Lnkage of Adrenoleukodystrophy to a Polymorphc DNA Probe

Patrick R. Aubourg, MD,+ George H. Sack, Jr, MD, PhD,"-FS§ Deborah A. Meyers, PhD,t John J. Lease, BA,+I and Hugo W. Moser, MD"'I

Linkage studies between X-linked adrenoleukodystrophy and a cloned deoxyribonucleic acid fragment (St14), which detects polymorphisms in the distal end of the long arm of the X chromosome (Xq27-28), have shown no recombina- tion in six families. The Iod score for these data (and another kindred reported earlier 12) is 13.766 at recombination fraction (6) = 0.0. These data permit assignment of adrenoleukodystrophy carrier status in family members at risk, supplementing the chemical measurement of very-long-chain fatty acids.

Aubourg PR, Sack GH Jr, Meyers DA, Lease JJ, Moser HW: Linkage of adrenoleukodystrophy to a polymorphic DNA probe. Ann Neurol 21:349-352, 1987

Adrenoleukodystrophy (ALD), an X-linked recessive disorder mainly involving neural white matter and the adrenal cortex {24], is one of the most frequent inher- ited neurodegenerative disorders. Due to defective peroxisomal beta-oxidation of very-long-chain fatty acids (VLCFA) {25], the levels of these fatty acids, particularly hexacosanoic acid (C26:0), rise in plasma {13], fibroblasts [l2], cultured amniotic fluid cells {14], and chorionic villi [23. Thus, measuring VLCFA levels permits reliable identification of 93% of obligate heterozygotes {l6] and affected fetuses. Childhood ALD and adrenomyeloneuropathy (AMN) are the most common forms of ALD. However, it is clinically variable and other manifestations include isolated adre- nal insufficiency, adult ALD, and "asymptomatic" ALD-persons without symptoms who have the biochemical changes of ALD { 151. In addition, 70% of female heterozygotes have neurological signs or symp- toms, but few develop severe disease [227. The mecha- nisms underlying this broad clinical spectrum are un- known, but different phenotypes can occur in the same

Since the gene responsible for ALD has not yet been isolated, we have begun analysis of polymorphic markers linked to ALD using restriction endonuclease digestion and probe hybridization [5, 281. The probes are deoxyribonucleic acid (DNA) clones of known genes or anonymous fragments without known func- tion that are located close to the disease locus. Probe markers already have been characterized for several X-linked disorders, including Duchenne muscular dystrophy (DMD) [41, fragile X-mental retardation

family.

syndrome (fra{X]) 13, 211, and X-linked retinitis pigmentosa { 11. Previous studies have shown close ge- netic linkage between ALD and the glucose-6-phos- phate dehydrogenase (G6PD) locus locating the ALD gene in the Xq28 region of the X chromosome [lo]. A recombinant fragment of human DNA (St14) that recognizes very polymorphic loci in the Xq28 region has been isolated recently [ 191; this recombinant frag- ment has been designated DXS52 in the catalog of cloned human DNA segments 1261. We now have performed linkage analysis in six families at risk for ALDIAMN and have found that St14 and ALDIAMN are present within a short genetic distance. This finding should permit better identification of carriers and also should be helpful in identifying the ALD gene itself.

Materials and Methods Afcertainment of Families at Risk Four families from the United States, one from England, and one from Nova Scotia were included in the study (Fig). We deliberately chose families with a large sibship to get the most efficient data collection. The male subjects included patients with ALD or AMN. Carriers were identified accord- ing to family history, clinical findings, and assays of VLCFA levels in plasma and/or fibroblasts. As reported previously 1161, VLCFA levels were considered abnormal if they were greater than two standard deviations above the laboratory mean; discriminant analysis was used for equivocal levels.

DNA Analysis Total genomic DNA was extracted from leukocytes in whole blood collected in ethylenediaminetetraacetic acid (EDTA) or cultured fibroblasts [8] , digested to completion with re-

From the 'John F. Kennedy Institute and the Johns Hopkins Uni- versity School of Medicine, Departments of ?Medicine, $Pediatrics, $Biological Chemistry, and "Neurology, Baltimore, MD 21205.

Received July 1, 1986, and in revised form July 30. Accepted for publication July 3 1, 1986. Address correspondence to Dr Sack, John F. Kennedy Institute, Room 418, 707 N Broadway, Baltimore, MD 21205.

349

I

I I

111 3 * 3

I v 3

2 2 B

A

C

I ;Qh, ;;sb II ';A* 111

111 II 2A 2A 2 A dyb 4 Zbir

3 4 E

5 2A 2A 2A 2A 2 8 5 3 D

Pedigree data for the six families studied (A-F); adreno- leukodystrophy (ALD) status is indicated for affected males and carrier females. St l4 distal X-chromosome alleles are indicated by smll numbers based on the allele system reported earlier 120); numbws in parentheses were derived fmm analysis of other mem- bers of the kindred, since deoxyribonucleic acid was unavailable. Note the consistent segregation of ALD and Sr14 marker in each kindred.

striction endonuclease Taq I, separated by electrophoresis on 1% agarose gels, and blotted onto nitrocellulose paper [27]. The probe was a 3.0-kilobase (kb; equal to 1,000 base pairs) DNA fragment derived by Eco RI digestion of the original St14 recombinant and labeled with 32P by nick trans- lation [9], or as described elsewhere [GI. Hybridizations were carried out at 37°C for 48 hours in 50% formamide, 0.45 M sodium chloride, 45 mM sodium citrate, 0.1% sodium dodecyl sulfate, 0.296 Ficoll, 0.2% polyvinyl pyr- rolidone, and 10 mM Hepes (pH, 7.4). Filters were washed at room temperature or at 51°C in 0.3 M chloride, 30 mM sodium citrate, 0.1% sodium dodecyl sulfate, 0.02% Ficoll, and 0.02% polyvinyl pyrrolidone. The filters were exposed

to preflashed x-ray film at -70°C with an intensifying screen.

Linkage Analysis Detection of linkage and estimation of the recombination fraction were performed with the computer program LIPED 1231 on an IBM-PC(AT). This program uses the method of maximum likelihood to calculate lod scores (log of odds of linkage versus independent assortment [I 1)) at selected re-. combination fractions for each pedigree, and also permits summing lod scores at each value for individual pedigrees.

Results Genetic Linkage between the St14 Polymorphisms and ALD Locus In human DNAs digested with the restriction en- donuclease Taq I and analyzed by Southern blotting technique, the St14 probe reveals a polymorphic sys- tem with at least ten allelic fragments, in addition to fragments of constant size. Additional polymorphisms

350 Annals of Neurology Vol 21 N o 4 April 1987

LodScores at Recombination Fraction (0) = 0 (fibroblasts were not available). When she became ~ ~~

Family Lod

A 5.046 B 0.718 C 0.857 D 2.329 E 1.204 F 2.408 Bou6 [2} 1.204

Total 13.766 ~ ~~ ~

Led = log of odds of linkage versus independent assortment.

also are revealed, as in Family E (see Figure) and other studies C191. In Caucasian populations, St14 patterns are informative in more than 90% of the cases when DNAs are analyzed with both Taq I and Msp I en- donucleases 1203, (that is, the presence of two differ- ent St14 alleles permits distinction between the X chromosome bearing the ALD mutation and the nor- mal chromosome in carriers).

All of our six families were informative for Taq I polymorphisms. Four of these families were three- generation nuclear families, while the other two had a more complex pedigree (see Figure). To estimate the recombination fraction, we used all family data includ- ing obligate carriers as well as females considered to be heterozygotes by VLCFA measurement in their plasma or fibroblasts. We found concordant segrega- tion between St14 alleles and the ALD trait in all six families. For example, in Family A, the Taq I allele 2 always was associated with the ALD gene through four generations. The lod score for this family alone was 5.046 at zero recombination. In other words, the odds in favor of linkage between ALD and St14 were over 100,000 to 1 (105.046 to 1). Allelic haplotypes of other families are given in the Figure. The results obtained from all the families were pooled by summing the lod scores for individual families (Table). We have also included data from a previous study showing linkage between the Msp I polymorphism of St14 and the ALD locus in a French family 121. For the seven families studied, the total lod score was 13.766 at a maximum likelihood estimate of recombination (0) of 0%-that is, the odds were 10'3.766 to 1 in favor of linkage. No evidence for recombination between ALD and St14 was found (confidence interval = 0-4%).

Application to Carrier Detection Since no evidence of recombination between St14 and ALD has been found, prediction of the carrier status of women at risk, using linkage analysis, has become pos- sible. An example of this is seen in Family B (see Figure). One sister (111-6) had a normal plasma C26: 0 level (0.256 pdml; normal control subjects, 0.330 f 0.180 pglml), which did not establish her carrier status

pregnant, amniocentesis was performed at 16 weeks' gestation. The C26:O level in the cultured amniotic cells was ten times higher than that of control subjects and the C26 : OK22 : 0 ratio was eight times higher than that of control subjects. The pregnancy was inter- rupted and postmortem analysis of the fetal adrenal cortex confirmed the diagnosis of ALD and showed that 111-6 was a carrier.

DNA analysis with the St14 probe showed that her sister (111-2), an obligate heterozygote, and her af- fected brother (111-4) both inherited allele 3 from their mother (11-3). Based on the pedigree and segregation of the ALD trait with St14 allele 3, we concluded that, prior to her pregnancy, 111-6 had a 96 to 100% proba- bility (using a confidence interval of 0-4s) of being a carrier, although her VLCFA plasma levels were nor- mal. For this particular woman, subsequent assays of VLCFA levels in fibroblasts showed results in the het- erozygous range, but at least 7% of obligate hetero- zygotes have both normal plasma and fibroblast VLCFA levels {16, 22). Thus, a problem encountered with biochemical assays in the detection of carrier status could be circumvented with St 14 linkage information.

Discussion Seven families segregating ALDIAMN have shown no recombination between the polymorphic St 14 marker and ALD. Thus, linkage analysis could be used to de- termine carrier status. The main disadvantage of link- age analysis is the need for family testing to determine the alleles present in each family member. Since a plasma VLCFA assay followed by fibroblast studies when plasma levels are normal identifies 93% of obli- gate heterozygotes without testing other family mem- bers {lb}, we currently recommend DNA analysis for carrier detection only when the biochemical assays are negative or difficult to interpret.

Similarly, since the prenatal diagnosis of ALD can be made reliably by assay of VLCFA in cultured am- niocytes or chorionic villi, analysis at the DNA level should be most useful when the biochemical assay is not available for technical and/or practical reasons.

Our studies also extend the previous observation that the ALD locus is in the Xq28 region of the X chromosome [lo]. The parent St14 DNA fragment of 9 kb, from which our 3-kb probe is derived, originated from the Xq26-Xqter region of the X chromosome 120, 26). By linkage analysis between fra(X), St14, and the coagulation factor IX gene (FIX; located on Xq27), Oberl6 and colleagues have established the following order: centromere-FIX-Stl4-Xqter 12 11. Furthermore, two other studies have shown very close linkage between St14 and hemophilia A (HEMA) {7, 191. Linkage analysis also has shown that the St14 locus is very close, in terms of recombination distance,

Aubourg et al: ALD Linkage to St14 351

to locus DX13 {20}. Thus, the subtelomeric region of the X chromosome includes a large cluster of loci: HEMA, G6PD, CBP and CBD (protan and deutan color blindness, respectively), ALD, hypoxanthine phosphoribosyl transferase (HPRT), FIX, fra(X), and the anonymous fragments DX13, 52 A, and St14. Since we have not seen any recombinations between St14 and ALD, it is not yet possible to determine whether ALD is distal or proximal to St14, but this should become clear with further studies.

Recently Nathans and associates isolated genomic and complementary DNA clones encoding the apo- proteins of the three (blue, red, green) light-sensitive pigments 117, 181. Green- and red-pigment genes are proposed to reside in a head-to-tail tandem array in the Xq22-Xq28 interval, segregating with GGPD activity. In Western Europeans, about 8% of males are color blind; about 75% of these have a defect in the deutan series, and about 25% have a defect in the protan series. Preliminary studies (in preparation) in some ALDIAMN families suggest linkage between ALD and color blindness. Thus, multipoint linkage analysis with protan and deutan color blindness, ALD, St14, and other loci may help establish the most likely linear order for these markers.

Supported by USPHS grants AM31745-01 and HD10981-9 and grants from the March of DimedBirth Defects Foundation and the United Leukodystrophy Foundation. Dr Aubourg was supported by the Foundation pour la Recherche Medicale Franpise.

We are grateful to Dr J-L Mandel for the gift of recombinant St14 plasmid, Nancy E. Maestri for assistance with the linkage analysis, and Mrs Mary A. Mix for secretarial assistance. Dr Sack and Mr Lease are grateful for generous gifts from Mr and Mrs William M. Griffin and Mr Daniel M. Kelly.

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352 Annals of Neurology Vol 21 No 4 April 1987