V Chan, M Tang, TK Chan

Introduction

Many serious birth defects, including inherited genetic disorders, are now diagnosable in utero. They account for 20% of the deaths during newborn period and a higher percentage of serious morbidity in infancy and childhood.¹ The incidence and type of severe inherited single gene disorders vary in different ethnic groups and occur in up to 2% of the newborns.² Besides the high financial cost incurred by such newborns for hospital intensive care, maintenance therapy and rehabilitation programs for their severe handicap, the physical and psychological sufferings of the affected and their families are immeasurable. With the technological advances in cytogenetic analysis, accurate DNA diagnosis and improved methods of fetal cell procurement, prenatal diagnosis of many of these fetal abnormalities can be performed and has become an important aspect of management in at risk pregnancies.

The Prenatal Diagnosis Laboratory for chromosomal abnormalities at Tsan Yuk Hospital (Wu Chung Prenatal Diagnosis Laboratory) and the DNA Diagnostic Laboratory at Queen Mary Hospital were established jointly by the Department of Obstetrics and Gynaecology and the Department of Medicine, University of Hong Kong in 1982 and have continued to develop for the past 14 years. This article reviews the prenatal diagnosis programme to-date.

Cytogenetic Analysis for Down's Syndrome and Other Chromosomal Abnormalities

It is now known that chromosome disorder forms a major category of fetal abnormality, accounting for a large proportion of reproductive wastage, congenital malformation and mental retardation. Complete karotyping of the fetus is made for pregnancy of all mothers of advanced maternal age (above 35 years). Early methodology involved culture of fetal cells from either chorionic villi (CVS) biopsied at 9-11 weeks gestation or amniotic fluid obtained by amniocentesis at 16-18 weeks gestation; harvesting of cells at metaphase, photomicroscopy and laborious identification of individual chromosomes from the enlarged photographs. Since it took 2-3 weeks before the cells were ready for harvesting and a further few days for photography and subsequent karyotype analysis, the number of samples which could be handled each month was limited. With the development of CCT camera for capture of microscopic images, dedicated computer software for karyotype analysis, automated metaphase finder and the ability to perform cytogenetic analysis from a direct cell preparation (without the need for culture), a karyotype result can now be obtained within a few days. In addition, the complete chromosomal arrangements can be archived onto optical disk without the need for photography. Currently, in Hong Kong, at least 2500 karyotype analyses are performed each year. Apart from mothers of advanced age, women with past or family history of chromosomal abnormalities are counselled for prenatal testing. For pregnancies at risk for Klinefelter's syndrome, X and Y chromosome numbers may be quantitated by slot-blot hybridization of the fetal DNA with the corresponding probes and subsequent measurement of the radioactive intensities of the X and Y bands using a laser densitometer³ or a Phosphor-Imager. The ratio of these X, Y bands are compared to that of a normal control processed simultaneously on the same slot-blot membrane for calculation of chromosome numbers.

More recently, fluorescence in situ hybridization (FISH) technique has been developed for chromosome detection in metaphase or interphase cells.⁴ This method, whilst allowing detection of abnormality in chromosome numbers (e.g. trisomy 21 and 18, monosomy X, XXY and XXX), is unable to detect translocation Down's syndrome or mosaicism. Thus, whenever possible complete karotyping should still be the method of choice for prenatal testing of chromosomal abnormalities. Since 1982, 399 cases of abnormal fetus have been identified (Table).

DNA-Based Prenatal Diagnosis of Single-Gene Defects

The advent of recombinant DNA technology in the 1970's and 1980's has advanced our knowledge of the molecular pathology of diseases, offered new methods for preparation of therapeutic materials as well as new strategies for therapy. It has also increased the accuracy for diagnosis of severe single gene defects and made detection at the DNA level possible, that is, even before expression of the gene. Kan and Dozy5 were the first to apply DNA technology for the prenatal diagnosis of sickle cell anaemia. In Hong Kong, the DNA Prenatal Diagnosis Laboratory was established in 1982, with 1075 diagnoses made and 218 affected pregnancies confirmed to-date (Fig).

Fig DNA-based prenatal diagnoses performed in Hong Kong since 1982.

(I)

α and β thalassemias

The globin genes, defects of which are responsible for causing thalassemias, were the first genes to be cloned. Thalassemia (thal) is the commonest congenital disease in Southeast Asia. In Hong Kong, with a 6% incidence of thal trait and an annual birth rate of 80,000, there should be 280 pregnancies at risk for homozygous α or β thal. A prenatal diagnosis programme using DNA techniques has been established since 1982 for the diagnosis of these conditions in Hong Kong.

Screening

95% of the local population is ethnic Chinese, thus one has essentially a homogeneous genetic group. Couples who are both α or β thal trait run a 25% risk of having a homozygous fetus with each pregnancy. Such fetus would either die in late pregnancy or at birth, as in the case of homozygous α thal 1 (Hb Barts' hydrops fetalis), or develop severe anaemia in infancy and require lifelong transfusion, as in the case of homozygous β thal. Thus prenatal diagnosis is offered for all pregnancies at risk. Screening for thal trait is carried out at the antenatal clinic of major hospitals throughout Hong Kong, based on low mean corpuscular volume (MCV: < 75 fl).⁶ If a pregnant woman has a low MCV, the husband will then be assessed and if both have low MCV, they will be tested for α or β thal trait by the presence of occasional red cells with H inclusions and ζ gene map or elevated Hb A2 level respectively. Since six years ago, screening is extended to couples seeking premarital counselling at the Family Planning Association Clinics throughout the territory. If both partners are found to have α or β thal trait, they are counselled regarding the mode of inheritance and the reproductive options available to them. Thus they would be forewarned of the need for prenatal diagnosis before marriage and well before pregnancy.

Prenatal Testing

Prenatal diagnosis of α thal is performed by the detection of α genes in fetal DNA extracted directly from CVS or amniocytes.⁷ Although it is possible to employ polymerase chain reaction (PCR) technique for amplification of the α thal 1 chromosome,⁸ Southern blot or dot-blot is still the method of choice because of the definite risk of false negative with PCR due to maternal DNA contamination of even 0.01%. With β thal, a reverse dot blot procedure with allele specific oligonucleotide hybridization allows direct detection of the mutation.⁹ It is possible to screen simultaneously for all the twelve known Chinese β thal mutations in a single strip and obtain a result within six hours of fetal sampling.

(II)

Haemophilias

Haemophilia A is the commonest congenital bleeding disorder, affecting approximately 1:10,000 live-born males. Females with family history of the disease are offered carrier testing and prenatal diagnosis if required. The FVIII gene is 186 kb and direct detection of each point mutation, although possible, using PCR of the gene fragments, screening of the abnormal fragment by denaturing gradient gel electrophoresis, followed by direct genomic sequencing,¹⁰ is nonetheless, an extremely laborious and time-consuming procedure. Fortunately, 40% of those with severe haemophilia have the same molecular defect due to gene inversion in intron 22, which can be detected by gene mapping using Southern blot of Bcl I digested genomic DNA and hybridization to an intron 22 probe.¹¹ For the other cases, linkage analysis using three common restriction fragment length polymorphisms (RFLPs), viz. Bcl I, Kpn I/Xba I (A, B and C sites) and Taq I - St 14, will be informative in 98% of the Chinese haemophilia A families.¹² Use of the multi-allelic microsatellite repeat polymorphisms (MRPs) located in intron 13 and intron 22 will add to the accuracy and informativeness of linkage analysis.^13,14 In addition, the recently developed semi-automated method for MRPs using fluorescein-labelled primers for PCR and automated analysis by laser fluorometry will provide a quick haplotype analysis.¹⁵

Haemophilia B is less common and affects 1:30,000 male live births. Chinese and other Orientals lack heterozygosity for the RFLP sites commonly found in Caucasians.¹⁶ The two most useful sites, the 5' Mse I and 3' Hha I sites, only give a heterozygote rate of 0.33 and 0.28 in Thais and Chinese respectively. Detection of the mutation by PCR of the exons and their splice junctions and direct genomic sequencing had been employed.¹⁶ When the mutation affects an enzyme recognition site, subsequent prenatal testing can be made by direct restriction enzyme analysis of the PCR gene fragment. This strategy is still feasible given the relative small size of the FIX gene with exon length of 2.8 kb.

(III)

Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) and its milder form, Becker muscular dystrophy (BMD), are caused by mutation of the dystrophin gene located on the short arm of the X chromosome at Xp21. This X-linked recessive disorder affects 1:3,500 live-born males. Patients with DMD usually present with proximal muscular weakness in infancy, progressing to loss of ambulation by 12 years of age and eventual death in the second to third decade of life from respiratory failure or cardiomyopathy. Recent efforts of gene therapy using myoblasts transplant failed to live up to expectations. Thus secondary prevention is the mainstay of management for families at risk. Prenatal diagnosis and carrier testing for this condition has been available in Hong Kong since 1989 and although there is no established screening for at-risk females except for increased serum creatinine kinase in about 60% of carriers, anyone with a family history will be eligible. Since the indexed patient may die within the third decade, it is our practice to advise storing the propositus' DNA by establishing a permanent cell-line of each affected subject by Epstein-Barr Virus transformation of their lymphocytes.

Approximately 50-60% of DMD are due to gene deletion, which can be identified by PCR of deletion-prone exons^17,18 or Southern blot hybridization with cDNA probe. For the non-deletion cases, linkage by RFLP¹⁹ or MRP^20,21 can be used. However, with the huge size of the DMD gene (~2000 kb), multiple intragenic sites as well as sites on the 5' and 3' flanking regions should be used to reduce the risk of error due to meiotic recombination.

In prenatal cases where linkage analysis is employed, it is customary to obtain a haplotype of the DMD gene using multiple (8-10) RFLP or MRP sites. The development of multiplex PCR using fluorescein-labelled primers and automated analysis allows a comprehensive haplotype analysis of the entire DMD family within 1 - 2 days.

(IV)

Huntington's Disease

Huntington's Disease (HD) is a neurodegenerative disorder which manifests in mid-life, presenting with involuntary choreic movement, cognitive and psychiatric disturbances and dementia. There is currently no effective treatment nor means to arrest or delay the inexorable progression to death, which usually occurs 10-20 years after onset of disease. The identification of the HD gene (IT 15) and the detection of the defect, which is an expansion of the 5' trinucleotide repeat sequence (CAG), has allowed more accurate diagnosis of the condition.²² It is now possible for us to offer presymptomatic testing for at risk individuals²³ as well as prenatal diagnosis of this condition. However, various ethical and psychosocial issues need to be considered. Testing and results should only be made known to the concerned individuals with counselling from the geneticist, neurologist as well as psychiatrist.

Future Prospects

Continued improvements in the methods of procurement of fetal tissue have been made since we started the prenatal diagnosis programme in 1982. Now, amniocentesis in early gestation and trans-abdominal CVS are safe under experienced hands and can be applied in the intermediate gestational period between classical CVS at 9-11 weeks and amniocentesis at 18-22 weeks. Noninvasive methods are being developed including enrichment of fetal cells from maternal blood²⁴ or cervical swabs²⁵ and, if these procedures are reliable, would increase the safety of the prenatal diagnosis procedure even further. DNA diagnosis can be made in a single cell and would allow preimplantation diagnosis using cells from the blastomere of an in-vitro fertilised ovum.²⁶ Pre-implantation diagnosis is particularly important in assisted pregnancy programme.

New genes responsible for severe incurable defects as well as genes responsible for susceptibility of late-onset diseases are being discovered. Prenatal diagnosis for the former defects are well justified, while those for the latter categories are more contentious and requires further public discussion and policy decisions. As for the DNA Diagnostic Laboratory, a constant challenge to expand the repertoire of genetic testing has to be met. Since many of these defects have unique ethnic variations, research and development work are required locally. Resources are required for these efforts as well as for the prenatal and carrier testing programmes in order for us to keep up with the increasing demands and expectations of paediatricians, physicians, obstetricians and their patients.

Conclusion

Before the availability of gene therapy as a treatment modality for common genetic diseases, prevention by prenatal diagnosis and therapeutic abortion of the affected fetus remains the best alternative for families at risk. Whenever possible, first trimester testing should be advocated as this will reduce risk and psychological trauma associated with second trimester termination, should the fetus be diagnosed as severely affected. In our experience, prenatal testing is a well-accepted procedure amongst the local Chinese population, provided adequate counselling has been given. In the fourteen years since the prenatal diagnosis service has been established in Hong Kong, there are many females who have returned for multiple prenatal diagnosis, with one having undergone testing for seven pregnancies. At centres where fetal sampling is performed by a dedicated and experienced team of obstetricians, fetal loss due to the procedure should be negligible. Future advances in fetal DNA procurement and DNA diagnostic techniques will make prenatal testing safer and more accurate.

Acknowledgement

We would like to thank the Croucher Foundation for continued financial support for the R & D work vital for the success of the DNA based Prenatal Diagnosis Programme in Hong Kong. The efforts and contributions of Professor HK Ma, Professor Sir D. Todd, the Wu Chung Foundation and the Wideland Foundation are also grateful acknowledged.

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