Detection of point mutations associated with genetic diseases by an exon scanning technique

GENOMICS

8,656-663

(1990)

Detection of Point Mutations Associated with Genetic Diseases by an Exon Scanning Technique DANIEL

L. KAUFMAN, *rl VIJAYA RAMESH,t ANDREA I. MCCLATCHEY,~ JOHN H. MENKES,?~ AND ALLAN J. ToBIN**I’,‘IT,*

*Department of Biology, *Department of Neurology, §Department of Pediatrics, llMolecular Biology Institute, and BBrain Research Institute, University of California, Los Angeles, California 90024; and tDepartment of Neurology and Genetics Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 Received

April

18, 1990;

INTRODUCTION

Determining the genetic basis of inherited human diseases remains a major challenge to contemporary genetics. Linkage analysis and the isolation of specific “candidate genes” can provide suspect sequences for evaluation in disease states. The availability of a complete human genome sequence will aid in the identification of culpable mutations, provided that

osss-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

July 13, 1990

sequence alterations can first be identified in disease states. Detecting genetic lesions in suspect genes may be a formidable task (reviewed in U.S. Congress Office of Technology Assessment, 1986; Caskey, 1987). Southern and Northern blotting often cannot detect small genetic lesions. Synthetic oligonucleotides, which are useful for detecting the presence of known polymorphisms, are unsuited for screening large genes or mRNAs for unknown sequence alterations, and DNA sequencing, even in its most automated forms, may be impractical for evaluating genes or mRNA in disorders of unknown etiology. The ability of RNase A to cleave RNA probes at positions where it is mismatched to a target DNA or mRNA sequence provides a powerful method for surveying large regions of DNA or mRNA for small mutations (Myers et al., 1985; Winter et al., 1985). However, because many genes are expressed only in inaccessible tissues, such as the brain, mRNAs from a specific gene are often unobtainable. On the other hand, genomic DNA, which is readily available by blood sampling or amniocentesis, contains all genes and can be analyzed for sequence alterations independently of the site or timing of gene expression. Myers et al. (1985) used RNase A to detect point mutations in genomic DNA using colinear RNA probes, i.e., probes transcribed from cloned genomic DNA. Analysis of large nuclear genes by this method would require many contiguous RNA probes from cloned genomic DNAs to span the length of a suspect gene. Furthermore, the presence of repetitive sequences in probes derived from genomic DNA would hinder this analysis. We have therefore devised a strategy that uses RNA probes derived from cDNA templates (cRNAs) to detect lesions in suspect genes. The cRNA probes form heteroduplexes with the exons of the target

A major challenge in genetics is identifying the basis of human heritable disease. We describe an “exon scanning” technique which surveys exons in genomic DNA for sequence alterations. By hybridizing genomic DNA to RNA probes derived from cDNAs, we can use RNase A to survey entire coding regions, comprising exons spread across extensive regions of genomic DNA, for mutations associated with genetic disease. Exon scanning of the B-globin locus in the DNA of patients with 12 different hemoglobinopathies detected all of the culpable single base substitutions and deletions, but not single base insertions. Our analysis also revealed unsuspected polymorphisms and corrected a diagnosis originally based on hemoglobin electrophoresis. Exon scanning of the ornithine aminotransferase gene in a gyrate atrophy patient detected and localized a mutation in the sixth exon. Subsequent PCR amplification and sequencing characterized this as a missense mutation (proline + glutamine). Exon scanning of genomic DNA for sequence alterations, in combination with PCR amplification and sequencing, should be a generally useful strategy for evaluating suspect genes in disorders of unknown etiology, as well as for clinical diagnosis. o m90 Academia PWLX, IIIC.

’ Present address: Molecular Genetics stitute, P.O. Box 85800, San Diego, CA ’ TO whom correspondence should be of Biology, University of California, Los

revised

Laboratory, The Salk In92138. addressed at Department Angeles, CA 90024-1606.

656

DETECTION

OF

POINT

MUTATIONS

S 5*

3’

Exon

I

Exon

2

Exon -denature

genomic

-hybridize transcribed

to cRNA from

3

DNA probe cDNA

I

n

Exp”’ 0 EXO”k /L”~Genomic DNA ---

\\/

Ribonuclease cleavage

c RNA

probe

A sites WT

S

FIG. 1. Flow diagram of exon scanning. A labeled cRNA probe, transcribed from a wild-type cDNA, is hybridized to denatured genomic DNA. In the resulting cRNA/DNA hybrid, the exon sequences of the DNA base pair with the cRNA probe. The intron sequences, which may constitute most of the gene, loop out as single-stranded DNA. The RNA-DNA hybrid is then treated with RNase A, which digests ail unhybridized probe and cleaves the cRNA at exon/intron junctions, generally producing exon-sized cRNA fragments. RNase A also cleaves at mismatched base pairs. The cRNA fragments are fractionated by size by electrophoresis under denaturation conditions and visualized by autoradiography. The example shows a @-globin gene from a HbS/HbS homozygote. The loss of exon 1 and the appearance of two new bands localizes the HbS mutation relative to the ends of exon 1.

gene, while the introns, which may constitute most of the gene, loop out as single-stranded DNA (Fig. 1). We can therefore use RNase A to survey an entire coding region, composed of exons scattered over many kilobases, for point mutations. Since only exons are analyzed for mutations, we call this technique “exon scanning.” MATERIALS

AND

METHODS

All genomic DNA samples were pretreated with RNase A (Sigma, 5 pg/ml) for l-3 h (37’C) to remove RNA that might hybridize with the probe. The RNase A was removed by proteinase K digestion (0.3 mg/ml, 1 h at 37’C), followed by phenol and chloroform extractions and ethanol precipitation. Human @globin sense and antisense cRNA probes were transcribed from a HindIII-EcoRI subclone of SP64-HpAG-IVS

BY

EXON

657

SCANNING

1,2 in Bluescribe (Stratagene) (Krainer et al., 1984). These transcripts contain all of exons 1 and 2 and part of exon 3 (50 bases) of the j3-globin gene. Human OAT sense and antisense cRNA probes were transcribed from a 2.0-kb EcoRI subclone of HOATl in Bluescribe (Ramesh et al, 1986). Probes were synthesized according to the directions of the RNA polymerase supplier (BRL), using [(x-32P]CTP (400 Ci/mmol), ranging from 30 j&f (for @globin probes of 420 bp) to 70 PM (for OAT probes of 2.0 kb) as the only dCTP source in the transcription reaction. Following transcription and DNase treatment, the full-length transcript was purified by gel electrophoresis in low-melting-point agarose gels containing ethidium bromide. The full-length probe was excised and placed in an Eppendorf tube, 10 pg of carrier tRNA was added, and the volume was brought to 400 ~1 with TE + 0.1% SDS. The sample was heated to 65°C for 5 min and then extracted with phenol and chloroform. Following removal of a portion to determine [32P]CTP incorporation, the cRNA was precipitated with Na-acetate and isopropanol. Following centrifugation, a 70% EtOH wash, and vacuum drying, the sample was resuspended in TE + 0.1% SDS. Approximately lo6 cpm of probe was hybridized to 6-15 pg of genomic DNA using the method of Myers et al. (1985). The RNase A reaction and subsequent steps followed those of Myers et al., with the exception of using more stringent RNase A cleavage conditions, generally, 1 h at 30°C.

RESULTS

P-Hemoglobinopathies To evaluate the ability of RNase A to recognize point mutations in cRNA/DNA heteroduplexes, we examined the @globin locus in 12 patients with hereditary diseases that affect hemoglobin synthesis. Hemoglobinopathies are the most common genetic disorders worldwide, and early detection and clinical intervention are often crucial (reviewed in Bunn and Forget, 1986). We studied genomic DNA from (1) five P-thalassemia patients, each with a different single base substitution which causes a nonsense mutation: nonsense 15,17, 37,39, and 43 (the numbers refer to the codon at which the mutation occurs); (2) a HbS and a HbC patient, in which a missense mutation caused by single base substitution leads to an altered structure and function of the ,&globin chain; and (3) five different patients with different frameshift mutations: 6(-l), 8(-2), 41-42(-4), 8-9(+1), and 106107(+1) (the numbers in parentheses indicate the

658

KAUFMAN

ET AL.

106-107 3'

Exon 1

Exon 2

Exon

3 100 ni

Sense

Antisense

200-

237 43

154-

l-

142-

17 15

'b

75-

(B)

r\

()~RI

Exon 1 39139

IV17

5w+

C/IVSlJ

Exon 3 Sense -

+

-

+

-

II----

-.-

-

(2$)

*

-

-

6/+

---

-

-

8/8 -y-

-

-

(2s

-

-

-

s/s

-

-

-

HPFH

-

-.-

-

37H

-----

15/15

-

-

-

2ty---

Antisense

v-

c2n

41s42/41-42 15/15

-

Exon 2

+ +

+

+ +

+

+ +

+

-

DETECTION

OF

POINT

MUTATIONS

number of nucleotides lost or gained at a particular codon). The fl-globin gene consists of three exons of 142 (exon l), 223 (exon 2), and 261 (exon 3) bases which extend over 1.6 kb in the genome (Lawn et al., 1980). The &globin sense and antisense cRNA probes were 420 bases in length, containing all of exons 1 and 2 and the first 50 bases of exon 3. Exon scanning of the P-globin gene in these hemoglobinopathies showed that RNase A generally cleaved the cRNA probe at exon/intron junctions to produce exon-sized fragments, as well as at the mismatches caused by mutations in the genomic DNA (Fig. 2). Cleavage at a mismatch converted an exon-sized cRNA fragment into two new fragments, which were absent in wild-type. The sizes of these new fragments established the mutation site relative to the ends of the affected exon. Homozygous mutations displayed complete cleavage of the probe at mismatches, whereas heterozygote mutations showed about 50% cleavage (Fig. 2). As expected, RNase A readily cleaved mismatches involving pyrimidines in the cRNA probe (Table 1; Myers et al., 1985; Myers and Maniatis, 1986). For example, using the sense P-globin cRNA probe, RNase A easily detected the C/A mismatch (cRNA/ DNA) caused by the nonsense 39 mutation (Fig. 2, lane 39/39). However, the corresponding G/T mismatch produced in heteroduplexes with the antisense

BY

EXON

SCANNING

659

cRNA was not susceptible to RNase A cleavage. By using both sense and antisense probes, we could detect all of the known substitution and deletion mutations (Table 1 and Fig. 2). This efficiency in detecting point mutations in genomic DNA with RNase A is higher than that predicted by Myers et al. (1985) and Myers and Maniatis (1986), whose criterion for detectability was at least a 50% cleavage at a mismatch. We are able to detect cleavages that take place at low efficiency, as long as the background is low. Thus, several single base substitutions and all deletion mutations are visible with both sense and antisense probes (Fig. 2 and Table 1). Both sense and antisense probes revealed small deletion mutations, even when the mismatched RNA bases were purines (Table 1). The probe thus appears to be more vulnerable to RNase A at deletions than at substitutions. Perhaps greater “breathing” of the heteroduplex occurs at the cRNA loop out over a deletion than at the mismatched bases at a substitution mutation, which makes the adjacent pyrimidines more susceptible to RNase A cleavage. The converse situation occurs at insertion mutations: the extra nucleotides of the genomic DNA loop out as single-stranded DNA in the cRNA/DNA hybrid, whereas the cRNA probe remains entirely in duplex. This may be the reason that RNase A could not detect the two types of single base insertion muta-

FIG. 2. (A) Exon scanning of human hemoglobinopathies. All substitution and deletion mutations were detected with either sense or antisense probes, whereas the insertion mutations examined (106-107 and S-9) were not. To minimize the complexity of this figure, we show data only for the probes (sense or antisense) for which RNase cleaved at the mismatch. Detection of neutral polymorphisms: The DNAs designated 17/43,17/17 56/+, 15/l& 818, and C/IVS15 all contain a phenotypically silent mutation in codon 2 in addition to their diseasecausing mutations. This codon 2 mutation leads to cleavage of exon 1 with the sense probe, despite the fact that the sense probe does not reveal the disease-causing mutations (at codons 6,8, 15, and 17). The products of codon 2 cleavage are labeled 2 and 2b. The nonsense 17 mutation in exon 1 is detectable only with the antisense @-globin probe. However, exon scanning with the sense probe clearly showed that about 50% of the exon 2 fragment had been cleaved to produce two new fragments of 82 and 140 bases (labeled 56 and 56t,). These data imply that a sequence alteration occurs 82 bases in from one end of exon 2, corresponding to amino acid 56 or 77. PCR amplification and sequencing of exon 2 from this patient’s DNA confirmed that the patient was heterozygous for a new type of neutral polymorphism in the fl-globin gene at codon 56 (GGC to GGT). (H. Kazazian, personal communication). Artifacts: While the major bands observed correspond to the protected exon-sized fragments and the expected cleavage products due to mismatches, several types of artifacts were observed: (i) Cleavage at perfectly matched base pairs. Two unexpected bands (labeled *) are the product of RNase A cleavage within exon 2 at perfectly matched base pairs about 145 nucleotides from the 5’ end of this exon. This region appears sensitive to RNase cleavage even though it is not particularly rich in pyrimidines or in A and T. This artifact band is itself cleaved again by RNase A at the mismatches due to mutations producing the subfragments labeled 3gt, and 41-42, 43r,. The conditions for RNase treatment here are more stringent than those described by Myers et al. (3,9). Under the less stringent conditions used by Myers et al., these artifacts do not appear, but cleavage at mismatches is less than 100%. (ii) Partial cleavage at intron/exon junctions is observed with the antisense cRNA. For example, the mismatches due to nonsense 37 and 43 are readily cleaved by RNase A, but exon/intron junction between exon 1 and exon 2 is cleaved with only about 50% efficiency. When cleavage occurs at the mismatch, but not at the exon/intron junction, a fragment containing exon 1 linked to the nucleotides of exon 2 prior to the mutation is produced. These bands migrate just below the band labeled 37, in the nonsense 37 lane, and just above the band labeled 43, in the nonsense 43 lane (A). It is unclear why greater partial cleavage at intron/exon junctions occurs with the antisense probe. These junctions all involve pyrimidines in the antisense probe, while in the sense probe the exon l/exon 2 junction involves only purines and yet is readily cleaved. Perhaps conditions other than those used here could maximize cleavage of intron/exon junctions. (iii) RNase A may nibble the ends of cRNA fragments to produce a nested set of bands separated by a few base pairs. For example, using the sense cRNA, there are two minor bands immediately below the exon 1 band due to the loss of a few bases from this fragment. (B) A diagrammatic representation of the expected RNase A cleavage sites in the Hb cRNA probe. DNAs labeled with $ additionally contain a neutral codon 2 polymorphism which would produce the additional cleavage shown at the bottom of B.

660

KAUFMAN

TABLE Exon Sequence alterations Substitutions Nonsense 39 homozygote

Exon

2

Scanning

of Human

Sequence content cRNA/DNA

ET

AL.

1 Hemoglobinopathies Sense probe detectability

C CC AG GG TC A

+

G GU GG CA CC T

-

Sequence context cRNA/DNA

Antisense probe detectability

CUGGG GA CC T

C

Nonsense 15* homozygote

1

Nonsense 17* homozygote

1

56 polymorphism heterozygote

2

Nonsense 17/43* heterozygote

1

A GC AG CG TC A

-

U CU GC GA CG T

Nonsense 17/43* heterozygote

2

G UU AG AA TC A

+

C CUAA GA TT T

Nonsense 37 heterozygote

2

G UG AC AC TG T

-

C GU CA CA GT A

homozygote

A UG GG AC CC A

+/-

U CC CA GG GT T

t

HbC/IVS 1,5 heterozygote

G CU AG GA TC T

+/-

C CU AG GA TC A

+/-

2* polymorphism

C CA CU GT GA A

t

HbS

E homozygote

8 (-AA)*

t

G uu cc AAGG T

AGGUG TC AC T

2 GU’AG CA TC T

CU’AG GA TC A

cuuu

AAAG UC AA AG TT

UU AA

U CC CA GG GT

AA GU CA

A% TG GA

1

3

GA CT

A UG GG AC CC AG TC

Frame shift insertions 106-107(+G) heterozygote

CU’GC GA CG T

GGCAA CC TT A

heterozygote

homozygote

GCAAG CG TC A

Frame shift deletions 41-42 (CTTT) homozygote 6(-A)

CC AC GG TG A

UG GG AC CC C

-

CC CA GG GT G

DETECTION

OF

POINT

MUTATIONS

TABLE Sequence alterations 8-9 (+G) heterozygote

Exon

1

Sequence content cRNA/DNA AG AA TC TT C

BY

EXON

661

SCANNING

l-Continued Sense probe detectability -

Sequence context cRNA/DNA uu cu AA GA G

Note. The efficiency of RNase cleavage of various types of mismatches with cRNAs hybridized to R-looped genomic of Myers et al. using colinear RNA probes (3,9). The nonsense 43, HbS, HbC, and all three deletion mutations could sense and antisense probes, since even partial cleavage at these mismatches gave readily detectable bands. The ability at a mismatch in probes of both orientations may be due to the sequence context of these mutations and the pyrimidines. The nucleotides surrounding each mismatch in the RNA strand are indicated in a 5’ to 3’ direction. +/-, partial cleavage detected, -, undetectable. * indicates that these individuals also contain the neutral codon 2

tions examined. However, larger insertions might be detectable, since cleavage does occur at exon/intron junctions, where the DNA introns loop out. In addition to the disease-causing sequence alterations described above, exon scanning also detected several unexpected neutral polymorphisms in the pglobin genes. These sequence variations were not detectable with the small synthetic oligonucleotides used in the initial characterization of the disorders. One of these polymorphisms, labeled 56 and 56, in Fig. 2, was precisely characterized as a new polymorphism in codon 56 by PCR amplification and direct sequencing (H. Kazazian, personal communication; details in legend to Fig. 2) (Mullis and Faloona, 1987). We also detected the previously described neutral polymorphism in codon 2 in P-globin loci containing the 17/43,17/17 56/+, 15/15,8/8, and C/IVS15 mutations (labeled 2 and 2, in Fig. 2; Krainer et al., 1984). This is the first reported association of the codon 2 variant with the nonsense 15 mutation. Neutral polymorphisms such as these are useful in genetic analyses for predicting other mutations that may be present. Exon scanning of genomic DNA from a patient originally characterized as homozygous HbE by protein electrophoresis detected a sequence alteration that was indistinguishable from the nonsense 43 and 4142(-4) thalassemia mutations (Fig. 2, lane HPFH). Subsequently, a blood sample from the patient was recharacterized as HbE + @-thalassemia. We observed three general types of artifacts in exon scanning the P-globin gene: (a) partial cleavage of the cRNA probe at perfectly matched base pairs, (b) partial cleavage of the cRNA at intron/exon junctions, and (c) RNase nibbling at the ends of cRNA fragments to produce a nested set of bands separated by a few base pairs (details in legend to Fig. 2). Despite these artifacts, exon scanning allowed us to detect and diagnose the genetic lesion underlying most of the hemoglobinopathies tested and to identify other

Antisense probe detectability -

DNA agree with those be identified with both of RNase A to cleave availability of nearby +, readily detected; polymorphism.

unsuspected silent polymorphisms. These results suggest that exon scanning can be used to survey larger genes for mutations that may underlie inherited disorders of unknown etiology. Gyrate Atrophy Gyrate atrophy (GA) is a hereditary degenerative disease of the retina and choroid plexus that leads to progressive blindness. Patients with GA are severely deficient in enzyme activity for ornithine aminotransferase (OAT, EC 2.6.11.13) which catalyzes the interconversion of ornithine, glutamate, and proline (reviewed in Valle and Simell, 1983). Although the gene for human OAT has been cloned and found to be linked to GA, alterations in the OAT gene of GA patients have generally not been observable by Southern or Northern analysis (Ramesh et al., 1986,1988a; Mitchell et al., 1988a). These data suggest that the severely reduced OAT activity may arise from subtle sequence alterations that are not detectable by these types of analyses. We therefore decided to use exon scanning to examine the OAT gene in GA patients. The OAT gene lies on chromosome 10 and spans 21 kb (Mitchell et al., 1988b; Ramesh et al., 1987). There are also several nonfunctional pseudogenes on the X chromosome. For exon scanning we used an OAT cRNA probe of 2.0 kb which contains the entire OAT coding region as well as most of the 5’ and 3’ nontranslated regions (Ramesh et al., 1986). This cRNA probe should hybridize to 10 exons of the OAT gene, which are dispersed across 16 kb of genomic DNA (Mitchell et al., 1988b). Exon scanning of the OAT gene with the 2.0-kb sense cRNA showed a complex pattern that was only partly consistent with the expected exon sizes. Our data suggest some cleavage within exons. We nonetheless could identify a new fragment of 77 bases that appears in a GA patient (referred to as L99 in Ramesh et al., 1986), which is not observed using wild-type

662

KAUFMAN

-I30

128 _

FIG. 3. Identification of a point mutation in OAT in a gyrate atrophy patient. (A) A 2.0-kb sense probe derived from OAT cDNA was hybridized to genomic DNA from a gyrate atrophy patient (L99) and a normal (wt) control. Exon scanning gives a new RNA fragment at 77 bases, concomitant with a 50% loss of a 130base fragment. (B) A 500..base OAT antisense probe containing sequences that span exons 2-7 further localizes the new 77-base fragment to exon 6.

DNA (Fig. 3A). In the same sample, there was a 50% loss of a 130-base fragment. To verify this finding, we used a smaller 500-base antisense probe (corresponding to nucleotides 196704 of HOATl (Ramesh et al., 1986)). This probe more clearly revealed the L99 mutation (Fig. 3B). By using this smaller probe and by predigesting genomic DNA with restriction endonucleases to cut exons at specific sites, we mapped the mutation approximately to codon 199, within exon 6. RNase digestion of hybrids of the 500-base probe with L99 DNA produces a new 77-base fragment and a 50% loss of a 128-base fragment corresponding to exon 6. A loo-base fragment, caused by internal cleavage of the cRNA within this exon, is also reduced in the L99 sample. PCR amplification and sequencing of this region confirmed that the patient was heterozygous for a single base missense mutation 77 bases from the 5’

ET

AL.

end of exon 6 (codon 199; CCA to CAA, proline to glutamine). This mutation produces a pyrimidine mismatch only with the sense OAT cRNA; therefore, it is surprising that the antisense probe detects this mutation so effectively. Analysis of this pedigree shows that this mutation cosegregates with the disorder (V. Ramesh, unpublished results). The other OAT gene in this patient must contain a different mutation, i.e., the patient is a compound heterozygote. The second mutation may lie at an exon-intron boundary, in an intron, or in a nontranscribed regulatory sequence. Or this technique may simply not detect it. The three OAT pseudogenes on the X chromosome did not interfere with analysis of the functional OAT gene by exon scanning. The pseudogenes show about a 10% sequence divergence from the functional OAT gene (A. McClatchey, personal communication), so RNase A should cleave any probe that hybridized to the pseudogenes into small fragments. Exon scanning thus appears to be suitable for surveying individual members of multigene families. Several other groups have identified single base mutations in the OAT gene of GA patients (Mitchell et al., 1988a, 1989; Ramesh et al., 1988b). The mutation we identify here, however, has not been previously described. Gyrate atrophy appears to be genetically heterogeneous, with several alleles of the OAT gene leading to a deficiency in enzymatic activity. Diagnosis of the type of underlying lesion may be important for predicting the age of onset, severity, and effective therapy. Some GA patients for example, are responsive to pyridoxine administration, the cofactor for OAT (Valle and Simell, 1983; Ramesh et al., 198815).

DISCUSSION The heterogeneity of genetic lesions that underlie a single disease phenotype complicates the determination of both genetic etiology and diagnosis. When a gene is expressed only in an unavailable tissue, or only during embryonic life, direct study of mRNA or protein is not possible, and genetic analysis is limited to studying DNA. For a larger gene, this task is a formidable one, even with PCR and automated sequencing techniques. The strategy we describe here significantly reduces the magnitude of this task by first scanning suspect genes and then sequencing implicated gene regions after PCR amplification. This strategy allows a rapid survey of exons that extend over many kilobases in the genome. Exon scanning also focuses on coding region mutations that can alter protein structure and function.

DETECTION

OF

POINT

MUTATIONS

Our approach offers several advantages for the search for genetic lesions: (a) it requires small amounts of DNA which can be obtained from lymphocytes or amniotic fluid; (b) it can utilize both sense and antisense cRNAs to examine both DNA strands; (c) it can analyze many DNA samples on a single gel; (d) it avoids detection of the frequent polymorphisms in introns, whose significance may be harder to discern; and (e) it can map the position of a sequence alteration for precise characterization by PCR amplification and sequencing. Although exon scanning by its very nature cannot detect mutations in intron or promoter regions it is especially useful for testing hypotheses concerning candidate genes in heterogenous disorders and for surveying expressed genes identified by linkage analysis. The method also provides a new tool for clinical and prenatal diagnosis. Characterization of mutant alleles by this method should aid disease classification and will be valuable in associating specific allele types with clinical manifestations, such as severity, mode of expression, penetrance, and age of onset. ACKNOWLEDGMENTS We thank H. Kazazian, R. Myers, T. Maniatis, S. Orkin, T. Huisman, V. Vadakan, and Y. W. Kan for providing cloned and genomic DNA samples from hemoglobinopathies; H. Kazazian for PCR amplification and genomic sequencing of the @-globin codon 56 silent polymorphism; R. Myers, G. Evans, J. Gusella, and A. Berk for helpful advice; H. Kabe, N. Lee, and K. McKeown for help in manuscript preparation; M. Khrestchatisky, M. Erlander, and J. MacLennan for their comments on the manuscript; and other members of the A. Tobin and J. Gusella laboratories for many helpful conversations throughout the course of this work. This work was supported by grants to A.J.T. from NINCDS (NS 22256 and NS 20356) and a program project grant to A. V. Delgado-Escueta (NS 20198). D.L.K. was partially supported by a training grant in Genetic Mechanisms from NIGMS (GM 07185).

BY

BUNN, H. F., AND FORGET, B. G. (1986). “Hemoglobin: ular, Genetic andclinical Aspects,” W. B. Saunders, phia.

2.

CASKEY, C. T. (1987). Disease diagnosis by recombinant DNA methods. Science 236: 1223-1228. LAWN, R. M., EFSTRATIADIS, A., O’CONNELL, C., AND MANIATIS, T. (1980). The nucleotide sequence of the human b-globin gene. Cell 21: 647-651.

3.

4.

KRAINER, A. R., MANIATIS, T., M. R. (1984). Normal and mutant NAs are faithfully and efficiently 993-1005.

RUSKIN, human spliced

G. A., BRODY, L. C., LOONEY, J., STEEL, G., SuCHANEK, M., DOWLING, C., KALOUSTIAN, V. D., KAISERKUPFER, M., AND VALLE, D. (1988a). An initiator codon mutation in ornithine-&aminotransferase causing gyrate atrophy of the choroid and retina. J. Clin. Invest. 81: 630-633.

6. MITCHELL,

G. A., BRODY, L. C., SIPILA, I., LOONEY, J. E., WONG, C., ENGELHARDT, J. F., PATEL, A. S., STEEL, G., OBIE, C., KAISER-KUPFER, M., AND VALLE, D. (1989). At least two mutant alleles of omithine-6-aminotransferase cause gyrate atrophy of the choroid and retina in Finns. Proc. Natl. Acad. Sci. USA 86: 197-201.

7. MITCHELL,

G. A., LOONEY, J. E., BRODY, L. C., STEEL, G., SUCHANEK, M., ENGELHARDT, J. F., WILLARD, H. F., AND VALLE, D. (1988b). Human ornithine-d-aminotransferase. J. Biol. Chem. 263: 14,288-14,295.

8. MULLIS,

K., AND FALOONA, F. A. (1987). Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. In “Methods in Enzymology” (R. Wu, Ed.), Vol. 155, pp. 335350, Academic Press, New York.

9. MYERS, of single matches

R. M., LAF~IN, Z., AND MANIATIS, T. (1985). Detection base substitutions by ribonuclease cleavage at misin RNA:DNA duplexes. Science 230: 1242-1248.

10.

MYERS, R. M., AND MANIATIS, T. (1986). Recent advances in the development of methods for detecting single-base substitutions associated with human genetic diseases. Cold Spring Harbor Symp. Quant. Biol. 51: 275-284.

11.

RAMESH, V., BENOIT, L. A., CRAWFORD, P., HARVEY, P. T., SHOWS, T. B., SHIH, V. E., AND GUSELLA, J. F. (1988a). The ornithine aminotransferase (OAT) locus: Analysis of RFLPs in gyrate atrophy. Amer. J. Hum. Genet. 42: 365-372.

12.

RAMESH, V., EDDY, R., BRUNS, G. A., SHIH, V. E., SHOWS, T. B., AND GUSELLA, J. F. (1987). Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes. Hum. Genet. 76: 121-126.

13.

RAMESH, V., MCCLATCHEY, A. I., RAMESH, N., BENOIT, L. A., BERSON, E. L., SHIH, V., AND GUSELLA, J. F. (1988b). Molecular basis of ornithine aminotransferase deficiency in B-6-responsive and -nonresponsive forms of gyrate atrophy. Proc. Natl. Acad. Sci. USA 85: 3777-3780.

14.

RAMESH, V., SHAFFER, M. M., ALLAIRE, AND GUSELLA, J. F. (1986). Investigation using a cDNA clone for human ornithine DNA 5: 493-501.

15.

U.S. Congress “Technologies Beings,” U.S. D.C.

16.

VALLE, D., AND SIMELL, 0. (1983). In “The Metabolic Basis of Inherited Disease,” 5th ed. (J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, J. L. Goldstein, and M. S. Brown, Eds.), pp. 382-401, McGraw-Hill, New York.

17.

WINTER, E., YAMAMOTO, F., ALMOUGUERA, C., AND PERUCHO, M. (1985). A method to detect and characterize point mutations in transcribed genes: Amplification and overexpression of the mutant c-Ki-ras allele in human tumor cells. Proc. Natl. Acad. Sci. USA 82: 7575-7579.

MolecPhiladel-

B., AND GREEN, h-globin pre-mRin vitro. Cell 36:

663

SCANNING

5. MITCHELL,

REFERENCES 1.

EXON

Office of for Detecting Government

Technology Heritable Printing

J. M., SHIH, V. E., of gyrate atrophy aminotransferase. Assessment (1986). Mutations in Human Office, Washington,