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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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).
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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.
Thein
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