some clinical implications of recombinant dna technology with emphasis on prenatal diagnosis of...

Some Clinical Implications of Recombinant DNA Technology with Emphasis on Prenatal Diagnosis

of Hemoglobinopathies

A L A N A N D E R S O N

L a v a l U n i v e r s i t y C a n c e r R e s e a r c h C e n t e r at I ' H 6 t e I - D i e u de Q u e b e c a n d D e p a r t m e n t d e B i o l o g i e , U n i v e r s i t e Lava l , Q u e b e c , Q u e b e c

Recombinant DNA technology has made possible remarkable advances in understanding the molecular genetics of human and other eucaryotic cells. This technology also has clinical applications, some of which may soon involve clinical laboratories. Restriction endonucleases and cloned DNA probes permit the direct analysis of cellular DNA to detect sequence abnormalities associated with particular genetic disorders. Use of this approach in the antenatal diagnosis of hemoglobinopathies is now possible on a routine basis. The principles behind the methods are quite general and may be applied to other hereditary diseases once suitable DNA probes become available. The same approach may be used to detect carriers of recessive gene defects and so improve genetic counselling. Other clinically related applications of recombinant DNA technology include the production of antigens for vaccine preparation and of specific human proteins (e.g. interferon and human growth hormone) for therapeutic use, as well as the use of nucleic acid hybridization for identification of microbial pathogens. It seems likely that recombinant DNA technology will, in the future, play an increasingly important role in the diagnosis, prevention and treatment of human disease.

KEY WORDS: antenatal diagnosis, restriction endonuclease, molecular cloning

O ver the past ten years, several new and powerful techniques have become available to molecular bio-

logists. These techniques have made possible remarkable advances in understanding the molecular genetics of eucaryotic cells and organisms. They also have medical applications, for example: (i) in clinical research, leading to increased understanding of the molecular basis of human disease; (ii} in the production of clinically important proteins, such as hormones and vaccines; (iii) in prenatal diagnosis and genetic counselling; (iv) in molecular epidemiology and the identification of microbial pathogens.

Another potential medical application of the new recombinant DNA technology is in gene therapy, although other applications are much nearer at hand. The present summary concerns primarily points (iii) and (iv), which involve methods that have been successfully applied in clinical research and that may soon become routine clinical screening techniques (1-3}.

T h e ne w gene t i c s

The new genetics, which is characterized by the use of restriction endonucleases, DNA sequencing and mo-

Correspondence: Dr. Alan Anderson, Centre de Recherche, l'Hotel-Dieu de Quebec, 11 cSte du Palais, Quebec, Quebec, G1R 2J6. Telephone: (418) 694-5281.

This paper is based on a presentation by the author at the Joint Congress on Clinical Chemistry, Quebec City, June 26-30, 1983.

lecular cloning, permits molecular genetic analysis on a routine basis with any organism, including man.

Restriction endonucleases are enzymes that cut DNA at specific sequences. Over 350 are known (4) and about 80 are available commercially. They are powerful anal- ytical and synthetic tools for recombinant DNA technology (1, 5, 6). Analytically, they may be used to con- struct restriction maps of DNA, to isolate specific DNA fragments for sequencing or for use as molecular probes, and to perform DNA analysis by Southern blotting (5). Synthetically, restriction endonucleases are employed to obtain the specific DNA fragments, often with staggered and therefore "sticky" ends, used to make recombinant DNA for molecular cloning.

Molecular cloning (7) involves the construction in vitro of recombinant DNA molecules, followed by their introduction into suitable microbial hosts. Clones of the host are then isolated containing a single type of recombinant DNA molecule and hence a single "cloned" fragment of foreign DNA. Molecular cloning is, in es- sence, a powerful method of purifying specific DNA sequences.

In general, the DNA fragments to be cloned are of two types: (i) complementary DNA (cDNAI, i.e. double- stranded DNA synthesized from mature messenger RNAs;cDNAs contain sequences coding for proteins but do not contain introns; ( ii ) genomic DNA sequences, i.e. genes or pieces of genes which may contain introns, protein coding sequences and intergenic regions.

The expression of cloned genes may be studied by reintroducing them into living cells. In one notable recent example (8), the gene for rat growth hormone was attached to a highly active mouse promoter and the fusion sequence was injected into the male pronucleus of fertilized mouse eggs. Some of the transgenic animals obtained from the reimplanted eggs synthesized large amounts of rat growth hormone and grew up to twice as fast as their normal li t termates. Experiments such as this have potential for the development of ani- mal models of human genetic diseases. They could also lead to the farming of valuable proteins if stable lines derived from transgenic animals can be obtained.

Cloned cDNAs may be subjected to DNA sequence analysis and from this the encoded protein sequence may be predicted. Full-length cloned cDNAs may also be at tached to bacterial regulatory sequences and introduced into bacterial cells for the production of the corresponding proteins (e.g. interferons (9), human growth hormone (10)). In addition, cloned cDNAs, as well as cloned genomic fragments (see below}, are often used as

112 CLINICAL BIOCHEMISTRY, VOLUME 17, APRIL 1984

RECOMBINANT DNA IN PRENATAL DIAGNOSIS

EcoRI

~2 l f~ i n r -

Kb 3'0 2b 5"---,,3'

~-thoI I [

I0 6 DIGESTION

ISOLATED MIXTURE OF DNA RESTRICTION FRAGMENTS

BomHI BomHI

Ps st t

~ EEl C K'b ' & . . . . ~ . . . . ~o . . . . ~ . . . . 2'o . . . . ,b . . . . 6

5'-----*3' j3° -thai O

Figure 1 - - Maps of the human u-like (upper) and B-like qlow- er~ globin genes. Introns are represented in white and exons in black. There are 5 ¢,-like genes: a l and (,2 (the adult genes), dJtxl (a pseudogene)and C1 and C2 (the embryonic genes). The two Eco RI sites responsible for generating the 23 kb Eco RI fragment mentioned in the text are shown. Indicated also is the approximate extent of an c~-thalassemia 1 (¢,-thal 1) deletion found in Southeast Asian populations and discussed in the text: it removes both (,1 and ¢t2 and, when homozygous, causes hydrops fetalis (the rightward extent of this deletion is not defined). There are 7 [3-like globin genes: [3 and 5 (the adult genes), ~1~1 and dJ~2 (pseudogenes), ';~ and A~ (the fetal genes) and elan embryonic geneL The starred Hind HI sites in the (;~ and ..Ly genes are examples of polymorphic restriction sites ~14, 31). The restriction sites responsible for the gener- ation of the 1.8 kb Barn H1 fragment and the 4.4 kb Pst I fragment mentioned in the text are indicated. The approximate extent of a 13 gene deletion associated with a ~"-thalassemia I It'-thai L and discussed in the text is shown as well. At least a dozen other c~ and 13 gene deletions, responsible for various types of thalassemia, have been identified and mapped. See references 12, 20, 23 and 24.

molecular probes for the identification of homologous sequences by molecular hybridization.

Cloned genomic f ragments may be sequenced, but, because of their great length tup to 20 kilobases tkbl or more, and clones with overlapping insertions are often sought and obtained), total sequencing is rarely worth- while at present. Rather, part ial sequences are determined and physical maps are constructed, showing restriction sites and also the positions of genes of interest (Figure 1 ). The maps of the cloned genomic f ragments apply also to these same sequences within the cellular DNA. Knowledge of the restriction maps of the genes to be analysed for muta t ions is essential for prenata l diagnosis of genetic disorders. One mus t know what the normal genes look like in order to recognize the mod- ifications introduced by disease-causing mutations.

The h u m a n hemoglobins and their genes constitute a r emarkab le example of a system tha t has been successfully analysed using the new genetics I l l - 1 3 ; Figure 1). The fundamenta l knowledge so obtained has had rapid clinical application, notably for prenata l diagnosis of hemoglobinopathies (14-161. The c~-like globin genes are clustered in a 25 kb region on the short a rm of chromosome 16 and the [3-like globin genes are in a 50 kb region on the short a rm of chromosome 11 (12, 13; Figure 1). The DNA sequences of the 5 [3-like globin genes and their f lanking regions have been determined, as have those of the (x2 gene and portions of the 41 gene

~ 1. HYBRIDIZATION WITH 2P-LABELLED PROBE

2. AUTORADIOGRAPHY

AGAROSE GEL

ELECTROPHORESIS

SOUTHERN

I BLOTT NG

~ NITROCELLULOSE FILTER

Figure 2 - - Schematic diagram illustrating DNA analysis by Southern blotting. The agarose gel is represented as having been stained with ethidium bromide. Individual bands are not resolved because of the large number of different restriction fragments present. The horizontal arrow near the middle of the agarose gel, as well as of the nitrocellulose filter, represents a restriction fragment homologous to the probe. A vis- ible band is evident at the corresponding position on the autoradiography film.

/121. Fur thermore , the molecular nature of the genetic lesions (point mutat ions and deletions) in many different hemoglobinopathies, including s tructural var ian ts and ~- and [3-thalassemias, is also known (11-13 , 15).

Prenatal diagnosis by Southern blotting and hybridization with specific probes

The idea behind prenata l diagnosis by DNA analysis is s t ra ightforward enough. If a fetus is at risk for a genetic disease, specific fetal genes may be analysed to see if the mu tan t form is present and if so whether in the homozygous or heterozygous state. For disorders tha t are inheri ted in a simple Mendelian fashion, the disease s ta tus of the individual may then be predicted with essential ly 100% accuracy. Note, however, tha t this approach represents a radical depar ture from tra- ditional genetic methods where the presence of the different allelic forms of a given gene was vir tual ly always inferred from their phenotypic effects on the individual.

DNA analysis by Southern blott ing (Figure 2) involves, first, restriction endonuclease digestion of a few Ixg of cellular DNA. H u m a n genomic DNA is normal ly isolated from peripheral blood (15, 16). Fetal DNA may be isolated directly from amniotic fluid cells obtained by amniocentesis; amniocytes may also be cultured if grea ter quant i t ies of DNA are required (15, 16). The DNA f ragments are separated according to thei r size by agarose gel electrophoresis (7) and then t ransferred to nitrocellulose filters (5). The t ransferred DNA is immo- bilized in singled-stranded form on the filters, thus per- mi t t ing subsequent hybridization with a sui table a2P-labelled probe (Figure 3) specific for the gene under study. Out of the several hundred thousand or more restr ict ion f ragments present, only those tha t are homologous to the probe will hybridize with it and thus form dark bands on an X-ray film after autoradiography (Figure 2}.

CLINICAL BIOCHEMISTRY, VOLUME 17, APRIL 1984 113

ANDERSON

I. RESTRICTION ENDONUCLEASE DIGESTION

2. PURIRCATION OF RESTRICTION FRAGMENT

CLONED cDNA OR GENOMIC FRAGMENT IN RECOMBINANT PLASMID

ONose

DNA Polymerase dNT32p

(NICK TRANSLATION)

~P-LABELLED PROBE

Figure 3 - Schematic diagram summarizing the steps involved in the preparation, from a cloned sequence, of a ~nick translated" (57) x-'P-labelled probe. The two arrows on the recombinant plasmid define a restriction fragment within the cloned sequence.

Genotype AS AA SS

5 (I .8)

(I , 5 ) '

5' Flanking region I

i

I , 1.15

BAt ,.,5 ,.55

1" i IEZ] .I. 0.2 _, T

I

/3 globin gene Figure 5 - - Schematic representation of the expected results of a Southern blot of Mst II-digested genomic DNA from individuals with sickle-cell trait (AS), from normal indivuals (AA) and from individuals with sickle-cell anemia (SS). The 1.15 kb Mst II fragment may be used as the probe. The faint band marked at 1.8 kb comes from cross-hybridization of the probe with 5-globin sequences. See references 18 and 19.

Figure 4 - - Map showing the 3 Mst II sites (vertical arrows) in the ~-globin gene and its 5' flanking region, that are pertinent for the identification of the ~s allele. In white are represented the first ~-globin gene intron and part of the second intron; in black are the first two ~-globin exons. The starred Mst II site in the first exon, at codons 5, 6 and 7 of the ~-globin gene sequence, is destroyed by the ~s mutation. Distances are given in kb. Shown on the two lower lines are the 1.15 and 1.35 kb fragments produced by Mst II digestion of the ~:x and ~s alleles respectively. See references 17, 18 and 19.

Means of identifying mutant genes

TABLE 1 Eco RI Fragments of Fetal DNA Expected to Hybrid-

ize with a Probe Specific for the ~-Globin Genes '~

Genotype Disease Status Fragment Length

~ / ~ Normal 23 kb ~ / - - Heterozygous 23 kb

~-Thalassemia 1 - - / - - Hydrops fetalis None

"See reference 22 for an example of prenatal diagnosis of hydrops fetalis by this method.

1. DIRECT ANALYSIS WITH CLONED DNA FRAGMENTS AS PROBES

a. A point mutation alters a suitable restriction site

Point mutations that alter appropriate restriction sites are relatively easy to identify by DNA analysis (15). Although this situation would be expected to arise rather rarely, the ~s mutation, responsible for sickle- cell anemia, eliminates an Mst II site present in the sequence coding for amino acids 5, 6, and 7 in the normal ~A gene (17-19; Figure 4). This site, CCTGAGG in ~A, becomes CCTGTGG in ~s, and the latter-is not cleaved by the enzyme. (Actually Mst II cuts at CCTNAGG, where N is any base.).

The Mst II site in ~A codons 5, 6 and 7 is flanked by Mst II sites 1.15 kb away on one side (the upstream or 5' side) and 0.2 kb away on the other side (the down- stream or 3' side) (17-19; Figure 4). Hence, Mst II digestion of the ~A allele, and hybridization with a probe specific for this part of the ~A gene, yields two

hybridizing fragments of 1.15 kb and 0.2 kb, al though in practice the 0.2 kb fragment is normally not seen because of its small size (Figures 4, 5). On the other hand, Mst II digestion of the ~s allele yields a single fragment of 1.35 kb (Figures 4, 5), due to elimination of the Mst II site by the ~s mutation. The 1.35 kb and the 1.15 kb fragments will both be present in Mst II digests of DNA from individuals with sickle-cell trait (Figure 5).

Two groups (18, 19) have successfully performed prenatal diagnosis for sickle-cell anemia using Mst II digestion. Diagnosis should be possible in 100% of cases at risk for sickle-cell anemia and the analysis can be done on DNA isolated from cells present in as little as 8 mL of amniotic fluid (18).

b. A deletion alters the length(s) of restriction fragment(s) or completely eliminates genes

The thalassemias are a heterogeneous group of hemoglobinopathies in which the synthesis of one or more globin chains is reduced or absent (11, 13). Some of


IPD Scanner


TABLE 2 Hybridization of 1.8 kb Barn H1 Fragments of Genomic DNA with Synthetic

Probes Specific for Normal B-Globin and Mutant [3"-Thalassemia Genes"

Hybridization with a 1.8 kb [3-Globin Gene Fragment

Genotype Disease Status Normal Probe Mutant Probe

[3:~/[3 '~ Normal + - [Yx/B "-thai [3-Thalassemia Minor + + [3"-thal/[3"-thal [3-Thalassemia Major - +

"Results taken from reference 26. The sequences of the normal and mutant probes are given in Figure 6.

I NTRON I NT RON I 2

D - -

23, 24). Such a difference would permit prenatal diagnosis of this form of [3"-thalassemia (15, 23).

2. DIRECT ANALYSIS USING ALLELE-SPECIFIC SYNTHETIC PROBES

5'- CTGCCTATT[-G-IGTCTATTTT-5' normal probe

5'- CTGCCTAT T[A--]G TCTAT T T T- 3' mutant probe

Figure 6 - The sequence of synthetic allele-specific probes used to identify the common Mediterranean ~+-thalassemia allele (26L The "normal probe" is a 19-nucleotide sequence from near the end of the first intron. The "mutant probe" is the corresponding 19-nucleotide sequence from the ~'-thalassemia allele: it contains a single base substitution IG-A~ as shown.

these disorders are caused by point mutat ions and others by globin gene deletions (12, 13). An extreme example of the lat ter is hydrops fetalis due to homozygous ~- thalassemia 1. Normal individuals have dupli- cated c~-globin genes (12) and may thus be designated ~ /~c~. The two ~ genes are contained on a 23 kb Eco RI f ragment (20; Figure 1, Table 1 ). Fetuses suffering from hydrops fetalis contain no ~ genes (20, 21; Figure 1) and are therefore of genotype - - / - - ; they may be identified prenata l ly (22) because their DNA yields no Eco RI f ragments hybridizing to an ~ gene probe (Table 1 I. For such cases, a [3-globin gene probe is usually included in the hybridization mixture (20, 22), to provide assurance tha t the hybridization reaction itself is proceeding normally. Other forms of~- thalassemia, involving deletion of 1, 2 or 3 (~-globin genes (12), could be diagnosed prenata l ly by DNA analysis, but their clinical severi ty would rarely justify the procedure (16).

Similarly, partial or complete [3 gene deletions responsible for tha lassemias involving the 13-like globin chains may be detected directly by DNA analysis (15, 16). For example, in a rare [3°-thalassemia found among Asiatic Indians, 700 base pairs are deleted from the [3-globin gene (23, 24). P s t I digestion normally releases the gene on a 4.4 kb f ragment (Figure 1), but, in the case of the [3 ° deletion (Figure 1), the remaining [3 gene sequences are released on a 3.7 kb P s t I f ragment (15,

Many point mutat ions do not alter restriction sites and thus cannot be detected by method 1.a. A general approach to identifying any single-base change (or any other changes, for that matter) in the sequence of a gene is to synthesize synthetic oligodeoxynucleotides which may then be end-labelled with 3._,p for use as probes. This approach has now been used by two groups to identify mutan t [3-globin genes by Southern blot analysis of human DNA and it has been proposed for use in prenatal diagnosis (25, 26). Single base substi- tutions are carried by both mutan t genes studied; one was the [3s allele (25) and the other was the common Medi ter ranean [3 '- thalassemia allele (26), which has a G-A replacement in the first intron (27, 28).

The method involves preparat ion of two synthetic probes, one tha t will recognize only the mutan t form of the gene and another tha t will recognize only the normal form. Thus, to identify the [3 ' - thalassemia allele, two probes 19 bases long, bracket ing the site of the muta t ion and differeing only at that site (Figure 6), were synthesized (26). Hybridization was performed under s t r ingent conditions such tha t the single mis- match prevented reaction of the normal probe with the mu tan t gene sequence and of the mutan t probe with the normal gene sequence; note that, as expected, DNA from heterozygotes reacted with both probes (26; Table 2). (Barn H1 digests of human genomic DNA were used to test the method and a 1.8 kb [3 globin gene f ragment (Figure 1) was detected).

Allele-specific synthetic probes have the advantage of g e n e r a l i t y - - a n y mutan t gene is potentially detectable using them. This approach is, however, at the l imit of presently available technology for prenata l diagnosis and it requires, in addition, the preparat ion of two synthetic probes for each individual muta t ion to be analysed. Therefore, it will probably only be applied to prenata l diagnosis where other methods are not feas- ible (14). However, synthetic probes may also be useful in other ways. A recent example is provided by the identification (with yet another allele-specific synthetic probe) of the specific 13 gene muta t ion associated with a mild form of [3-thalassemia in Medi ter ranean pat ients (29).


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3. INDIRECT ANALYSIS USING LINKED RESTRICTION SITE

POLYMORPHISMS AND CLONED PROBES Prospects for prenatal diagnosis of other genetic diseases and for genetic counselling

Certain restriction sites are polymorphic (30), i.e. they may or may not be present in a given individual. Such restriction site polymorphisms are common in the region of the B-like globin genes (31, 32) and probably elsewhere in the genome as well (31}. Their presence may be recognized by variations in restriction fragment length, detectable by Southern blot analysis (30). Restriction site polymorphisms may serve as neutral genetic markers to indicate the presence of a particular mutant or normal allele to which they are closely linked.

Polymorphic restriction sites in the region of the [3-like globin genes have been used extensively in prenatal diagnosis of hemoglobinopathies 114, 33-36). This approach does not require detection (or even knowledge) of the basic genetic defect, but usually does require preliminary studies to establish, for each fami- ly, the linkage relationships between the polymorphic sites and the mutation responsible for the genetic disease. Typically, both parents and one affected or normal child are analysed (14, 35). The details of the approach have recently been summarized in a report on 95 pregnancies at risk for sickle-cell anemia or [3-thalassemia (14). Ten different polymorphic restriction sites in the B-globin gene region were used. Definitive prenatal diagnosis solely by analysis of these restriction site polymorphisms was possible in 82 cases (86 percent) and was proved correct in all 78 cases available for confirmation when the report was published (14).

It is worth re-emphasizing that there are pregnancies where prenatal diagnosis is possible by this indirect approach, as well as a minority of cases where diagnosis is not possible. This latter situation arises, for examle, when both parents are homozygous for all polymorphisms studied. But, when diagnosis is possible, it is essentially 100% accurate, both in theory 114, 35} and in practice (14).

4. PRESENT STATUS OF PRENATAL DIAGNOSIS OF

HEMOGLOBINOPATHIES BY DNA ANALYSIS

Prenatal diagnosis of hemoglobinopathies by DNA analysis is now a well-established experimental technique (15, 16). When the disease is caused by a deletion or by a point mutation that eliminates a suitable restriction site, the genetic lesion may be identified directly by hybridization with a cloned probe (18, 19, 22). For point mutations that do not affect a restriction site, the method of choice, at present, would appear to be that of indirect analysis with the aid of restriction site polymorphisms and cloned probes (14, 35). In those cases where diagnosis is not possible by the latter method, the use of synthetic, allele-specific probes may be proposed; it should be noted, however, that allele- specific probes have thus far been employed only to identify mutant [3 gene alleles in genomic DNA isolated from peripheral blood (25, 26, 29) and not to perform prenatal diagnosis.

The methods of prenatal diagnosis by DNA analysis described above are quite general and may be applied to other single gene disorders when suitable probes become available. Although no other genetic diseases are so well studied as the hemoglobinopathies, some recent preliminary results with two disorders showing an autosomal recessive mode of inheritance illustrate the kind of results that are now being obtained. Genomic DNA from patients suffering from type A isolated growth hormone deficiency {37), or from a deficiency in apolipoprotein A-I (38) was examined by restriction enzyme digestion, Southern blotting and hybridization with gene-specific probes. The results in each case were consistent with the basic defect in these disorders being a deletion within or near the corresponding gene sequences. Similar results were obtained with DNA from patients with hemophilia B and anti-factor IX antibodies, an inherited X-linked disorder (39). Clearly, prenatal diagnosis may soon be possible for these diseases and others like them.

Note, however, that in the case of inherited diseases for which no protein defect has been identified, progress toward identification of the genetic lesion will be slower (but see below}. This is so because protein sequence information provides the most direct route to the cloning of the corresponding cDNAs (91, and it is the availability of cloned cDNAs that opens the door to genomic cloning as well as to DNA analysis by Southern blotting.

Concerning genetic counselling, DNA lesions detectable by Southern blot analysis and serving for prenatal diagnosis can, in general, also serve to establish the genotypes of (adult) individuals. The quality of genetic counselling that could be offered to these individuals would thereby be improved. For example, several relatives of the apolipoprotein A-I deficient patients discussed above were found, by DNA analysis, to be previously unrecognized heterozygotes for the genetic lesions found in homozygous form in the patients (38).

From another point of view, the availability of prenatal diagnosis by any means, including DNA analysis, has put genetic counselling into a somewhat different perspective. Prospective parents who might previously have been unwilling to risk having children because of the danger of genetic disease may now feel free to do so, if they can be assured by prenatal diagnosis that their children will be normal.

Activation of cellular oncogenes

The activated form of the human c-Ha-ras oncogene is one member of a class of human transforming genes that cause a morphological transformation of NIH 3T3 cells in cell culture. This activated oncogene was origi- nally isolated from a human bladder carcinoma cell line. Remarkably, the mutation responsible for the activation of c-Ha-ras is a single base substitution in the coding sequence of the gene (40, 41). This mutation abolishes a restriction site for Hpa II and Msp I and can therefore be directly detected by Southern blot analysis



(41). Digestion with Hpa II and/or Msp I and hybridization with a probe specific for this part of the oncogene yields two fragments of 355 base pairs and 57 base pairs from the normal oncogene and a single fragment of 412 base pairs from the mutated oncogene (42). In a recent survey of 29 human tumors, no cases were found where the specific mutation in codon 12 ofc-Ha-ras was present (that is, in all cases the 355 base pair fragment, and not the 412 base pair fragment, was observed) (42).

These results, even though negative, are pertinent to this presentation. They illustrate that the techniques used to look for activated cellular oncogenes in human tumors are comparable to those presently applicable to prenatal diagnosis of genetic disease. Thus, the technology exists to identify specific activated cellular oncogenes in human tumors, should it become clinically pertinent to do so. For the moment, however, such studies are directed towards defining the role, if any, that activated cellular oncogenes may play in human cancer.

Use of nuc l e i c acid h y b r i d i z a t i o n for ident i f i ca t ion of microb ia l p a t h o g e n s and for m o l e c u l a r e p i d e m i o l o g y

Southern blotting, as well as colony and dot hybridization (two other methods involving nucleic acid hy- bridizations} have recently been employed to identify nucleic acids of viral or bacterial pathogens. For example, (i) Southern blotting has been used to identify patients in which the DNA of hepatitis B virus is integrated into liver cell DNA or in which the viral DNA is present in non-integrated form in liver cells (43-45); (ii) enterotoxigenic E. coli, carrying genes for the heat- labile or for the heat-stable toxins, have been identified by colony hybridization (46); (iiil proviral DNA of the human T-cell leukemia virus has been detected in lym- phocytes from 2 patients with AIDS by Southern blotting (47); (iv) human rotavirus has been detected in stool suspension by dot hybridization (48).

In the cases involving detection of viral nucleic acid sequences within cells, molecular hybridization of the type employed is clearly the method of choice. Also, conventional immunologic or bioassays for the heat- stable and heat-labile toxins of E. coli are costly and inconvenient for large scale epidemiological studies, such that DNA analysis is an attractive alternative method to be investigated. And finally, the filters on which the rotaviral RNA-containing stool samples were dotted could be stored for two months and could be transported by mail without loss of activity, again giv- ing the hybridization method a certain advantage for epidemiological studies over the presently used (immu- nological and electron microscopic) methods.

In any case, nucleic acid hybridzation assays seem likely to complement existing methods for the detection and study of microbial pathogens. These hybridization assays are evidently based upon the same principles of nucleic acid recognition as are the methods used to detect mutant and normal genes in human cells by DNA analysis. Furthermore, the two sorts of analysis, one in pathogenic microbiology and the other in human genetics, are performed in similar or identical fashion such

that they may be considered as variants of the same basic methodology.

Future p r o s p e c t s for D N A ana lys i s

Significant improvements in methods for DNA analysis may be forthcoming. Probes incorporating biotinylated derivatives of dUTP, and detectable by fluorescent antibodies or by an enzymatic assay, can now be prepared (49, 50). Should they prove re- liable and sufficiently sensitive, they could replace 32P-labelled probes. Not only would biotinylated probes be non-radioactive, but they would be more stable (32p has a 2-week half-life). Also, a method for obtaining chorionic villi biopsies at from 7 to 13 weeks of ges- tation has recently given favorable results (51, 52). This procedure, if proven safe, would be advantageous for prenatal diagnosis by DNA analysis. It gives gener- ous quantities (10 to 75 p.g) of fetal DNA and permits therapeutic abortion, when indicated, substantially earlier than when amniocentesis furnishes the fetal cells (52).

The prospects for further rapid advances in human genetics are bright, as witnessed by recent optimistic assessments (53-55). Indeed, a general approach has been suggested for mapping the whole human genome using restriction site polymorphisms as marker loci (53). The method would involve first the identification of polymorphic loci and then the establishment, by pedigree studies, of genetic linkage of these loci (at the level of - 10 centimorgans or roughly 10,000 kb) to known genes or to genes responsible for inherited diseases. Note that this approach is quite the opposite of that described above in relation to prenatal diagnosis using linked restriction site polymorphisms. There, polymorphic restriction sites were identified near (within about 10 kb) the previously cloned ~ globin gene. The difference of approximately 1000-fold in the degree of physical linkage involved in the two ap- proaches is formidable, but does not necessarily pose insurmountable problems for the long term (see references 53 and 55 for a full discussion). In any event, an example of linkage, at a distance of approximately 10 centimorgans, between a polymorphic restriction site on the X-chromosome and the locus for Duchenne muscular dystrophy has been reported (56). This may repre- sent the first of many examples where a previously unmapped mutant gene is mapped by linkage to a polymorphic restriction site.

A c k n o w l e d g e m e n t s

I thank C~line Dallaire for preparing the illustrations, Chantal B~dard for typing the manuscript and Markus Affolter and Luc B~langer for comments.

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some clinical implications of recombinant dna technology with emphasis on prenatal diagnosis of...

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