Prenatal diagnosis of hemoglobin H disease

278

Febrt~ary 1978 The Journal of P E D I A T R I C S

Prenatal diagnosis of hemoglobin H disease Hemoglobin H disease was diagnosed prior to the twenty-second week of gestation in a pregnancy at risk for homozygous a-thalassemia using the technique of DNA-DNA hybridization. Fetal DNA was obtained from amniotic fluid fibroblasts obtained during the thirteenth week of gestation and grown in culture. The fetal fibroblast DNA was hybridized to radioactive a-globin eDNA. The number of a-globin genes present in the fetus was determined by comparing results of hybridization studies on the fetal DNA to similar studies on subjects with well-defined a-thalassemia syndromes and with normal subjects. The diagnosis of hemoglobin H disease was confirmed at birth by studies of the cord blood. This study confirms the ability of DNA-DNA hybridization techniques to distinguish the three-gene deject of hemoglobin H disease from the lethal four-gene defect of homozygous a-thalassemia.

H. M. Koenig, Commander (MC) USN,* T. S. Vedviek, Ph.D., S a n Diego, Calif., A. M . Dozy, M . T . , M. S. Golbus, M . D . , and

Y. W . Kan, M.B:, B.S., S a n Francisco, Calif.

ALPHA-THALASSEMIA is a hereditary disorder of hemoglobin synthesis that occurs with high frequency in Asian populati0ns.l-3 infants with homozygous a-thalassemia are either stillborn in a hydropic state between 30 to 40 weeks' gestation or they die soon after birth. This condition is a major cause of fetal wastage in southeast Asia. In addition, severe maternal toxemia often complicates pregnancies in which the fetus has homozygous athalassemia? From the Department of Pediatrics, Naval Regional Medical Center," Department of Pathology, University of California San Diego," and the Departments of Medicine, Obstetrics and Gynecology, and Laboratory Medicine, University of California, and Hematology Service and Howard Hughes Medical Institute Laboratory, San Francisco General Hospital. Supportedin part by Bureau of Medicine and Surgery Clinical Investigation Program Project 3-16-039," United States Public Health Service Grants AM16666, GM-17702; The National Foundation-March of Dimes; Maternal and Child Health, State of California. The opinions or assertions expressed herein are those of the authors and are not to be construed as official or as reflecting the views of the Navy Department or the naval service at large. *Reprint address: Box 306 N.R.M.C., San Diego, CA 92134.

tlol. 92, No. 2, pp. 278-281

The molecular defects of the a-thalassemia syndromes have recently been delineated in the Asian population. I n each diploid cell the normal synthesis of the a-globin chains of hemogl0bin is directed by four structural genes. The common forms of a-thalassemia syndromes in Asians are due to deletion of one or more of these structural genes. 5-7 Thus, the increasing clinical severity of the silent Abbreviation used RBC: red blood cell DNA: deoxyribonucleic acid. carrier state (a-thalassemia-2), a-thalassemia trait (athalassemia-1), and hemoglobin H disease is the result of deletion of one, two, and three structural a-globin genes, respectively. When all four a-globin structural genes are deleted, the lethal homozygous state results. Since no aglobin chain is produced, "y-globin, the predominant globin chain synthesized during intrauterine development, forms tetramers (7~ or hemoglobin Bart's) that have a high oxygen affinity and are functionally ineffective in oxygen transport. The blood contains a small amount of ~ chain which forms hemoglobin Portland ( ~".,')t2) and, though functioning normally, is present in inadequate quantity to sustain life. The number of a-globin structural genes can be determined by DNA-DNA hybridization techniques#' 8 As all

0022-3476/78/0292-0278500.40/0 9 1978 The C. V. Mosby Co.

Volume 92 Number 2

Prenatal diagnosis o f hemoglobin H disease

80

a thai1

i

I

279

I

I

Hb-H disease

X

60 "10 Bo3 E Co

< Hydrops

a

thai-1

* Normal + Deletion Other possiblegenotypesin offspring

eo

o~

40

E

20

Family

I I Hb-Hdisease I I ~thal'2 Fig. 1. Pedigree of family in this report.

nucleated cells contain the full complement of DNA, any nucleated cells, including amniotic fluid fibroblasts, can be used for these studies2 The probe for a-globin structural genes, a radioactive copy of DNA (cDNA) complementary to a-globin mRNA sequences, is synthesized with reverse transcriptase. When the a-cDNA is incubated under controlled conditions with DNA from human cells, the extent to which the a-cDNA is annealed to the cellular DNA depends on the number of a-globin structural genes present2 We describe herein the use of this technique for prenatal diagnosis in a pregnancy at risk for homozygous a-thalassemia. MATERIALS AND METHODS A Filipino woman with hemoglobin H disease and her husband with a-thalassemia trait had had two previous pregnancies. The first pregnancy terminated at 34 weeks' gestation with a stillborn male infant with homozygous athalassemia. The second pregnancy was uneventful and resulted in a female infant with a-thalassemia trait (Fig. 1). The mother had previously been shown to have only one a-globin structural gene; the father and daughter had two a-globin structural genes. The couple requested prenatal diagnosis because the mother did not wish to carry another affected pregnancy to term. Fifteen weeks after the last menstrual period, amniocentesis was performed. Fetal fibroblasts were cultured for six weeks, and the DNA was extracted and depurinated as previously described" ~; 0.65 mg of DNA were obtained. Peripheral blood white cells from the parents as well as bone marrow from the parents and the child with

l

Controls

o3 D.

tested:

BM

WBC

Father

[3

B

M other

0

Child

/~

Fetus

I

I

I

HYDROPS

Hb-H DISEASE

(~ TH A L-I

0 I

NORMAL

Fig. 2. Percentage of a-cDNA annealed to the DNA's of the fetus, his family, patients with a-thalassemia syndromes, and nonthalassemia controls.

a-thalassemia trait were obtained for DNA extraction. Results of molecular hybridization studies in this family were compared to those of the DNAs obtained from placentas, fetal liver and spleen, bone marrow, and peripheral blood white cells of patients with a spectrum of a-thalassemia syndromes and from normal controls. All specimens were tested using the same cDNA probe. The a-cDNA probe was prepared with reverse transcriptase from 32P-deoxycytidine triphosphate as previously described5 The a-cDNA contained 75 to 80% a-globin sequences; the remainder represented fl-globin sequences. The a-cDNA (9.5 pg 1,000 cpm) was incubated at 78~ for 76 hours in duplicate 15/zl reaction mixtures containing 75/zg of cellular DNA, 0.04 M Tris-HC1 (pH 7.5), 0.5 M NaC1, and 0.001 M Na~ EDTA. 3H-labeled unique sequence DNA, (1,000 cpm) served as an internal control for hybridization. The percentages of the radioactive DNA annealed were quantitated by hydroxylapatite. The single-stranded DNAs were eluted with 0.16M and the double-stranded with 0.4M sodium phosphate at 60~ RESULTS The percentages of a-cDNA hybridized by cellular DNA from the amniotic fluid fibroblasts, the sibling's

280

Koenig et al.

The Journal of Pediatrics February 1978

Table I. Hematologic values in Filipino neonates: normal, alpha-thalassemia trait, and hemoglobin H disease No. Hgb (gm/dl) Normal infants a-thaiassemia trait Hgb H disease

141 6 1

RBC .O'2/,) IMCV( )

16.2 • 0.7 14.8_+ 0.6

4.37 _+ 0.19 5.37 _+ 0.20

12.8

5.61

108 + 3 85 _+ 2 71

Hgb = Hemoglobin;RBC = red bloodcells;MCV = mean corpuscular volume. bone marrow, and the parents' peripheral blood white cells and bone marrow are shown in Fig. 2. The data from control groups with well defined ~-thalassemia syndromes and nonthalassemic individuals are also shown. The father's peripheral blood White cells and bone marrow and the sibling's bone marrow DNA hybridized about 55% of the c~-cDNA, a level comparable to other patients with a-thalassemia trait. DNA from fetal liver and spleen of infants with homozygous a-thalassemia hybridized 25% of the c~-cDNA, whereas DNA from normal controls hybridized 65% of the c~-cDNA. The fetal fibroblast DNA obtained from the amniotic fluid hybridized 42% of the c~cDNA, identical to the amount of a-cDNA hybridized by the DNA from the mother and other patients with hemoglobin H disease. Chromosome analysis on the fibroblasts revealed that the fetus was a male, assuring authentic fetal origin of the fibroblasts. The fetus was believed to have hemoglobin H disease, and the pregnancy was not interrupted. The pregnancy continued uneventfully to term. At the fortieth week a male infant weighing 2,950 gm was delivered spontaneously. Physical examination was remarkable in that the baby was slightly pale, the liver edge was palpable 2 cm below the right costal margin, and the splenic tip was felt 3 cm below the left costal margin. The cord blood hematologic values were: hemoglobin, 12.8 gm/dl; red blood cells, 5.61 • 1012/1; mean corpuscular volume, 71 ft. The severity of the anemia and microcytosis is more evident when compared to hematologic values of normal infants and of infants with ~thalassemia trait (Table I). Review of the infant's peripheral blood smear revealed anisocytosis and poikilocytosis, 100 nucleated RBC per 100 WBC, and Howell-Jolly bodies in many RBC. The reticulocyte count was 7 9 7 x 10~/1 (normal < 200 x 10~/1). Hemoglobin electrophoresis on cellulose acetate demonstrated hemoglobin Bart's and F. The concentration of hemoglobin Bart's was 26% by DEAE Sephadex chromatography. Cord blood RBC were incubated with l~C-leucine and globin chains separated by

carboxymethyl-cellulose chromatography.l~ The ratio of a to non-a (fl + "D globin synthesis was 0.5. These findings confirm the diagnosis of hemoglobin H disease in the newborn infant. DISCUSSION In the case reported here, a fetus at risk for homozygous c~-thalassemia was correctly predicted to have hemoglobin H disease by the twenty-second week of gestation. The genotype presented in this family is unusual, since the combination of c~-thalassemia trait and hemoglobin H disease in the parents may result in offspring with silent carrier a-thalassemia, a-thalassemia trait, hemoglobin H disease, or homozygous c~-thalassemia. In most pregnancies at risk for homozygous a-thalassemia, both parents usually have a-thalassemia trait: the other possible offspring are ~-thalassemia trait and normal. Prenatal diagnosis of a-thalassemia trait with cDNA-DNA hybridization technique has previously been reported2 This study demonstrates that it is possible to distinguish a case of hemoglobin H disease, where only one intact a-globin structural gene is present, from homozygous a-thalassemia, and underscores the sensitivity of this technique. As homozygous a-thalassemia is invariably fatal, it does not pose the chronic medical and economic problems that sickle cell disease and homozygous/?-thalassemia do. In our experience, however, all couples who have previously given birth to a hydropic child, invariably request this procedure because they do not want to complete a similar pregnancy. An additional indication for prenatal diagnosis is the frequency of toxemia associated with such pregnancies. In fact, in Thailand, where the incidence of c~-thalassemia is high, pregnancies in parents who have previously had a hydropic infant have been terminated on the basis of toxemia alone, with the assumption that the fetus has homozygous a-thalassemia. Of course, such a practice may miss some hydropic fetuses as well as result in mistaken abortion of some unaffected fetuses. In a-thalassemia in which the homozygous state is fatal at birth, it is important that a procedure with minimal risks be utilized for prenatal diagnosis so that nonaffected fetuses will be unharmed. The method described herein satisfies this requirement since amniocentesis for fibroblast culture has been shown to be a safe procedure. 11 Although c~-thalassemia can also be diagnosed by fetal blood analysis,12fetal blood sampling, which is associated with significant risk to the fetus, should not be used. la-~4 At present there are very few disorders known to be caused by structural gene deletion. As more of these disorders are discovered and the cDNAs for the structural genes become available, prenatal diagnosis of these conditions by molecular hybridization will be possible.

Volume 92 Number 2

We thank the Office of Program Resources and Logistics, Viral Cancer Program, Viral Oncology, Division of Cancer Cause & Prevention, National Cancer Institute, Bethesda, Md., for the AMV polymerase.

Prenatal diagnosis o f hemoglobin H disease

8.

9.

REFERENCES

1. Wasi P, Na-Nakorn S, Pootrakul S, et al: Alpha- and betathalassemia in Thailand, Ann NY Aead Sci 165:60, 1969. 2. Koenig HM, and Vedvick TS: Alpha-thalassemia in American-born Filipino infants, J PEDIATR 87:756, 1975. 3. Lie-Injo LE: Alpha-chain thalassemia and hydrops fetalis in Malaya: Report of five cases, Blood 20:581, 1962. 4. Na-Nakorn S, and Wasi P: Alpha-thalassemia in northern Thailand, Am J Hum Genet 22:645, 1970. 5. Ottolenghi S, Lanyon WG, Paul J, et al: Gene deletion as the cause of a-thalassemia (hydrops fetalis), Nature 251:389, 1974. 6. Taylor JM, Dozy A, Kan YW, et al: Genetic lesion in homozygous alpha-thalassaemia (hydrops fetalis), Nature 251:392, 1974. 7. Kan YW, Dozy A, Varmus HE, et al: Deletion of a-globin genes in haemoglobin H disease demonstrates multiple aglobin structural loci, Nature 255:255, 1975.

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12. 13.

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Ramirez F, Natta C, O'Donnell JV, et al: Relative numbers of human globin genes assayed with purified a and fi complementary human DNA, Proc Natl Acad Sci USA 72:1550, 1975. Kan YW, Golbus MS, and Dozy AM: Prenatal diagnosis of a-thalassemia: clinical application of molecular hybridization, New Engl J Med 295:1165, 1976. Clegg JB, Naughton MA, and Weatherall D J: An improved method for the characterization of human hemoglobin mutants: identification of a~ fi2 95GLU, hemoglobin-N (Baltimore), Nature 207:945, 1965. NICHD National Registry for Amniocentesis Study Group: Midtrimester amniocentesis for prenatal diagnosis: Safety and accuracy, JAMA 236:1471, !976. Kan YW, Bellevue R, Rieder RF, et al: Prenatal diagnosis of a-thalassemia, Clin Res 22:374A, 1974. Alter BP, Modell CB, Fairweather D, et al: Prenatal diagnosis ofhemoglobinopathies: A review of 15 cases, New Engl J Med 295:1437, 1976. Kan YW, Golbus MS, Trecartin R, et al: Prenatal diagnosis of fi-thalassemia and sickle cell anemia: Experience with 24 cases, Lancet 1:269, 1977.