPFH phenotype in the English pedigree. The results of linkage analysis using the b-globin cluster microsatellite marker17 confirm the absence of linkage between the b cluster and the HPFH phenotype. It was hoped that the clear-cut phenotype of the proband in this pedigree would be present in other members of the extended family. However, the situation as described has established that this form of HPFH is indeed complex and genetically heterogeneous and that, apart from the b-globin locus and the 6q gene, there must be at least one other autosomal locus that controls Hb F levels in adults. The underlying genetic heterogeneity underscores the importance of analyzing data from one large pedigree, since interpretation of linkage analyses involving several different small families is difficult and could be misleading. ACKNOWLEDGMENT We thank Liz Rose and Milly Graver for preparation of the manuscript, Professor Peter Beverley for permission to use the anti – g-globin chain antibody, and Professor Sir D.J. Weatherall for encouragement and support. Linkage analyses were undertaken using programs provided by the UK Human Genome Mapping Project Resource Centre (funded by the MRC). REFERENCES 1. Stamatoyannopoulos G, Nienhuis AW, Majerus PW, Varmus E: The Molecular Basis of Blood Diseases. Philadelphia, PA, Saunders, 1994 2. Gianni AM, Bregni M, Cappellini MD, Fiorelli G, Taramelli R, Giglioni B, Comi P, Ottolenghi S: A gene controlling fetal hemoglobin expression in adults is not linked to the non– a globin cluster. EMBO J 2:921, 1983 3. Martinez G, Novelletto A, Di Rienzo A, Felicetti L, Colombo B: A case of hereditary persistence of fetal hemoglobin caused by a gene not linked to the b-globin cluster. Hum Genet 82:335, 1989 4. Thein SL, Sampietro M, Rohde K, Rochette J, Weatherall DJ, Lathrop GM, Demenais F: Detection of a major gene for heterocellular hereditary persistence of fetal hemoglobin after accounting for genetic modifiers. Am J Hum Genet 54:214, 1994 5. Giampaolo A, Mavilio F, Sposi NM, Care A, Massa A, Cianetti L, Petrini M, Russo R, Capellini MD, Marinucci M: Heterocellular HPFH: Molecular mechanisms of abnormal g gene expression in association with b thalassemia and linkage relationship with the b globin gene cluster. Hum Genet 66:151, 1984 6. Sampietro M, Thein SL, Contreras M, Pazmany L: Variation of HbF and F-cell number with the G-g XmnI (C-T) polymorphism in normal individuals. Blood 79:832, 1992 7. Zago MA, Wood WG, Clegg JB, Weatherall DJ, O’Sullivan M, Gunson H: Genetic control of F cells in human adults. Blood 53:977, 1979 8. Miyoshi K, Kaneto Y, Kawai H, Ohchi H, Niki S, Hasegawa K, Shirakami A, Yamano T: X-linked dominant control of F-cells in normal adult life: Characterization of the Swiss type as hereditary persistence of fetal hemoglobin regulated dominantly by gene(s) on X chromosome. Blood 72:1854, 1988 9. Thein SL, Weatherall DJ: A non-deletion hereditary persistence of fetal hemoglobin (HPFH) determinant not linked to the bglobin gene complex, in Stamatoyannopoulos G, Nienhuis AW, AID Blood 0019 / 5h38$$$362 (eds): Hemoglobin Switching, Part B: Cellular and Molecular Mechanisms. New York, NY, Liss, 1989, p 97 10. Cappellini MD, Fiorelli G, Bernini LF: Interaction between homozygous bo thalassaemia and the Swiss type of hereditary persistence of fetal haemoglobin. Br J Haematol 48:561, 1981 11. Craig JE, Rochette J, Fisher CA, Weatherall DJ, Marc S, Lathrop GM, Demenais F, Thein SL: Dissecting the loci controlling fetal haemoglobin production on chromosomes 11p and 6q by the regressive approach. Nat Genet 12:58, 1996 12. Dover GJ, Smith KD, Chang YC, Purvis S, Mays A, Meyers DA, Sheils C, Serjeant G: Fetal hemoglobin levels in sickle cell disease and normal individuals are partially controlled by an Xlinked gene located at Xp22.2. Blood 80:816, 1992 13. Thorpe SJ, Thein SL, Sampietro M, Craig JE, Mahon B, Huehns ER: Immunochemical estimation of haemoglobin types in red blood cells by FACS analysis. Br J Haematol 87:125, 1994 14. Antonarakis SE, Boehm CD, Giardinia PJV, Kazazian HHJ: Non-random association of polymorphic restriction sites in the bglobin gene cluster. Proc Natl Acad Sci USA 79:137, 1982 15. Gilman JG, Huisman THJ: DNA sequence variation associated with elevated fetal Gg globin production. Blood 66:783, 1985 16. Craig JE, Sheerin SM, Barnetson R, Thein SL: The molecular basis of HPFH in a British family identified by heteroduplex formation. Br J Haematol 84:106, 1993 17. Tautz D: Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res 17:6463, 1989 18. Wong Z, Wilson V, Patel I, Povey S, Jeffreys AJ: Characterization of a panel of highly variable minisatellites cloned from human DNA. Ann Hum Genet 51:269, 1987 19. Lathrop GM, Lalouel JM, Julier C, Ott J: Multilocus linkage analysis in humans: Detection of linkage and estimation of recombination. Am J Hum Genet 37:482, 1985 20. Lathrop GM, Lalouel JM, Julier C, Ott J: Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 81:3443, 1984 21. Cottingham RW Jr, Idury RM, Schaffer AA: Faster sequential genetic linkage computations. Am J Hum Genet 53:252, 1993 22. Hill AVS, Bowden DK, Weatherall DJ, Clegg JB: Chromosomes with one, two, three, and four fetal globins: Molecular and hematologic analysis. Blood 67:1611, 1986 23. Gilman G, Johnson ME, Mishima N: Four base-pair DNA deletion in human Ag globin gene promoter associated with low Ag expression in adults. Br J Haematol 68:455, 1988 24. Manca L, Cocco E, Gallisai D, Masala B, Gilman JG: Diminished AgT fetal globin levels in Sardinian haplotype II bo-thalassaemia patients are associated with a four base pair deletion in the AgT promoter. Br J Haematol 78:105, 1991 25. Beldjord C, Ducrocq R, Nadifi S, Lapoumeroulie C, Elion J, Labie D: A haplotype-linked four base pair deletion upstream of the A g globin gene coincides with decreased gene expression. Hum Genet 89:625, 1992 26. Gilman JG, Josifovska O, Erlingsson S, Milner PF, Nagel RL: Direct demonstration that the AgT globin gene is linked to the 4 bp promoter deletion in the bA chromosome of sickle cell traits. Am J Hematol 43:312, 1993 27. Trent RJ, Bowden DK, Old JM, Wainscoat JS, Clegg JB, Weatherall DJ: A novel rearrangement of the human b-like globin gene cluster. Nucleic Acids Res 9:6723, 1981 28. Wood WG: Increased HbF in adult life. Bailliere’s Clin Haematol 6:177-213, 1993 29. Rochette J, Craig JE, Thein SL: Fetal hemoglobin levels in adults. Blood Rev 8:213, 1994 30. Marti HR: Normale und anormale mensliche Haemoglobine. Berlin, Germany, Springer Verlag, 1963 08-14-97 10:32:48 bldal WBS: Blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1997 90: 428-434 Genetic Heterogeneity in Heterocellular Hereditary Persistence of Fetal Hemoglobin J.E. Craig, J. Rochette, M. Sampietro, A.O.M. Wilkie, R. Barnetson, C.S.R. Hatton, F. Demenais and S.L. 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