Association of heterocellular HPFH, -i-thalassaemia

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Journal of Medical Genetics, 1984, 21, 263-267

Association of heterocellular HPFH, -i-thalassaemia, and a 0-thalassaemia: haematological and molecular aspects L CIANETTI*, A CARE*, N M SPOS1*, A GIAMPAOLO*, M CALANDRINI*, M PETRINI*, A MASSA*, M MARINUCCI*, F MAVILIO*t, M CECCANTI4, AND G F SASSO+ From *thc Depar tment of -Haemnatology, Istituto Superiore di Sanita; tthe Institute of Experimental Medicine, Consiglio Nazionale delle Ricerch,e; and +the Cattedra di Patologia Speciale Medica II, University La Sapienza, Rome, Italy.

An Italian family in which heterocellular hereditary persistence of fetal haemoglobin (HPFH) interacts with both p3- and 6r-thalassaemia is described. The index case was an 8 year old girl who was presumed to inherit both heterocellular HPFH and r+-thalassaemia from her mother and 6,I-thalassaemia from her father. She was healthy and never needed blood transfusions. The possible contribution of heterocellular HPFH to the less severe expression of the compound 6p,P+thalassaemia heterozygosity is discussed. By DNA analysis the specific 6r-thalassaemia defect on the y6,3 globin gene region has been established. In addition, a previously unreported association of a polymorphic restriction site haplotype with a P+-thalassaemia mutation has been observed. SUMMARY

Hereditary persistence of fetal haemoglobin (HPFH) is a group of non-thalassaemic disorders of human haemoglobin synthesis characterised by Hb F production in adult life.' 2 Two main types of HPFH are commonly recognised, namely (1) pancellular HPFH, in which Hb F synthesis is increased in the whole RBC population, and (2) heterocellular HPFH. tn the latter condition the increased 1-lb F synthesis is restricted to an expanded F cell pool. The molecular defects underlying the former condition are, in most cases, extensive deletions in the y85 globin gene region.' 2 However, very little is known at the present about the molecular basis of the heterocellular forms of HPFH. In these forms no gross structural abnormality has been detected by gene mapping studies on the y8p globin gene region.30 The heterocellular HPFH determinant(s) would act on the erythropoietic bone marrow by enhancing the propensity of the erythroid progenitors to generate erythroblasts with the potential for y chain synthesis, that is, the precursors of F cells, thus increasing the H-b F level in peripheral blood. It is generally recognised that high Hb F levels in adult life may greatly ameliorate the clinical expression of Received for publicaition 7 October 1983. Accepted for publication 28 Decenmber 1983.

a number of inherited disorders of haemoglobin synthesis,' like sickle cell anaemia. In particular, the interaction of heterocellular HPFI4 with r-thalas-

saemia has been reported to be responsible for an almost complete suppression of the clinical symptoms in homozygous 3-thalassaemia.5-8 In the present report we describe a Sicilian family with a previously undescribed interaction of heterocellular HPFH with both 53- and 8p-thalassaemia. The index case, who presumably carried a heterocellular HPFH/P+-thalassaemia gene complex associated with a 8r-thalassaemia determinant, was apparently healthy.

Material and methods

Standard haematological methods were used. Hb A. level was estimated by an elution method after cellulose acetate electrophoresis of haemolysates.' I-b F level was determined by alkali denaturation (FAD)9 or by DEAE-cellulose (DE-52) column chromatography.'0 The intracellular distribution of Hb F was evaluated on peripheral blood smears by indirect immunofluorescence using anti-y chain antibodies.1' Determination of Gy/Gy + Ay globin chain ratios was carried out by densitometric tracing of globin chain electrophoresis on Triton-urea 263


L Ciacitli et ilt

264

polyacrilamide gel slabs.' Chain biosynthesis ratios were determined on fractionated reticulocytes as previously described.High molecular weight DNA was prepared from peripheral blood leucocytes by standard techniques1:3 and digested with several restriction endonucleases according to the supplier's specifications. DNA fragments were separated by 0 8 to I 000 agarose gel electrophoresis and transferred to nitrocellulose filters according to Southern." Filter hybridisation, washing, and autoradiography were carried out as previously described." Plasmid JW 102, JW 151, and JW 101 containing huliman 1, y, and x c-DNA sequences'" were 32p labelled by nick translation to a specific activity of 2 to 6 108 dpm/p.g and used as hybridisation probes.

These findings strongly suggested the presence of a heterocellular H PFH determinant coexistent with ,3-thalassaemia in this subject. The father (1.1) had a normal Hb A., level (2-66%), increased I-b F percentage (7-8 0/), and a thalassaemia like o/3 chain synthesis ratio (1 * 93). By indirect immunofluorescence assay nearly all his RBCs appeared to be F cells. These findings indicated that he was a 8P-thalassaeilia carrier. A second daughter (11.2) showed slightly decreased MCV and MCH values, but normal Hb A.,, Hb F, and serum iron levels. allele was ruled out in all The presence of the family members by peptide mapping carried out on AI

-

Results

The haematological and haemoglobin findings and the results of the in vitro haemoglobin synthesis of the family members are listed in the table. The index case (11.1, fig 1) was an 8 year old girl who showed moderate pallor. The spleen and liver were palpable 4 cm and 2 cm below the costal margin respectively. Her growth was normal and she never received blood transfusions. Her parents and sister were asymptomatic. She showed abnormal RBC indices and morphology. The Hb A.2 level was in the normal range (2 82°, of the total Hb), and Hb F was largely prevailing in peripheral blood (82 1 %), whereas Hb A accounted for 15 1 %. All RBCs were F cells, and the mean Hb F/F cell content was 19 1 pg. The proband showed an unbalanced x/non-x chain synthesis ratio (I 72). The y/non-x chain synthesis ratio (O 76) was roughly of the same order of magnitude as the 1-b F level observed in the red cell haemolysate. Both parents (L. I and 1.2) showed abnormal RBC indices and morphology. In particular, her mother (1.2) was shown to be a '-thalassaemia carrier. In -V), addition, she showed an increased Hb F level (38 consistently higher than that usually observed in - 004 °", n - 32, in 13-thalassaernia carriers (I1 23 our laboratory). H'b F was heterogeneously distributed in RBCs with a high F cell count (15 30)

A

p

+

ha plotypes -

-

-

p

+

5p-thal

+

pth

-

-

A2 '/° Hb F'/o

2 66 7. 77

3 81

F

100

1 5,3

Hb

cells I!,

+

+

5 61

4~~~~~~~2

4 1

II

--pth haplotypes -

-

Hb A2 'IO

Hb F '/o F

cells'/.

5p-thal

-

2.82

2.74 1 93

+

+

4.0

0P-thalassaemia heterozygote

ete rozygote

FIGi

pA

-

82.0 100.0

P- thalassaemia

heterocelHu H PFH

A_ +

+ _

+_

lar

norm

a

Fainil/,' pe(ligi-ee.

[ABLE Hacetn.cito/a/icall(Id g//lobitl c/1lill synthesis (Iclt(1. .SIbject

Age (cc)

1.1

1.2 11.1

11.2

40 38 8 6

RBC

HA) (/1)

15-0

12'4 10.8

12 6

6 52 5 13

4-62 5.48

(p,g)

u'o'z- s MXICHC Gy ,) (g, (l)

67.5

23.0

34-1

290

71.5

24-2

24-4

655 70-3

23.4

338 35 6 32 7

MtC I

Hct

)

( /12/

44.0 36-7 30.3

385

MCH

23.()

'

p,j

y

0-56

0-52

0.04

-

0-65

44.8

0580X14

0-44

40-7

0-93

0-91

0-02

Hb FE cell (pg)

6-0 19-2 10-9


Associationt of heter-ocellular HPFH, F t-thalassaenia, and 8 i°-thalassaemnlia

265

B

A

Gy 6.5--.. _-

5P 5.2 -

3p 3.2 -W

5y 2.5 - 5 3 2.2 - _

_

3S 1 6- _wa

'!

,0

88B

p

S

AY

G

FI(i 2 Electrophoretic patterns (top) of

LCeo RI fragments from a normal sub]ect

B

E

E

E E E

0

B

pA H

HH HH HH

A

A A

22 1.8

88

E

B

B

52

A;A 32

(A) and I.1 (B). DNA y8p globin gene maps (bottom) with the studied restriction sites in normal (PA) and 8P°-thalassaemia chromosomes. In bot/i cases the expected Eco RI fragments are indicated below the map. B= Bam HI, H= Hind III, E= Eco RI, A = A va II. Asterisks above a letter meant polymorphic site.

Eco RI fra gments

E 5 Kbp

- ht 53tha H

HH

-

H

Eco RI tragment

purified y chains. Gy/Gy+A ratio values are listed in the table. The structure of the y8p globin gene region was studied by restriction endonuclease mapping on genomic DNA obtained from peripheral WBCs from all family members. The restriction patterns obtained by Eco RI, Pst 1, and Bgl II digestion of DNA from subject 1. and 11. indicated that a deletion, starting from the 8 IVS-2, typical of the so-called southern ftalian 8,0-thalassaemia,17 18 occurred heterozygously in both of them (fig 2). However, normal restriction patterns were observed in the y8p region in subjects 1.2 and 11.2. In all subjects the chromosomal linkage of the four polymorphic sites studied (Hind III Gy, Hind fIl Ay, Ai'a It 13, and Bamn 141 PI) was established (fig I). In particular, subject I1.2 was shown to have received the 'normal' chromosomes from her parents. The ,3--thalassaemia chromosome in both subjects 1.2 and 11. carried a Hind III Gy Hind 111 Ay- Al'a Il Bamn H l 3 haplotype. The Bani HI map on the x globin gene region ruled out the presence of gross deletions or rearrangements in all subjects. y

,

Discussion A number of cases of compound heterozygosity for 5- and 83-thalassaemia have been reported. 1 19 The clinical expression of this condition is very hetero-

geneous, ranging from severe to moderate anaemia. In most cases the disorder is milder than in homozygous 3-thalassaemia and patients show the lhaematological and clinical picture of thalassaemia intermedia. Although the reasons for this wide clinical variability are not quite clear, some genetic determinants, which are known to reduce the overall globin chain imbalance in red cells, could be regarded as responsible for some, at least, of the milder conditions observed in this compound heterozygosity. x-thalassaemia and heterocellular HPFH have repeatedly been reported, when found in association with homozygous 3-thalassaemia,1 5-8 to produce clinical pictures much milder than Cooley's disease (that is, moderate anaemia without need of transfusion). In the present family, x-thalassaemia can be ruled out on the grounds of globin chain synthesis ratios and genomic DNA mapping. However, heterocellular HPFI1 is reasonably associated in cis with a 3-thalassaemia determinant in one of the patient's parents (1.2). Indeed, analysis of haplotype segregation indicated that the maternal chromosome inherited by the non-thalassaemic daugher (11.2) does not carry HPFH and ~-thalassaemia (normal Hb A. and Hb F levels, normal F cell count). Evidence has been provided that the heterocellular HPFH determinant is, at least in most cases, linked to the y8~globin gene cluster.4-6 8 Therefore, both


266

L Cianetti et al

HPFH and f-thalassaemia determinants could be 3-thalassaemia mutation appears to be a P+ mutation. associated in cis in 1.2. Alternatively, the normal Hb This P+-thalassaemic haplotype has never been F and F cell count observed in subject 11.2 may be described before. It could have arisen either by an accounted for assuming that (1) HPFI4 and P- independent mutation on the same haplotype or by thalassaemia determinants lie in trans in 1.2 and (2) a crossing over within the y8g gene region, involving a recombination event between these determinants is different r+-thalassaemic haplotype. responsible for the normal chromosome in 11.2. Nevertheless, a number of families have been This work was supported in part by CNR grant reported in which heterocellular ffPFH determinant (contribute No 82.02429.04). is linked in cis to a P-thalassaemia mutation, this References situation being not uncommon in southern Italy.5 Weatherall DJ, Clegg JB. The thalassaemia syndromes. 3rd ed. Oxford: Blackwell Scientific Publications, 1981. Thus, it may reasonably be suggested that both 2 Peschle C, Migliaccio G, Migliaccio AR, et al. Haemodeterminants are inherited together by the patient globin switching in humans. In: Dunn C, ed. Current (11.1), although it is not possible to be positive, since concepts in erythropoiesis. London: Wiley, 1983. 3 Jones RW, Old JM, Wood WG, Clegg JB, Weatherall DJ. the expression of 8r-thalassaemia trait would hide Restriction endonuclease maps of the 3-globin gene the haematological expression of HPFH (that is, cluster in the British and Greek forms of HPFH, and for increased Hb F level and F cell percentage). If this is one example of Gyp+HPFH. Br J Haematol 1982;50: the case, the almost complete suppression of any 415-22. 4 Wood WG, Old JM, Darbre PD, Clegg JB, Weatherall severe clinical manifestation in 11. 1 could be attributDJ, McRae IA. The British type of HPFH: cellular and able, in the absence of cx-thalassaemia, to the molecular analysis of increased Hb F production. In: presence of heterocellular 1TPFH determinant in the Weatherall DJ, Fiorelli G, Gorini S, eds. Advances in red cell biology. New York: Raven Press, 1982:249-61. P+-/ 83-thalassaemia condition. 5 Marinucci M, Mavilio F, Gabbianelli M, et al. Cellular In fact, the determinant for heterocellular HPFH mechanisms of increased Hb F production in the interseems to act first by enhancing both the average F action between heterocellular HPFH and P-thalassaemia. of the in and the level marrow cell production bone In: Weatherall DJ, Fiorelli G, Gorini S, eds. Advances in red cell biology. New York: Raven Press, 1982:263-70. physiological response of the erythropoietic compart6 Wood WG, Weatherall DJ, Clegg JB. The interaction of ment to an erythropoietic stimulus. However, when heterocellular hereditary persistence of fetal haemoglobin the erythropoiesis is more stressed, it would also enwith r-thalassaemia and sickle cell anaemia. Nature 1976; the erythroid hance the cellular y chain synthesis in 264:247-9. 7 Marinucci M, Mavilio F, Giuliani A, et al. P-thalassemia precursors. The amount of Hb F produced in this associated with increased Hb F production. Evidence for way would be capable in homozygous r-thalassaemia the existence of a heterocellular hereditary persistence of or in other related thalassaemic conditions of fetal hemoglobin (HPFH) determinant linked to Preducing the globin chain synthesis imbalance in red thalassemia in a Southern Italian population. Hemoglobin 1981 ;5:1-17. cells and to reach haemoglobin (chiefly Hb F) levels 8 Cappellini MD, Fiorelli G, Bernini LF. Interaction beonly slightly below normal in peripheral blood. This tween homozygous PO-thalassaemia and the Swiss type of would be obtained in the absence of severe bone hereditary persistence of fetal haemoglobin. Br J Haematol 1981 ;48:561-72. marrow expansion or dramatic increase of the Pembrey ME, McWade P, Weatherall DJ. Reliable ineffective erythropoiesis.5 routine estimation of small amounts of foetal haemoAs far as Gy/Gy +Ay ratios are concerned, a 0-290 globin by alkali denaturation. J Clin Pathol 1972;25: ratio observed in the patient's father (1.1) is compat738-41. ible with the Gy/Gy +Ay ratio range found in southern '0 Huisman THJ, Jonxis JHP. The hemoglobinopathies. Techniques of identification. New York: Marcel Dekker, Italian 83-thalassaemia (0 142 to 0 317, unpublished 1977. data), but in the absence of AyT chains it is not possible Wood WG, Stamatoyannopoulos G, Lim G, Nute PE. F cells in the adult: normal values and levels in individto conclude that the considerable y chain output uals with hereditary and acquired elevations of Hb F. observed in patient 11. 1 is sustained by both chromoBlood 1975;46:671-82. somes and to what degree. 12 Alter BP, Goff SC, Efremov GD, Gravely E, Huisman It is worthy of note that in both members 1.2 and THJ. Globin chain electrophoresis: a new approach to the determination of the Gy/Ay ratio in fetal haemoglobin 11.1 the f-thalassaemic chromosome carries the and to studies of globin synthesis. Br J Haematol 1980;44: Hind II Gy-, HindIII Ay-, Ava I 3+,Bam HI r3haplotype. This pattern has been reported in 13 527-34. Britten RJ, Graham DE, Henfeld BR. Analysis of association with a G--A substitution in IVS-1 or repeating DNA sequences by reassociation. In: Grossman L, Moldave K, eds. Methods in enzymology. Vol XXIX. IVS-2 5' splice junction, thus giving a PO-thalassaemia York: Academic Press, 1974:363-418. mutation.20 21 In subject 11.1 the r-thalassaemia is 14 New Southern EM. Detection of specific sequences among inherited in trans to a 83 gene deletion, and yet there DNA fragments separated by gel electrophoresis. J Mol is some expression of Hb A (15- 1 ). Therefore, the Biol 1975;98:503-17.


Association of heterocelliular HPFH, r3-thalassaemia, and 8 F-thalassaernia 1; Mavilio F, Giampaolo A, Care A, Sposi NM, Marinucci M. The 83 crossover region in Lepore Boston (887 Glu "116 His) hemoglobinopathy is restricted to a 59 base pairs region around the 5' splice junction of the large globin gene intervening sequence. Blood 1983 ;62:230-3. 16 Wilson JT, Wilson LB, deRiel JK, et al. Insertion of synthetic copies of human globin genes into bacterial plasmids. Nutc leic Acids Res 1978 ;5 :563-81. 17 Ottoleilghi S, Giglioni B, Comi P, et al. Globin gene deletion in HPFH, 80P° thalassaemia and Hb Lepore disease. Natlure 1979;278:654-6. 18 Bernards R, Kooter SM, Flavell RA. Physical mapping of the globin gene deletion in (8,5)0 thalassaemia. Gene 1979 ;6 :265-80. 19 Kattamis C, Metaxotou-Mavromati A, Karomboula K, Nasika E, Lehmann H. The clinical and haematological

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findings in children inheriting two types of thalassaemia: high-A2 type r-thalassaemia, and high-F type or 8pthalassaemia. Br J Haemnatol 1973 ;25:375-84. 20 Giampaolo A, Mavilio F, Massa A, et al. Molecular heterogeneity of thalassaemia in the Italian population. BrJ Haematol 1984;56:79-85. 21 Orkin SH, Kazazian HH Jr, Antonarakis SE, et al. Linkage of P thalassaemia mutation and globin gene polymorphisms with DNA polymorphisms in human 3 globin gene region. Natuire 1982;296 :627-3 1.

Correspondence and requests for reprints to Dr Luciano Cianetti, Laboratorio di Ematologia, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Roma, Italy.


Association of heterocellular HPFH, beta(+)-thalassaemia, and delta beta(0)-thalassaemia: haematological and molecular aspects. L Cianetti, A Care, N M Sposi, et al. J Med Genet 1984 21: 263-267

doi: 10.1136/jmg.21.4.263

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