Prenatal Diagnosis of Thalassemia in South China" JIZENG ZHANG,b SHIPING CAIbJ AND YUET WAI K A N ' J ~ , ~ bNan Fang Hospital First Military Medical University S h a h 510515 Guangzhou, Guangdong China and CDepartmentsof Medicine and Laboratory Medicine, and Howard Hughes Medical Institute Laboratory University of California San Francisco, California 94143-0724
INTRODUCTION Both a- and b-thalassemias are common genetic disorders in South China. The gene frequency of each of these disorders is estimated to be 3-5% in the province of Guangdong, which has a total population of over 50 million. The gene frequencies in other provinces in South China are similar or even higher. It is now possible to obtain cells of fetal origin either by amniocentesis or by chorionic villus biopsy. In addition, recent advances in molecular biology have simplified considerably the DNA diagnostic techniques for the thalassemias.' A prenatal diagnosis laboratory for thalassemias was established in the Nan Fang Hospital of the First Military Medical University in Guangzhou, the capital of Guangdong Province, in South China. This laboratory serves Guangdong, Guangxi, Fujian, and Hainan provinces. This paper reports the results of the diagnostic work carried out in this laboratory during 1989.
DIAGNOSIS OF a-THALASSEMJA The a-thalassemia of clinical significance in South China is usually due to the deletion of the Southeast Asian type, involving all +a- and a-globin genes on the short arm of chromosome 16. This deletion is commonly referred to as --SEA/ and can be clearly demonstrated by Southern blot analysis.' In 1987, Chehab and colleagues3 devised a polymerase chain reaction- (PCR) based method for the diagnosis of homozygous a-thalassemia, utilizing primers which would amplify 136 bp of DNA normally present between the +al- and a2-globin genes. This DNA
aThis work was supported in part by the Guandong Provincial Scientific Committee and NIH Grant DK 16666. dY. W. Kan is an investigator of the Howard Hughes Medical Institute. eAddress correspondence to Dr. Y. W. Kan, Howard Hughes Medical Institute, U-426, University of California, San Francisco, CA 94143-0724. 264
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segment is deleted in a-thalassemia of the --SEA/ type. Concomitantly, a 110-bp DNA fragment of the p-globin gene is also amplified, which serves as the control for the PCR. In homozygous a-thalassemia, otherwise known as hemoglobin (Hb) Bart's hydrops fetalis syndrome, the 110-bp P-globin gene fragment is present after PCR, whereas the 136-bp a-globin gene fragment is absent.' This method, which is relatively simple and rapid, has been adopted by our laboratory as the diagnostic test. We have performed 38 prenatal diagnoses on pregnancies at risk for homozygous a-thalassemia, and 5 fetuses with homozygous a-thalassemia were identified.
DIAGNOSIS OF P-THALASSEMIA There are now 15 known P-thalassemia point mutations in the Chinese p ~ p u l a t i o n .In ~ order to identify the more commonly encountered mutations in South China, we have studied 236 chromosomes with P-thalassemia by the technique of allele-specific oligonucleotide hybridization to amplified fragments of the P-globin gene. The data indicate that four mutations, a 4-bp deletion in codon 41-42 (-CTTT), a C+T mutation in IVS-2 at position 654, an A+T nonsense mutation in codon 17 and an A+G mutation at TATA box position -28, account for over 90% of all the mutant alleles studied (TABLE 1). For prenatal diagnosis of pregnancies at risk for homozygous or compound TABLE 1.
B-Thalassemia Mutations and Their Frequencies in South China Mutation" Codon 4 1 4 2 (- CTTT) IVS-2 nt 654 (C-+T) Codon 17 (A+T) TATA box nt -28 ( A & ) TATA box nt -30 (T+C) Codon 71-72 (+A)
Frequency (%) 49.6 18.8 15.8 9.0 1.5 0.8
"Numberof chromosomes = 236. nt, nucleotide.
heterozygous P-thalassemias, DNA was extracted from the peripheral blood cells of the couples under investigation. DNA was also extracted from chorionic villi obtained at 7-11 weeks of gestation or from amniotic fluid cells obtained at 16-20 weeks of gestation. In vitro amplification of DNA was performed as described using two sets of primers to amplify the P-globin gene regions5Amplified DNA was dotted onto nylon filters, which were then hybridized with horseradish peroxidase- (HRP) labeled allele-specific oligonucleotide probes. Six pairs of probes were used, four corresponding to the four common P-thalassemia mutations found in the Chinese, as mentioned above, as well as pairs of probes for the frameshift mutation in codon 71-72 (+A) and the G-C mutation in IVS-1 at position 5. The presence of an oligonucleotide hybridized to amplified DNA is detected by histochemical staining for presence of the enzyme.6 Thirty-four cases of prenatal diagnoses of P-thalassemia were performed. The results are shown in TABLE2. Nine fetuses inherited p-thalassemia alleles from both of their parents.
ANNALS NEW YORK ACADEMY OF SCIENCES
266
DIAGNOSIS OF P-THALASSEMZA BY DNA SEQUENCING When the P-thalassemia mutation is not detected with oligonucleotide probes specific for the six common mutations, the entire P-globin gene can be amplified and sequenced directly to identify the mutation. During the course of prenatal diagnosis for P-thalassemia in a Fujian couple in 1988, we encountered a mutation that was not detectable by oligonucleotides for the Chinese mutations then known. Amplification of the p-globin gene and direct D N A sequencing revealed a previously undescribed T+C TATA box mutation, which was carried by the father but was not inherited by the fetus. Even in this complicated case, prenatal diagnosis was accomplished within two weeks.'
DISCUSSION Carrier detection, genetic counseling, and prenatal diagnosis are indispensable components in the prevention of thalassemia major among populations in which the gene frequency for thalassemia is high, as it is in South China. The introduction of the PCR technique offers several advantages over previous prenatal diagnostic TABLE 2.
Prenatal Diagnosis in 34 Fetuses at Risk of p-Thalassemia
Diagnosis Normal Heterozygote (total)
Mutations -
fl
5 20 11
Codon 41-42 1VS-2nt 654 Codon 17 TATA box nt -28
3
4 2
Homozygote or Compound Heterozygote (total)
9
Codon 4142iIVS-2 nt 654 Codon 4142icodon 17 Codon 41-42icodon 41-42 Codon 4142RATA box nt
-- 28
3 3 2 1
methods.""' In vitro D N A amplification can be performed rapidly. The increased number of target sequences following amplification permits the use of less sensitive probes, i.e., 3SS-labeled oligonucleotides or non-radioactive probes such as the horseradish peroxidase-labeled oligonucleotides used in the present study. "Plabeled oligonucleotides, despite their short half-life, can also be used for u p to two to three months. Thus, the PCR technique has significantly simplified the D N A diagnostic procedure. More recently, we have shown that denaturing gradient gel electrophoresis also offers another non-radioactive means of detecting multiple mutations for P-thalassemias and other genetic disorders." The success of implementing carrier detection and prenatal diagnosis in decreasing significantly the number of live births affected with thalassemia major has been amply demonstrated in a number of Mediterranean regions.'* The present report clearly demonstrates that prenatal diagnosis for both a- and P-thalassemias can be successfuliy carried out in South China. With the appropriate funding and resources,
ZHANG et al.: PRENATAL DIAGNOSIS IN SOUTH CHINA
267
a more comprehensive program on screening, genetic counseling, and prenatal diagnosis can be established in South China. With these genetic services in place, it is anticipated that much progress can be made in the prevention of thalassemia major in South China. ACKNOWLEDGMENT We thank Mrs. A. Folkes for preparation of the manuscript. REFERENCES 1.
2. 3. 4. 5.
6. 7.
8. 9. 10.
11.
12.
ALTER,B. P. 1984. Advances in the prenatal diagnosis of hematologic diseases. Blood 6 4 329-340. A. 0. M. WILKIE, I. M. PRETORIUS, A. P. JARMAN & D. J. HIGGS,D. R., M. A. VICKERS, WEATHERALL. 1989. A review of the molecular genetics of the human a-globin gene cluster. Blood 73: 1081-1104. CHEHAB,F. F., M. DOHERTY, S. P. CAI,Y. W. KAN,S. COOPER& E. M. RUBIN.1987. Detection of sickle cell anemia and thalassemia. Nature 329 293-294. KAZAZIAN,H. H., JR. 1990. The thalassemia syndromes: Molecular basis and prenatal diagnosis in 1989. Semin. Hematol. 27: 209-228. CAI,S. P., J. Z. ZHANG, D. H. HUANG, Z. X. WANC& Y . W. KAN.1988. A simple approach to prenatal diagnosis of P-thalassemia in a geographic area where multiple mutations occur. Blood 71: 1357-1360. CAI,S. P., C. A. CHANG, J. Z. ZHANG,R. K. SAIKI,H. A. ERLICH & Y. W. KAN. 1989. Rapid prenatal diagnosis of P-thalassemia using DNA amplification and nonradioactive probes. Blood 73: 312-314. CAI,S. P., J. Z. ZHANG,M. DOHERTY & Y. W. KAN. 1989. A new TATA box mutation detected in prenatal diagnosis. Am. J. Hum. Genet. 4 5 112-1 14. SAIKI,R. K., S. SCHARF, F. FALOONA, K. B. MULLIS,G. T. HORN,€1. A. ERLICH& N. ARNHEIN. 1985. Enzymatic amplification of p-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354. SAIKI,R. K., T. C. BUGAWAN, G. T. HORN,K. B. MULLIS& H. A. ERLICH. 1986. Analysis of enzymatically amplified P-globin and HLD-DQ (Y DNA with allele-specific endonucleotide probes. Nature 324 163-166. SAIKI,P. K., C. A. CHANG,C. H. LEVENSON, T. C. WARREN,C. D. BOEHM,H. H. KAZAZIAN, JR. & H. A. ERLICH.1988. Diagnosis of sickle cell anemia and p-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes. N. Engl. J. Med. 319 537-541. CAI,S. P. & Y. W. KAN. 1990. Identification of the multiple p-thalassemia mutations by denaturing gradient gel electrophoresis. J. Clin. Invest. 85: 550-553. CAO,A,, C. ROSATELLI, R. GALANELLO, G. MONNI,G. OLLA,P. Cossu & M. S. RISTALDI. 1989. The prevention of thalassemia in Sardinia. Clin. Genet. 36: 277-285.
INTRODUCTION Both a- and b-thalassemias are common genetic disorders in South China. The gene frequency of each of these disorders is estimated to be 3-5% in the province of Guangdong, which has a total population of over 50 million. The gene frequencies in other provinces in South China are similar or even higher. It is now possible to obtain cells of fetal origin either by amniocentesis or by chorionic villus biopsy. In addition, recent advances in molecular biology have simplified considerably the DNA diagnostic techniques for the thalassemias.' A prenatal diagnosis laboratory for thalassemias was established in the Nan Fang Hospital of the First Military Medical University in Guangzhou, the capital of Guangdong Province, in South China. This laboratory serves Guangdong, Guangxi, Fujian, and Hainan provinces. This paper reports the results of the diagnostic work carried out in this laboratory during 1989.
DIAGNOSIS OF a-THALASSEMJA The a-thalassemia of clinical significance in South China is usually due to the deletion of the Southeast Asian type, involving all +a- and a-globin genes on the short arm of chromosome 16. This deletion is commonly referred to as --SEA/ and can be clearly demonstrated by Southern blot analysis.' In 1987, Chehab and colleagues3 devised a polymerase chain reaction- (PCR) based method for the diagnosis of homozygous a-thalassemia, utilizing primers which would amplify 136 bp of DNA normally present between the +al- and a2-globin genes. This DNA
aThis work was supported in part by the Guandong Provincial Scientific Committee and NIH Grant DK 16666. dY. W. Kan is an investigator of the Howard Hughes Medical Institute. eAddress correspondence to Dr. Y. W. Kan, Howard Hughes Medical Institute, U-426, University of California, San Francisco, CA 94143-0724. 264
ZHANG et ai.: PRENATAL DIAGNOSIS IN SOUTH CHINA
265
segment is deleted in a-thalassemia of the --SEA/ type. Concomitantly, a 110-bp DNA fragment of the p-globin gene is also amplified, which serves as the control for the PCR. In homozygous a-thalassemia, otherwise known as hemoglobin (Hb) Bart's hydrops fetalis syndrome, the 110-bp P-globin gene fragment is present after PCR, whereas the 136-bp a-globin gene fragment is absent.' This method, which is relatively simple and rapid, has been adopted by our laboratory as the diagnostic test. We have performed 38 prenatal diagnoses on pregnancies at risk for homozygous a-thalassemia, and 5 fetuses with homozygous a-thalassemia were identified.
DIAGNOSIS OF P-THALASSEMIA There are now 15 known P-thalassemia point mutations in the Chinese p ~ p u l a t i o n .In ~ order to identify the more commonly encountered mutations in South China, we have studied 236 chromosomes with P-thalassemia by the technique of allele-specific oligonucleotide hybridization to amplified fragments of the P-globin gene. The data indicate that four mutations, a 4-bp deletion in codon 41-42 (-CTTT), a C+T mutation in IVS-2 at position 654, an A+T nonsense mutation in codon 17 and an A+G mutation at TATA box position -28, account for over 90% of all the mutant alleles studied (TABLE 1). For prenatal diagnosis of pregnancies at risk for homozygous or compound TABLE 1.
B-Thalassemia Mutations and Their Frequencies in South China Mutation" Codon 4 1 4 2 (- CTTT) IVS-2 nt 654 (C-+T) Codon 17 (A+T) TATA box nt -28 ( A & ) TATA box nt -30 (T+C) Codon 71-72 (+A)
Frequency (%) 49.6 18.8 15.8 9.0 1.5 0.8
"Numberof chromosomes = 236. nt, nucleotide.
heterozygous P-thalassemias, DNA was extracted from the peripheral blood cells of the couples under investigation. DNA was also extracted from chorionic villi obtained at 7-11 weeks of gestation or from amniotic fluid cells obtained at 16-20 weeks of gestation. In vitro amplification of DNA was performed as described using two sets of primers to amplify the P-globin gene regions5Amplified DNA was dotted onto nylon filters, which were then hybridized with horseradish peroxidase- (HRP) labeled allele-specific oligonucleotide probes. Six pairs of probes were used, four corresponding to the four common P-thalassemia mutations found in the Chinese, as mentioned above, as well as pairs of probes for the frameshift mutation in codon 71-72 (+A) and the G-C mutation in IVS-1 at position 5. The presence of an oligonucleotide hybridized to amplified DNA is detected by histochemical staining for presence of the enzyme.6 Thirty-four cases of prenatal diagnoses of P-thalassemia were performed. The results are shown in TABLE2. Nine fetuses inherited p-thalassemia alleles from both of their parents.
ANNALS NEW YORK ACADEMY OF SCIENCES
266
DIAGNOSIS OF P-THALASSEMZA BY DNA SEQUENCING When the P-thalassemia mutation is not detected with oligonucleotide probes specific for the six common mutations, the entire P-globin gene can be amplified and sequenced directly to identify the mutation. During the course of prenatal diagnosis for P-thalassemia in a Fujian couple in 1988, we encountered a mutation that was not detectable by oligonucleotides for the Chinese mutations then known. Amplification of the p-globin gene and direct D N A sequencing revealed a previously undescribed T+C TATA box mutation, which was carried by the father but was not inherited by the fetus. Even in this complicated case, prenatal diagnosis was accomplished within two weeks.'
DISCUSSION Carrier detection, genetic counseling, and prenatal diagnosis are indispensable components in the prevention of thalassemia major among populations in which the gene frequency for thalassemia is high, as it is in South China. The introduction of the PCR technique offers several advantages over previous prenatal diagnostic TABLE 2.
Prenatal Diagnosis in 34 Fetuses at Risk of p-Thalassemia
Diagnosis Normal Heterozygote (total)
Mutations -
fl
5 20 11
Codon 41-42 1VS-2nt 654 Codon 17 TATA box nt -28
3
4 2
Homozygote or Compound Heterozygote (total)
9
Codon 4142iIVS-2 nt 654 Codon 4142icodon 17 Codon 41-42icodon 41-42 Codon 4142RATA box nt
-- 28
3 3 2 1
methods.""' In vitro D N A amplification can be performed rapidly. The increased number of target sequences following amplification permits the use of less sensitive probes, i.e., 3SS-labeled oligonucleotides or non-radioactive probes such as the horseradish peroxidase-labeled oligonucleotides used in the present study. "Plabeled oligonucleotides, despite their short half-life, can also be used for u p to two to three months. Thus, the PCR technique has significantly simplified the D N A diagnostic procedure. More recently, we have shown that denaturing gradient gel electrophoresis also offers another non-radioactive means of detecting multiple mutations for P-thalassemias and other genetic disorders." The success of implementing carrier detection and prenatal diagnosis in decreasing significantly the number of live births affected with thalassemia major has been amply demonstrated in a number of Mediterranean regions.'* The present report clearly demonstrates that prenatal diagnosis for both a- and P-thalassemias can be successfuliy carried out in South China. With the appropriate funding and resources,
ZHANG et al.: PRENATAL DIAGNOSIS IN SOUTH CHINA
267
a more comprehensive program on screening, genetic counseling, and prenatal diagnosis can be established in South China. With these genetic services in place, it is anticipated that much progress can be made in the prevention of thalassemia major in South China. ACKNOWLEDGMENT We thank Mrs. A. Folkes for preparation of the manuscript. REFERENCES 1.
2. 3. 4. 5.
6. 7.
8. 9. 10.
11.
12.
ALTER,B. P. 1984. Advances in the prenatal diagnosis of hematologic diseases. Blood 6 4 329-340. A. 0. M. WILKIE, I. M. PRETORIUS, A. P. JARMAN & D. J. HIGGS,D. R., M. A. VICKERS, WEATHERALL. 1989. A review of the molecular genetics of the human a-globin gene cluster. Blood 73: 1081-1104. CHEHAB,F. F., M. DOHERTY, S. P. CAI,Y. W. KAN,S. COOPER& E. M. RUBIN.1987. Detection of sickle cell anemia and thalassemia. Nature 329 293-294. KAZAZIAN,H. H., JR. 1990. The thalassemia syndromes: Molecular basis and prenatal diagnosis in 1989. Semin. Hematol. 27: 209-228. CAI,S. P., J. Z. ZHANG, D. H. HUANG, Z. X. WANC& Y . W. KAN.1988. A simple approach to prenatal diagnosis of P-thalassemia in a geographic area where multiple mutations occur. Blood 71: 1357-1360. CAI,S. P., C. A. CHANG, J. Z. ZHANG,R. K. SAIKI,H. A. ERLICH & Y. W. KAN. 1989. Rapid prenatal diagnosis of P-thalassemia using DNA amplification and nonradioactive probes. Blood 73: 312-314. CAI,S. P., J. Z. ZHANG,M. DOHERTY & Y. W. KAN. 1989. A new TATA box mutation detected in prenatal diagnosis. Am. J. Hum. Genet. 4 5 112-1 14. SAIKI,R. K., S. SCHARF, F. FALOONA, K. B. MULLIS,G. T. HORN,€1. A. ERLICH& N. ARNHEIN. 1985. Enzymatic amplification of p-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-1354. SAIKI,R. K., T. C. BUGAWAN, G. T. HORN,K. B. MULLIS& H. A. ERLICH. 1986. Analysis of enzymatically amplified P-globin and HLD-DQ (Y DNA with allele-specific endonucleotide probes. Nature 324 163-166. SAIKI,P. K., C. A. CHANG,C. H. LEVENSON, T. C. WARREN,C. D. BOEHM,H. H. KAZAZIAN, JR. & H. A. ERLICH.1988. Diagnosis of sickle cell anemia and p-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes. N. Engl. J. Med. 319 537-541. CAI,S. P. & Y. W. KAN. 1990. Identification of the multiple p-thalassemia mutations by denaturing gradient gel electrophoresis. J. Clin. Invest. 85: 550-553. CAO,A,, C. ROSATELLI, R. GALANELLO, G. MONNI,G. OLLA,P. Cossu & M. S. RISTALDI. 1989. The prevention of thalassemia in Sardinia. Clin. Genet. 36: 277-285.
