Am. J. Hum. Genet.

49:951-960, 1991

Carrier Detection and Prenatal Diagnosis in Duchenne and

Becker Muscular Dystrophy Families, Using Dinucleotide
Repeat Polymorphisms
P. R. Clemens,* R. G. Fenwick,* J. S. Chamberlain, R. A. Gibbs,* M. de Andradet
R. Chakrabortyt and C. T. Caskey*,t
*Institute for Molecular Genetics and tHoward Hughes Medical Institute, Baylor College of Medicine, Houston; tGenetics Centers, University
of Texas Graduate School of Biomedical Sciences, Houston; and Department of Human Genetics, University of Michigan Medical School, Ann
Arbor

Summary
To improve carrier detection and prenatal diagnosis for Duchenne and Becker muscular dystrophy families,
we determined allele frequencies and measures of variation for four (dC-dA)n * (dG-dT)n loci identified within
a deletion-prone region of the human dystrophin gene. The loci are highly polymorphic, with predicted
heterozygosities of 71.6%-93.3%. Direct DNA sequence analysis of the (dC-dA)n * (dG-dT)n locus in intron
49 revealed an additional length polymorphism which varies by single-basepair increments, is adjacent to
the dinucleotide repeat block, and enhances the polymorphic content of this marker. The four
(dC-dA)n * (dG-dT)n loci are each easily amplified by PCR in two diplex reactions. The variability of allele
lengths at these loci makes them ideal for carrier detection and prenatal diagnosis, often providing diagnostic
information when RFLP analysis is uninformative. These markers have aided in identification of deletion
mutations, exclusion of maternal cell contamination of chorionic villus samples, confirmation of paternity,
and mapping of gene recombinations. The allele identification of these loci can be performed either with
a radiolabel or with an automated, nonradioactive, fluorescent gel detection system.

Introduction

Duchenne and Becker muscular dystrophies (D/BMD)

are allelic forms of an X-linked recessive disease characterized by absent or abnormal dystrophin in skeletal
muscle, resulting in early muscle degeneration and
death. DMD is a common disease that affects 1 /3,500
male births, which has led to an emphasis on the development of simplified tools for carrier detection and
prenatal diagnosis (Chamberlain and Caskey 1990).
Direct mutation identification in the 2.3 x 1 06-bp
dystrophin gene is possible in 65%-70% of cases of
D/BMD by multiplex exon amplification using PCR
(Chamberlain et al. 1988; Beggs et al. 1990) and
Received April 8, 1991; revision received July 3, 1991.
Address for correspondence and reprints: P. R. Clemens, Institute for Molecular Genetics, Baylor College of Medicine, Houston,
TX 77030.
i 1991 by The American Society of Human Genetics. All rights reserved.
0002-9297/91 /4905-0005$02.00

Southern-based analysis (Koenig et al. 1987; Den

Dunnen et al. 1989; Hu et al. 1990). In the remainder
of cases without an identifiable mutation, however,
carrier detection and prenatal diagnosis depend solely
on DNA linkage studies. To date, linkage analysis
in D/BMD has been performed using diallelic RFLP
markers which may be limited by small family size,

inadequate family-member cooperation, and deceased

DMD males (Ward et al. 1989).
There are approximately 50,000-100,000 (dCdA)n (dG-dT)n (hereafter designated (CA)W) loci in
the human genome (Hamada and Kakunaga 1982;
Tautz and Renz 1984). (CA)n loci are a subclass of all
short tandem repeat (STR) sequences. The presence
of these frequently polymorphic loci can be exploited
to improve DNA linkage studies. In addition, STR
loci are of great diagnostic utility because they can
be easily assayed by PCR. One (CA)n locus has been
described in the 3' untranslated region of the human
dystrophin gene (Beggs and Kunkel 1990; Oudet et al.
951

Clemens et al.

9S2
Table I
Oligonucleotide Primers for PCR Amplification and Direct DNA Sequence
Analysis of (CA)n Loci in Human Dystrophin Gene (5' to 3')
Intron and Primer (type)

44:
DMD-44/A (PCR)
DMD-44/B (PCR)
45:
DMD-45/A (PCR)
DMD-45/B (PCR)
49:
DMD-49/A (PCR)
DMD-49/B (PCR)
DMD-49/C (sequencing)
50:
DMD-50/A (PCR)
DMD-50/B (PCR)

..........................

..........................

..........................

..........................

..........................

..........................

.................

..........................

..........................

1990), and four others have been identified in the 5'

terminus (Feener et al. 1991). J. S. Chamberlain (unpublished data) has identified and sequenced six others
and has designed flanking oligonucleotide PCR primers for their amplification. We describe here (a) allele
frequencies and measures of variation of four of these
six (CA)n loci (these four being located in introns 44,
45, 49, and 50 of the human dystrophin gene) and (b)
the use of these markers to improve the accuracy of
linkage analysis for D / BMD carrier detection and pre-

natal diagnosis.
Methods
DNA Samples

Samples for DNA extraction included 15-20 cc of

peripheral blood collected in sodium EDTA, cultured
amniotic fluid cells, and chorionic villus samples.
DNA was isolated using the ABI 340 DNA extractor
(Applied Biosystems, Foster City, CA).
Oligonucleotide Primers
Primers for PCR and direct DNA sequencing were
synthesized on an ABI 380B DNA synthesizer (Applied Biosystems, Foster City, CA), deprotected, evaporated to dryness, and used for PCR and sequencing
without further purification. Oligonucleotide primer
sequences are shown in table 1. For detection using
an automated fluorescent gel detection system, one
primer from each PCR primer pair was fluoresceinlabeled at the 5' terminus by a slight modification of
the method described by Gibbs et al. (1990). Oligonu-

Oligonucleotide Sequence
TCCAACATTGGAAATCACATTTCAA
TCATCACAAATAGATGTTTCACAG
GAGGCTATAATTCTTTAACTTTGGC
CTCTTTCCCTCTTTATTCATGTTAC
CGTTTACCAGCTCAAAATCTCAAC
CATATGATACGATTCGTGTTTTGC
GAGGCTTTATCAGAAGGGTGCAGA
AAGGTTCCTCCAGTAACAGATTTGG
TATGCTACATAGTATGTCCTCAGAC

cleotides to be labeled were synthesized using cyanoethyl phosphoramidites (Applied Biosystems) and
Aminolink II (Applied Biosystems). Following deprotection and evaporation to dryness, 0.2 gmol of the
amino-modified oligonucleotide was dissolved in 200
il of 150 mM sodium carbonate/bicarbonate, pH
9.0. After addition of 1 mg of the succinimidyl ester
of 5- (and 6-) carboxyfluorescein (Molecular Probes,
Inc) mixed with 100 gl dimethylformamide, the oligonucleotide-dye mixture was held at room temperature
for 16 h. Following purification by sodium acetate/
ethanol precipitation, the oligonucleotide was dissolved in 50% formamide, denatured by heating to
90C for 2 min, and further purified by electrophoresis through a 20% denaturing polyacrylamide gel.
Bands containing labeled oligonucleotide were excised
from the gel and soaked in 1.5 ml water at 37C for
16 h. The supernatant was aspirated and concentrated
to dryness.
In Vitro DNA Amplification and Electrophoresis

PCR was performed essentially according to the

method of Saiki et al. (1988). Twenty nanograms of
genomic DNA was mixed with 15 pmol of each PCR
primer in a total volume of 15 pl containing 10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2
mM each deoxyribonucleotide triphosphate, 2 iCi
deoxycytidine 5'-[a-32P] triphosphate, and 0.75 units
of Amplitaq DNA polymerase (Cetus). Samples were
heated to 94C for 4 min, followed by 18-25 cycles
of DNA denaturation (940C for 30 s), annealing
(620C for 30 s), and polymerization (650C for 2 min).
The final 650C incubation was extended to 7 min.

(CA), Polymorphisms in Dystrophin

The (CA). blocks in introns 44 and 49 can be amplified
in the same reaction. The (CA)n loci in introns 45
and 50 can also be amplified simultaneously, with the
exception that the amount of each DMD-50 primer is
decreased to 2.5 pmol. Aliquots of PCR products were
mixed with one-half volume of formamide dye solution (98% formamide, 10 mM EDTA, 0.1% bromophenol blue, and 0.1 % xylene cyanol), heated to 90'C
for 3 min, and electrophoresed on a 6% denaturing
polyacrylamide sequencing gel. Following electrophoresis, each gel was fixed for 30 min in a bath of 10%
methanol and 10% acetic acid. Autoradiography of
the dried gels with an intensifying screen was performed for 12-72 h. Alleles were sized by comparison
with an M13mp18 sequencing standard electrophoresed on the same gel.

953
C C?_

STR-

Cc
I

STR-49

STR-44

STR-4!

Direct DNA Sequencing

DNA sequencing of PCR products was performed

essentially according to the method of Gibbs et al.
(1989). Primers DMD-49A and DMD-49B were used
to amplify genomic DNA in a reaction volume of 50
jil by using the PCR conditions described above and
omitting deoxycytidine 5'-[a-32P] triphosphate. For
the single-strand-producing reaction, 20 pmoles of
primer DMD-49A was used to amplify 1 11 of the
double-strand PCR product in a total volume of 100
p1. The annealing and elongation times were increased
to 1 and 4 min, respectively. DMD-49C was used as
the sequencing primer.
Fluorescent PCR Product Detection

PCR was performed essentially as described above,

except that one of the oligonucleotide primers used for
each amplification was labeled with fluorescein. An
M13mpl8 DNA sequencing standard was generated
using a fluorescein-labeled Universal sequencing
primer. Aliquots of PCR products were mixed with an
equal volume of deionized formamide with 8.3 mM
EDTA, pH 8.0. Following denaturation for 3 min at
90C, samples were electrophoresed on a 6% denaturing polyacrylamide sequencing gel by using an automated laser fluorescent (ALF) DNA sequencer detection system (Pharmacia LKB Biotechnology).
Results
In Vitro (CA)n Amplification

To facilitate the rapid assignment of alleles, the

primers were designed such that the alleles of the intron 45 (CA)n block migrated to a different region of

Figure I
Autoradiograph showing alleles at 4 (CA)n loci in
human dystrophin gene for members of DMD family (pedigree and
allele assignments are shown in fig. 8). Each locus is labeled as a
STR followed by the number of the intron in which the (CA)n block
is found.

the gel than did the alleles of the intron 50 (CA)n block.
These two loci could then be amplified simultaneously. Similarly, the primers for the (CA)n loci in introns 44 and 49 were designed such that both loci
could be amplified in the same reaction. An autoradiogram of the electrophoretic pattern of DNA from four
members of a DMD family amplified at the four (CA)n
loci in two diplex reactions is illustrated in figure 1.
The electrophoretic pattern of each allele consists of
two or more bands spaced at 1- or 2-bp intervals.
The complexity of the electrophoretic band pattern is
minimized by reducing the number of PCR amplification cycles to the smallest number required to detect
the product. The band pattern produced for a given
allele is highly reproducible, and one band is most
intense in a homozygote or hemizygote (or two in a
heterozygote). Different alleles can be easily distinguished and assigned a size by comparison with an
Ml3mpl 8 sequencing standard. We have assigned letters for the alleles at each locus, beginning with A and
incrementing at 2-bp intervals. Rare alleles of STR-49
that occur between the 2-bp interval alleles are indicated by the letter of the preceding allele 1 bp smaller
followed by "1." Allele labeling begins at 174 bp for
STR-44, at 156 bp for STR-45, at 227 bp for STR-49,
and at 233 bp for STR-50.

Table 2
Allele Frequencies and Predicted Heterozygosity

To determine the informativeness of each (CA). locus, we analyzed 57 unrelated Caucasian males unaffected by D/BMD. For this segment of the population, we determined frequencies of observed alleles
for each of the (CA)n loci (table 2). Since males are
hemizygous for these loci, the allele frequencies are
directly obtained from their relative counts (yielding
maximum likelihood estimates). Measures of variation for the four loci are summarized in table 3, in
which (a) the heterozygosity is estimated by Nei's
(1978) method and (b) the numbers of alleles expected
on the basis of estimates of heterozygosity are computed according to the method of Chakraborty and
Griffiths (1982).
Direct DNA Sequence Analysis

All alleles observed for each of the (CA)n loci in

introns 44, 45, and 50 varied in length by 2-bp intervals, presumably because of a variable number of tandem CA blocks. However, alleles at 1-bp intervals
were found for the intron 49 (CA)n locus. To explore
this observation further, we sequenced several different alleles at this locus by using a primer complementary to DNA sequences between the PCR primers for
the sequencing primer and single-strand PCR product
generated from the double-strand PCR amplification
for the template. We identified polymorphism in the
number of C's adjacent to the (CA)n block in intron 49.
An example of the analysis for three males is shown in
figure 2.
DIBMD Carrier Detection and Prenatal Diagnosis
Haplotypes of allele lengths at four (CA)n loci in the
human dystrophin gene were determined in selected
D / BMD familieis in order to perform linkage analysis.
Standard Southern-based RFLP haplotyping for linkage analysis and both Southern analysis using cDNA
probes and exon amplification analysis for deletion
detection were also performed in each family, for confirmation of the STR analysis data and for comparison
of the techniques. In all cases, results of STR analysis
were compatible with results of both Southern-based
RFLP haplotyping and presence of the adjacent exons.
Of 38 affected males studied thus far, failure of DNA
amplification at one or more of the (CA)n loci has
always correlated with a deletion of the region (P. R.
Clemens and R. G. Fenwick, unpublished observations). STR-based haplotypes for carrier detection
(figs. 3-6), prenatal diagnosis (fig. 7), and gene recoin-

Allele Frequencies at (CA), Loci in Dystrophin

Gene

Allele Label (sizea)

STR-44:
A (174) .............................
E (182) .............................
G (186) .............................
H (188) .............................
I (190) ..............................
J (192) ..............................
K (194) .............................
L (196) ..............................
M (198) ............................
N (200) .............................
0 (202) .............................
P (204) ..............................
STR-45:
A (156) .............................
B (158) ..............................
E (164) .............................
F (166) ..............................
G (168) .............................
H (170) .............................
1 (172) ..............................
J (174) ..............................
K (176) .............................
L (178) ..............................
M (180) ............................
N (182) .............................
0 (184) .............................
STR-49:
A (227) .............................
B (229) ..............................
C (231) .............................
D (233) .............................
E (235) .............................
F (237) ....:.........................
G (239) .............................
G1 (240) ...........................
H (241) .............................
I (243) ..............................
J (245) ..............................
K (247)..............................
L (249)...............................
Li (250).............................
M (251) .............................
Ml (252)............................
N (253)..............................
0 (255)..............................
P (257)...............................
STR-50:
A (233)..............................
E (241)..............................
F (243)...............................
G (245)..............................
I (249) ...............................
J (251) ...............................

Frequency SE
(%)
1.8
5.3
5.3
3.5
19.3
3.5
14.0
22.8
1.8
3.5
3.5
15.8

1.7
2.4
2.4
4.8

5.3
12.3
5.3
3.5
12.3
10.5
22.8
15.8
3.5
1.8
3.5
1.8
1.8

2.9
4.3
2.9
2.4
4.3
4.0
5.5
4.8
2.4
1.7
2.4
1.7
1.7

3.5
1.8
1.8
7.0
1.8
1.8
10.5
1.8
8.8
3.5
12.3
7.0
14.0
1.8
10.5
1.8
1.8
3.5
5.3

2.4
1.7
1.7
3.3
1.7
1.7
3.7
1.7
3.7
2.4
4.3
3.3
4.6
1.7
4.0
1.7
1.7
2.4
2.9

17.5
47.4
10.5
7.0
15.8
1.8

5.0

1.7
2.9
2.9
2.4
5.2
2.4
4.6
5.5

6.6
4.0
3.3
4.8
1.7

a
Determined by comparison writh M13mpl8 sequenced with
Universal sequencing primer.

955

(CA). Polymorphisms in Dystrophin

Table 3
Summary of Measures of Variation

Locus Label
(sample size)
STR-44
STR-45
STR-49
STR-50

(57)

(57)
(57)
(57)

Heterozygosity SE
(%)
87.0
88.7
93.3
71.6

........
........

........

........

No. of Alleles
Observed

No. of Alleles
Expecteda

12
13
19
6

13.96
15.48
21.79
7.14

2.1
1.9
1.3
4.6

Based on heterozygosity and sample size and obtained by following Chakraborty and Griffiths' (1982)
method.
a

bination studies (fig. 8) in D/BMD families are illustrated.

In the DMD family illustrated in figure 3, the
affected male (III-4) was known to be deleted for exons
46-50 (data not shown). STR haplotyping confirmed
the deletion, since STR-49 failed to amplify while
STR-50 (3' to the exon) was detected. Six females in
this pedigree have the at-risk STR haplotype. For each
of these females, apparent homozygosity at STR-49
reflects deletion of one allele; densitometry of Southern analysis data (not shown) confirmed the deletion
in each case.
The pedigrees shown in figures 4 and 5 illustrate
carrier detection in families in which all DMD males
were deceased. In figure 4, the carrier status for II-1
was not resolved by two-allele RFLP haplotyping of
the available family members. However, on STR analysis, II-1 inherits the maternal haplotype which is opposite from the haplotype inherited by II-3, her unaffected brother. In figure 5, 1-2, 11-2, and III-1 each
have the same deletion beginning in exon 12 and extending through exon 52 (determined by densitometry

T C AG

T C AG

G
G
G
,~G

1,

of Southern analysis; data not shown). Therefore,

each female is hemizygous for the STR haplotype.
Figure 6 shows the use of STR haplotypes to detect
gonadal mosaicism. A female sibling (IT-1) of a deceased affected DMD male failed to inherit either maternal STR-45 allele. A deletion in this region was
confirmed by densitometry of the Southern analysis,
which shows hemizygosity.
The pedigrees of two DMD families requesting prenatal diagnosis are illustrated in figure 7. In family 1
the affected male (II-1) was found to have DMD when
his mother (1-2) was in the second trimester of pregnancy. On STR assay, the male fetus had the opposite
materal haplotype from his affected brother, a rapid
indicator that the fetus was unlikely to be affected.
Subsequent studies, using Southern analysis and multiplex exon amplification, showed a deletion mutation
removing exons 52-54 of dystrophin in 1-2 and II-1
but not in 11-2. In family 2, carrier testing and pregnancy risk were also performed simultaneously be-

/GG

G
III

IL
E

Allele Gi

AN.. ..A-%

Allele G1l

Allele J

Direct DNA sequence analysis of amplified alleles

Figure 2
of STR-49. Note variation in number of C's adjacent to the (CA)n
repeat (opposite strand shown).

t I

aI

ENE

'A I

F E

E E

Figure 3
Pedigree of DMD family showing STR haplotypes.
DLN = deletion. The affected male is deleted for the STR-49 locus.
Females with the at-risk haplotype are hemizygous for the STR-49
locus. Note that the daughters of II-1 have different paternal haplotypes, demonstrating that STR haplotyping is an excellent method
for confirming paternity within a family.

956

Clemens et al.
2

Ii-}

1 L

1'

E
l

3.4 3.6
7.8 8.3

-~3

11

l I

1'

PROBE ORDER
STR-44
STR-45
STR-49

1 '
E K
H DLN

STR-50

J M

I I
(3.6/ 3.4)
7.8
IE

3.4

7.8

7.8

E E

RFLP
PROBE ORDER
87.15 Taql
C7 EcoRV

Figure 4
Pedigree of DMD family in which all affected males
are deceased. II-1 is likely to be a carrier because she did not inherit
the same maternal STR haplotype as did her unaffected brother.
STR haplotypes are boxed, and RFLP haplotypes are in italics. An
additional 10 RFLP markers were uninformative in 1-2.

Figure 6
mosaicism

G
B
B
E
2

~~~1~

K
I
M
E
3

PROBE ORDER
STR-44
STR-45
STR-49
STR-50

E G
H B
E E

Pedigree of DMD family demonstrating gonadal

cause the patient, 11-2, was referred for DNA testing

when she was in the second trimester of pregnancy.
II-2 is unlikely to be a carrier for DMD, because she
inherited the same maternal haplotype as did her unaffected brother. Had sufficient time been available for
carrier testing for 11-2 first, prenatal testing for 111-1
would not have been necessary.
STR markers can also be used to define regions of
gene recombination. The pedigree illustrated in figure
8 shows a recombination, between STR-44 and STR45, occurring between individuals 11-3 and III-3.
Allele Assignment by Fluorescence Detection

PROBE ORDER

STR-44
STR-45

III

Figure 5

STR-49

STR-50
DLN
DLN
DLN
DLN
Pedigree of three-generation DMD family in which

I
G
L
F

all affected males are deceased. Deletion of the entire region encompassing the STR markers is evidenced by apparent homozygosity of
all loci in females and by failure to inherit maternal markers.

With minor modifications allele assignment can be

performed using a nonradioactive, automated system.
A typical example showing allele detection by the ALF
sequencing apparatus is illustrated in figure 9. Alleles
are assigned on the basis of their relationship to adjacent M13mpl8 sequencing peaks. Shadow bands of
the PCR products are easily distinguished because they
are significantly less intense than the principal peaks.
Discussion
Of 57 unrelated Caucasian males unaffected by D/
BMD who were studied to determine allele frequencies
of four (CA). loci in the human dystrophin gene, no
two males had the same haplotype. Study of other

(CA)n Polymorphisms in Dystrophin

957

Family 1

F
PROBE ORDER
STR-44
STR-45
STR-49
STR-50

11

11
Family 2

ll
A
D
i

H
I
H
A

AH

D
E

G
L
D
E

J
M
G
A

Figure 8
Pedigree of DMD family showing recombination
between II-3 and 111-3. The arrow marks the site of recombination.

Ill
L

J
JI

Figure 7
Prenatal diagnosis in two DMD families. The male
fetus is unlikely to be affected in either family; in family 1 the fetus
did not inherit the same haplotype as did his affected brother, and
in family 2 the fetus inherited the grandpaternal haplotype.

ethnic groups may show differences in degree or distribution of polymorphism of these (CA)n loci. The most
polymorphic locus in our study population is STR-49,
with 19 alleles and a predicted heterozygosity frequency of 93.3%. The least polymorphic locus is
STR-5O, with six alleles and a predicted heterozygosity frequency of 71.6%. The agreement between the
observed and expected numbers of alleles at each ofthe
four (CA)n loci indicates that the population genetic
assumptions of homogeneity and genetic equilibrium
that are required for using these markers for linkage
analyses are adequately justified by these data.
STR locus amplification can be used to identify deletions in males affected by D/BMD. To date, we have
identified and characterized 181 dystrophin deletion
mutations by multiplex exon amplification and Southern analysis (R. G. Fenwick, unpublished observations). The four (CA)n markers described here are located within a dystrophin gene region frequently

involved in deletion mutation (Chamberlain et al.,

submitted), and so a high proportion (71/181, or
39% of the total deletions) would be predicted to be
detected by failure of amplification of one or more of
the four (CA)n loci.
PCR artifacts create a potential problem for determination of STR haplotypes. Other investigators have
reported two bands for each (CA)n allele and have
demonstrated differing electrophoretic mobilities of
the complementary strands of amplified DNA by using
end-labeled primers (Weber and May 1989). We did
not observe a significant improvement in the complex
band pattern when we used end-labeled primers as
an alternative to deoxycytidine 5'-[a-32P] triphosphate
incorporation in the assay. This finding, together with
our observation that the complexity of the band pattern is diminished by performing fewer cycles of DNA
amplification, suggests that there are in vitro-generated minor sequence variants (shadow bands), possibly due to replication slippage and 3' base addition
by the thermostable DNA polymerase. The shadow
bands created during PCR amplification were not
eliminated by a variety of other changes in reaction
conditions. The shadow band pattern is relatively constant at a given locus and is reproducible and, therefore, did not interfere with allele assignment. The
addition of the unlabeled deoxyribonucleotide tri-

958

Clemens et al.

Detection of fluorescein-labeled (CA), alleles on ALF sequencing apparatus (Pharmacia LKB Biotechnology). For each group
Figure 9
of four sequencing lanes, designated as a "clone" by the ALF software, two lanes of M13mpl8 sequencing standard and two lanes of PCR
product were loaded. Alleles were assigned by visual comparison of PCR peak position relative to M13mpl8 sequencing-standard peaks.
STR-49 alleles are in red, and STR-50 alleles are in black. M13mpl8 sequencing-standard "A" and "C" reactions are in green and blue,
respectively.

phosphates to the reaction mixture only after the initial denaturation of the template at 940C eliminated
nonspecific amplification products which migrate in
other regions of the gel when present but did not affect
the pattern of shadow bands.
For three of the (CA), loci in introns 44, 45, and 50
of the human dystrophin gene, alleles vary by 2-bp
intervals, most likely because of a variable number of
dinucleotide tandem repeats, as observed by others
(Litt and Luty 1989; Tautz 1989; Weber and May
1989). At one (CA)n locus, Tautz (1989) cloned and
sequenced several alleles and showed that the variation was indeed solely due to variable numbers of dinucleotide repeats. Some alleles of the (CA)n locus in
intron 49 of dystrophin, however, differ by 1 bp. Direct DNA sequencing of PCR products revealed variation in the number of bases in a short run of C's adjacent to the (CA)n block. Therefore, this locus is a
composite of two different classes of length polymorphism, each consistent with the hypothesis that simple
sequence hypervariability results from slippage during
DNA replication or repair (Tautz and Renz 1984;
Tautz et al. 1986; Levinson and Gutman 1987; Tautz

1989). The presence of two different length polymorphisms within the same marker increases the heterozygosity, thereby enhancing the utility of the marker.
To our knowledge, this is the first time that polymorphism at 1-bp intervals has been described for a (CA)n
locus, implying either that polymorphism in the number of C's adjacent to a (CA), block is not common
or that 1-bp increments of allele lengths have gone
undetected.
Our retrospective study of selected D /BMD families
highlights the informative power of STR haplotyping.
Cases were chosen for the present study because important diagnostic questions could not be answered
with certainty by Southern-based RFLP analysis. For
example, in the family illustrated in figure 3, six females were determined to be carriers because they
were hemizygous at STR-49 and inherited the disease-associated haplotype of the other markers. This
family illustrates the fact that a female appearing to
be homozygous for a low-frequency STR allele may
carry a deletion (e.g., see II-1, 11-6, and 111-3 in fig. 3).
Furthermore, this family shows the ease of detecting
nonpaternity with STR haplotypes.

(CA)n Polymorphisms in Dystrophin

In D / BMD families in which affected individuals
unavailable, carrier detection or exclusion fre-

are

quently relies on linkage analysis to determine whether

the female family members have the same or opposite
haplotype as do unaffected male family members. In
this setting, STR haplotypes are more likely to be informative than are RFLP haplotypes. If the sister of a
deceased affected male is found to be at high risk to
be a carrier for DMD (e.g., see fig. 4), then she has the
option of prenatal diagnosis via STR haplotyping of
a chorionic villus sample or cultured amniocentesis
cells in the future.
Figure 5 illustrates a DMD family with no living
affected males in which we accurately identified deletion carriers by apparent homozygosity of the paternal
alleles for the four (CA)n loci. The high degree of polymorphism of these loci decreases the likelihood of homozygosity at all four loci in a normal female. As
demonstrated in this family, failure to inherit a maternal STR marker is readily apparent and provides
strong evidence for a deletion mutation in a carrier
daughter.
The high degree of heterozygosity of STR loci also
increases the likelihood for identification of gonadal
mosaicism. An example is illustrated in figure 6. Although the male with DMD is deceased, a female sibling inherits the paternal allele (but neither maternal
allele) of STR-45. The simplest interpretation of these
data is that the mother is a gonadal mosaic for the
deletion which was transmitted to her affected son and
carrier daughter.
For prenatal diagnosis, STR haplotyping provides
a marked improvement over Southern-based RFLP
analysis, because of the informativeness and speed of
the assay. Crucial diagnostic data can be obtained
even before extracting DNA, because DNA amplification of (CA)n loci can be performed on boiled cell
lysates. Furthermore, the STR assay can often exclude
maternal cell contamination of a chorionic villus sample, because of differences in the STR haplotype between mother and fetus. We have observed one family
in which a female fetus was predicted to carry a deletion mutation which included the STR-49 and STR-50
loci, because of failure to inherit the maternal alleles
for these markers (data not shown).
STR analysis permits more precise localization of
some recombination events within the dystrophin gene
than can be achieved with RFLP markers alone. Of 13
cases of gene recombination distal to pERT87 that
were identified by our DNA diagnostic laboratory, the
recombination was localized between intron 44 and

959

intron 50 in three cases by using STR analysis (P. R.

Clemens, unpublished observations). An example of
a recombination between STR-44 and STR-45 is illustrated in Figure 8. We do not propose that the recombination is the cause of the disease in the affected male
(111-3), because his mother and sister, who do not have
recombinant haplotypes, have elevated levels of serum
creatine kinase and mild myopathy.
We performed allele identification by electrophoresis either of radiolabeled PCR product on standard
denaturing sequencing gels or of fluorecein-labeled
product on an automated, fluorescent detection system. The principal advantages of the fluorescent detection system are use of nonradioactive substrates and
elimination of the need to fix, dry, and expose gels
after electrophoresis. Currently available computer
software for the automated, fluorescent system, which
is designed to analyze raw sequencing data generated
by the ALF sequencer, can perform only a crude analysis of STR amplification products. As additional software is developed to size alleles directly and to enter
allele assignments into a data base, the labor required
to interpret the gels and assemble the data for linkage
analysis could be markedly reduced.
In summary, STR analysis using (CA)n repeats
within the human dystrophin gene is well suited for
routine use in clinical laboratories engaged in linkage
studies for carrier detection and prenatal diagnosis in
D/BMD families, because the STR assay relies on
PCR amplification, which is simple to perform, provides reproducible results, and is highly informative.
With the addition of other hypervariable loci, the development of software to facilitate allele assignment
on an automated fluorescence sequencing apparatus,
and the capability to determine dosage, STR haplotyping may well replace the more cumbersome Southernbased studies as the standard technique for linkage
analysis in D/BMD families.

Acknowledgments
We thank Pat Ward, Nikki Nguyen, Maria Saluta, and
Al Edwards for valuable discussions; Susan Smith and Reza

Malek for technical assistance; and Pharmacia LKB Biotechnology for loan of the ALF DNA sequencer detection system.
P.R.C. is a recipient of a Muscular Dystrophy Association
postdoctoral research fellowship, and C.T.C. is an investigator of the Howard Hughes Medical Institute. This work
was supported by a Task Force on Genetics grant from the
Muscular Dystrophy Association.

960

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