Am. J. Hum. Genet. 51:721-729, 1992

Fluorescent Multiplex Linkage Analysis and Carrier Detection for Duchenne/Becker Muscular Dystrophy Lisa S. Schwartz, * Jack Tarleton,t Bradley Popovich,4 William K. Seltzer,§ and Eric P. Hoffman* Departments of Molecular Genetics and Biochemistry, Human Genetics, and Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh; tDepartment of Medical Genetics, Self Memorial Hospital, and Greenwood Genetic Center, Greenwood, SC; $Molecular Genetics Department, Children's Hospital and Health Center, San Diego; §Department of Pediatrics, University of Colorado Health Sciences Center, School of Medicine, Denver

Summary We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.

Introduction

Duchenne muscular dystrophy (DMD) is the most common lethal neuromuscular genetic disease, affecting 1 /3,500 males. Both DMD and the allelic, milder, and less common Becker muscular dystrophy (BMD) are inherited as X-linked recessive disorders; however, 30% of all cases are de novo mutations occurring in families with no previous family history of a neuromuscular disorder. Creatine phosphokinase (CPK)-level measurements traditionally have been the most commonly used test for carrier detection: 70%-75% of carriers have CPK levels above the upper limit of normal (Gruemer et al. 1985). However, CPK levels can vary in the same subject when measured at different times, and the CPK Received December 12, 1991; revision received May 27, 1992. Address for correspondence and reprints: Eric P. Hoffman, Biomedical Sciences Tower W121 1, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.

o 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5104-0004$02.00

level distributions of normal females and obligate carriers overlap; thus the results can only provide relative probabilities that the female is or is not a carrier (Hodgson and Bobrow 1989). With both the complete cloning of the gene responsible for DMD/BMD (Koenig et al. 1987) and the identification of the gene's product, dystrophin (Hoffman et al. 1987), molecular and biochemical techniques have been developed for the improved diagnosis of DMD/BMD patients, as have methods for more accurate carrier detection and prenatal diagnosis (Hoffman and Kunkel 1989; Hoffman and Schwartz 1991). Using cDNA probes spanning the entire dystrophin gene, deletion analysis can detect 55% of DMD mutations, by Southern analysis (Koenig et al. 1987; Hodgson et al. 1989; Hu et al. 1990; Hoffman, in press). Using the same cDNA probe deleted in the affected male, Southern analysis can quantitatively detect whether a female relative is hemizygous for the mutation and thus a carrier. This technique requires careful comparison of signal intensities of deleted and nonde721

722

leted exons in the female. The results are sometimes subjective, and the analysis is relatively time consuming and expensive to perform. Multiplex PCR analysis has been developed to amplify 18 selected exons of the dystrophin gene and is capable of detecting nearly all deletion mutations in affected males (Beggs et al. 1990; Chamberlain et al. 1990). A recent report has shown that the very rapid PCR analysis is probably more accurate than Southern-based deletion screening (Abbs et al. 1991). While PCR-based deletion screening may preclude the use of Southern analysis in affected probands with DMD/BMD, the nonquantitative nature of standard PCR reactions has limited the utilization of PCR for detection of female carriers of deletions. For the remaining 40% of patients in which no deletion or duplication can be found, linkage analysis must be performed to indirectly assess the carrier status of female relatives. This has traditionally been performed by Southern blot analysis using intragenic or flanking RFLPs. Any single RFLP is often not informative, which requires the use of numerous RFLPs across the dystrophin gene (Bakker et al. 1985; Hodgson and Bobrow 1989). Moreover, it is not uncommon for a family to prove entirely uninformative after dystrophin gene linkage analysis. Finally, because of the enormous size (11-12 cM) of the dystrophin gene, there is a relatively high likelihood that a recombination event could have occurred between the mutation and any single intragenic RFLP. The recombination frequency within the dystrophin gene has been estimated to be 10% (Abbs et al. 1991). Several dinucleotide sequences (CA repeats) have been identified within the human genome which are highly polymorphic and therefore useful in genetic linkage analysis. It is estimated that there are 50,000-100,000 CA repeats interspersed throughout the human genome. Their function is unknown, but it has been hypothesized that they act as hot spots for recombination or participate in gene regulation (Weber and May 1989). These CA repeat-length polymorphisms have been found to be inherited in a Mendelian fashion (Weber and May 1989). Recently, nine CA repeats have been identified within the dystrophin gene, one at the 3' untranslated region (Beggs and Kunkel 1990; Oudet et al. 1990), four at the 5' end (Feener et al. 1991), and one each in introns 44, 45, 49, and 50 (Clemens et al. 1991). These polymorphic loci can be amplified using PCR and can be used effectively for linkage analysis within DMD/BMD families. However, the alleles often differ

Schwartz et al.

by as few as 2 bp, making their detection by standard agarose gel electrophoresis difficult. Finally, the pattern seen for specific CA repeat-containing PCR products can be complex, making unambiguous interpretation problematic (Clemens et al. 1991). The use of fluorescently labeled primers analyzed on an automated DNA sequencer can allow for the rapid detection of fluorescent PCR products which differ in only a few base pairs. This method of analysis can be extended to the detection of exons amplified by PCR using fluorescently labeled primers for the direct detection of females heterozygous for a deletion of the particular exon. We describe the development of these techniques, which are completely PCR based, for unambiguous genetic analysis of DMD/BMD families. We show examples of cases in which our analysis accurately detected carriers and provided prenatal diagnosis where standard protocols proved inadequate. Material and Methods Material from Patients DNA was prepared, in several different laboratories, from peripheral blood of affected probands and family members by using procedures described by Higuchi (1989), Miller et al. (1988), or Kan et al. (1977).

Oligonucleotide Primers Primers for deletion analysis of affected males by standard gel electrophoresis were synthesized on an ABI 370 DNA synthesizer (Applied Biosystems, Foster City, CA), were deprotected in ammonium hydroxide, and were purified through an NAP-10 column (Pharmacia). For those PCR primers whose products were analyzed using an automated DNA sequencer, one primer of each pair was fluorescein labeled at the 5' end by using a slightly modified version of the procedure described in the Applied Biosystems User Bulletin (Applied Biosystems 1989). Oligonucleotides to be labeled were synthesized using an aminohexyl linker, Aminolink 2 (Applied Biosystems), at the 5' end, were deprotected, and were evaporated to dryness. The crude oligonucleotide (0.2 imol) was resuspended in 120 gl of 1 M NaCl and precipitated with 2.3 vol of 95% ethanol to remove salts. The dried precipitate was dissolved in 133.3 1l of 0.5 M NaHCO3/Na2CO3 (pH 9.0) buffer. The fluorescent dye, 5(and-6)-carboxyfluorescein, succinimidyl ester (Molecular Probes), was dissolved in anhydrous dimethylsulfoxide (DMSO) (Fluka) to a final concentration of 5 mg/ 60 1l. Six

Fluorescent Linkage Analysis and Carrier Detection microliters of the dye/DMSO solution was added to 20 pl of the dissolved oligonucleotide and was allowed to react, from 2 h to overnight, at room temperature in darkness. After the addition of 0.1 M TEAA (pH 7.0) (Applied Biosystems) to a final volume of 100 jl, unincorporated dye was removed using two sequential Sephadex G-25 spin columns (Boehringer-Mannheim). The labeled oligonucleotide was diluted in 1,400 jl of 1/10 TE, and 2 pl /25 gl PCR reaction was used directly from aliquots. Oligonucleotides which were not labeled were deprotected in ammonium hydroxide. A 0.09-imol portion in 1 ml of water was purified through a NAP-10 column, and 0.5 pl/25 pl PCR reaction was used directly from aliquots. Deletion Analysis of Affected Males The multiplex PCR was used to identify possible deletions in the affected males of the family. The primer sets and PCR conditions used were those described by Chamberlain et al. (1990) and Beggs et al. (1990) and correspond to 18 exons of the dystrophin gene. The PCR products were visualized on a 1 x TBE

(Tris-borate), 1% agarose/2% NuSieve gel stained with ethidium bromide. Carrier Detection by Deletion Analysis

Multiplex PCR reactions (25 pl) using fluorescently labeled primers specific for selected deleted and nondeleted exons in the affected male were done as follows: 940C for 3 min, to denature; followed by 20 cycles of 940C for 30 s and 650C for 4 min (step cycle), with a last annealing/elongation cycle of 940C for 1 min and 650C for 10 min. Two microliters of the PCR product was added to 5 pl of G-505 loading solution (DuPont) containing 0.2 M EDTA, 2 mg crystal violet/ml, and a G-505 fluorescently labeled 20-mer ohgonucleotide diluted in deionized formamide. After denaturation for 2 min at 950C, 3 gl of the sample was electrophoresed on a sequencing gel by using an automated laser fluorescent DNA sequencer detection system (Genesis 2000 DNA Analysis System; DuPont). For any individual sequencer run, each lane contained samples from multiplex PCR reactions using the same primers. In order to inactivate the reactive aminohexyl linker on the unincorporated primers, an excess of 1 M Tris (pH 8.0) was added to the mixture of primers for multiplex PCR. (The labeled primer [F primer]:unlabeled primer [R primer]:1 M Tris [pH 8.0] ratio was 9:2.25:1.)

723 Carrier Detection by Linkage Analysis

Multiplex PCR using fluorescently labeled primers for four CA repeat regions-3' CA (Beggs and Kunkel 1990; Oudet et al. 1990), 5'-DYS II (Feener et al. 1991), and STR-45 and STR-49 (Clemens et al. 1991)- was performed. The amounts of reagents and the PCR conditions used were as described elsewhere (Chamberlain et al. 1990) and were for 25 cycles. Two fluorescently labeled PCR products corresponding to dystrophin gene exons were used as internal size standards (exon 52, 113 bp; and exon 50, 271 bp). These standards were amplified in a normal female and were added to each multiplex CA repeat sample prior to being loaded on the sequencer. Samples were diluted with G-505 loading solution and were denatured and electrophoresed on a sequencer as described above. Results

Validity of Using the Automated Sequencer for Dosage Determinations The high sensitivity of the sequencer allowed us to keep the cycle number low (20 cycles), thereby remaining within the exponential portion of the PCRamplification curve. Thus, comparing the peak heights of PCR products for deleted and nondeleted exons in potential female carriers provides a means of measuring dosage and, thus, carrier status. To determine the validity of using the sequencer for dosage determinations, we tested both intrasample and intersample variability. Different exons showed different fluorescent intensities after 20 cycles of PCR. This was due to different efficiencies of fluorescent labeling and was reproducible for any single exon. To normalize the data for each pair of exons, we derived a factor by which each peak height was multiplied. For comparing exon 47 and exon 60, the relationship of signal intensities in normal individuals (equal dosage) was (voltage exon 47)(0.67) = voltage exon 60. Thus, voltage 47 . (voltage 60 x 0.67) = dosage exon 47. For 19 normal females, intersample variability for the dosage of exon 47 was mean + SD = 1.00 ± 0.18 (range 0.76-1.35). For 10 female carriers of a deletion of exons 45-48 (half dosage of exon 47), thedosageofexon47wasmean + SD = 0.51 + 0.07

(range 0.41-0.64). To determine intrasample variability, we tested four independent PCR reactions for six carriers and four normal individuals. This analysis measures the variability introduced by the PCR reaction and the vari-

724

ability introduced by the automated sequencer. For normal individuals the values were as follows: individual 1, 0.94 ± 0.10 (range 0.84-1.03); individual 2, 0.94 ± 0.08 (range 0.84-1.04); individual 3, 1.08 + 0.45 (range 0.76-1.73); and individual 4, 0.86 + 0.10 (range 0.76-.99). For six carriers of a deletion of exon 47, the values were as follows: individual 1, 0.50 ± 0.17 (range 0.34-0.70); individual 2, 0.41 + 0.09 (range 0.33-0.52); individual 3, 0.45 + 0.08 (range 0.36-0.54); individual 4, 0.42 ± 0.07 (range 0.33-0.50), individual 5, 0.49 + 0.05 (range 0.420.52); and individual 6, 0.50 ± 0.06 (range 0.460.58). From these results, we found no overlap in values between carriers and normal individuals. This shows that fluorescence intensity of 20-cycle PCR products is an accurate method for determining dosage. Carrier Detection by Deletion Analysis The proband (III-1) in figure 1 was determined by

standard multiplex PCR to have a deletion of exons 45-48 (data not shown). Deletion analysis of female relatives at risk to be carriers was performed using fluorescently labeled primers for exons 47 (test exon) and 60 (control). The dosage of exon 47 in the at-risk females (fig. 1) was compared with those of both normal controls and females known to be heterozygous for exons 45-48. The dosage of exon 47 was determined for each female, as described above. The dosage of exon 47 was calculated for 11-2 (0.50), II-5 (0.49), III-2 (0.61), III-3 (0.53), carrier control 1 (0.57), and carrier control 2 (0.44). Given the half dosage of these pedigree members, they were all judged to be both heterozygous for the deletion and, thus, carriers. Ratios for individuals 1-2 (0.99), II-3 (1.05), and II-4 (1.05) were similar to that for the normal control (0.98), and thus these pedigree members were deemed not to be carriers. Carrier Detection and Prenatal Diagnosis by Linkage Analysis Multiplex PCR using fluorescently labeled primers

for four CA repeat regions - one at the 3' untranslated

Schwartz

et

al.

region (Beggs and Kunkel 1990; Oudet et al. 1990), one at the 5' end (Feener et al. 1991), and one located in each of introns 45 and 49 (Clemens et al 1991)were performed to determine the haplotypes of the affected male and several informative family members, including those females at risk of being carriers. These four CA repeat loci were chosen because they were the most informative for the specific region in which they were located; and the size ranges of the alleles at each locus did not overlap, so they could be amplified and analyzed simultaneously. In addition, the CA repeat loci were distributed throughout the dystrophin gene, to reduce the uncertainties caused by possible recombination.

The PCR products were visualized by computerized traces of voltage readings (fluorescence intensity) as a function of time. The haplotypes of family members were determined by aligning the internal size markers between the lanes. There was some variability in mobility between different runs of the sequencing gels. For this reason, samples from all family members were run on the same gel. PCR-based linkage analysis was performed to determine the haplotypes of family members shown in figure 1 (data not shown). Consistent with the exondeletion analysis, an STR-45 allele was absent in III-1. Haplotyping of the female relatives of the proband confirmed the results of quantitative multiplex PCR analysis. This family also illustrates the use of CA repeat haplotyping in the detection of gonadal mosaicism. 1-2 is heterozygous at the STR-45 locus and is therefore not a carrier. Her daughters-II-2, II-4, and II-5 -all received the same maternal X chromosome. II-2 and II-5 are hemizygous for the deletion at STR-45 and therefore are carriers; this finding is consistent with the quantitative PCR exon data. On the other hand, 11-4 is not a carrier, on the basis of quantitative exon data, and therefore is homozygous at the STR-45 CA repeat locus. Because I-2 was informative at all CA repeat loci, there is no ambiguity in the risk assignments by CA repeats: undetected recombination between polymorphic loci and the disease is not possible.

Quantitative fluorescent multiplex PCR detection of DMD carriers. Shown is the pedigree of a DMD family with CA repeat Figure I haplotypes indicated (sequencer traces are not shown). The affected male was shown to be deleted for exons 45-48 and the STR-45 CA locus. Multiplex PCR of exons 47 and 60 was done with fluorescently labeled primers for 20 cycles, and 1 pil of the PCR product was analyzed on an automatic sequencer. The sequencer traces and the calculated exon dosages are shown for each female member of the pedigree. Females 11-2, II-5, 111-2, and III-3 are heterozygous for the deletion of exon 47. II-4 received the same maternal haplotype as did II-2 and II-5; however, she is not a carrier, on the basis of exon dosage: this shows that 1-2 is a gonadal mosaic. The detection of gonadal mosaicism is further verified by 1-2 being heterozygous for the STR-45 locus, although two of her daughters- 11-2 and II-5 - are hemizygous for the locus.

Family #28

1' 111 III a d

a

3

I

d d b a --

a

a

b

del 47/+

del 45-48

Normal Control

- J.

1.391 Vots

+/+

JX+1 i

1-2

+/+

1.405 Voks

1.524 Vols

11-2

1.825

111-2

Vofts

+/+

del 47/+

del 47/+

3.138 Vols

11-3++

11-4

+I+~~~+1 EXON 60

EXON 47

1.581 Vols

111-3

+/+ |

Carrier Control #2

1.903 Vols

~~~del 47/+

1.825 VoRs

+/+ del 47/+

EXON 60

EXON 47

726

Figure 2 illustrates a BMD family in which no deletion was found in the affected males-III-I and III-3 by standard multiplex PCR or Southern analysis with the complete cDNA. In addition, this family had proved refractory to standard RFLP analysis (data not shown). Individual 1-2 was uninformative for each of 11 RFLP loci tested by Southern blot. When the four CA repeat loci were used, she was informative at three ofthe four repeats. Individuals 11-2 and 11-3 were informative for only one RFLP locus but were informative at three different CA repeat loci (STR-45, STR-49, and 5' end of the gene). Individual III-4 dearly received the maternal haplotype that was the opposite of that received by her affected brother (III-3). She was uninformative for the 3' CA repeat. If it is assumed that the 10% recombination is equally divided between the first half and second half of the gene, I11-4 has 90% risk of being affected with DMD. Individual III-3 received the same maternal haplotype as did her unaffected brother (III-2) and therefore had