American Journal of Medical Genetics 52:146-150 (1994)

Arylsulfatase A Pseudodeficiency: A Common Polymorphism Which Is Associated With a Unique Haplotype

Joel Zlotogora, Yael Furman-Shaharabani, Sandra Goldenfum, Bryan Winchester, Kurt von Figura, and Volkmar Gieselmann Department of Human Genetics, Hadassah Hospital and Medical School, Hebrew University, Jerusalem, Israel (J.Z., Y.F.-S.); Diuision of Biochemistry and Genetics, Institute of Child Health, London, United Kingdom (S.G., B. W.); and Department of Biochemistry, Georg-August University, Giittingen, Germany (K.v.F., V.G.)

The allele for pseudodeficiency (PD) of the lysosomal enzyme arylsulfatase A (ARSA) is a common polymorphism in all populations. The PD allele frequency in different Israeli ethnic groups was found to range from 9.2-22.7%. The PD allele includes two differ- ent mutations PD( 1) and PD(2) in an aprox- imatively 1 Kb interval. In this study we confirmed that while PD(1) may be found alone as a polymorphism, PD(2) is always associated with the PD allele (660 alleles screened). Analysis of three ARSA intra- genic polymorphisms showed a complete linkage disequilibrium between the PD al- lele and an haplotype defined by the three polymorphic restriction sites. The results suggest that the origin of the PD polymor- phism may be a common founder, or recur- rent mutations which are occurring in a unique haplotype. @ 1994 WiIey-Liss, Inc.

KEY WORDS: arylsulfatase A, founder ef- fect, selection, polymor- phism, pseudodeficiency

INTRODUCTION Genetic polymorphisms are responsible for a large

amount of human diversity. Blood groups were among the first common polymorphisms described; they often allow differentiation of one individual from another, and their distribution is usually different from one pop- ulation t o another. Recently, many polymorphisms have been discovered at the DNA level; they allow the determination of the haplotypes in which mutations re-

Received for publication August 9, 1993; revision received November 22, 1993.

Address reprint requests to Joel Zlotogora, M.D., PbD., Department of Human Genetics, Hadassah Medical Center, P.O.B. 12000, Jerusalem, Israel 91120.

0 1994 Wiley-Liss, Inc.

sponsible for inherited disorders occurred, and aid at- tempts to understand their origin. For instance, it was suggested that AF508, the most frequent cystic fibrosis (CF) mutation among Caucasians, originates from a common founder since in most of the cases the muta- tion is found in a similar haplotype [European Working Group on CF Genetics: 19901. However, mutations re- sponsible for diseases are relatively rare and an unre- solved question concerns the origin of the common poly- morphisms: are they due to recurrent mutations at a “hotspot” or to a single or few common founder muta- tions? We tried to answer this question by studying a common polymorphism in the gene coding for the lyso- soma1 enzyme arylsulfatase A (ARSA).

The ARSA gene is located at 22q13 and its complete genomic sequence is known [Stein et al., 19891. Muta- tions in the ARSA gene may lead to the deficiency of ARSA and to metachromatic leukodystrophy (MLD), a severe degenerative disease of the nervous system [Kolodny, 19891. Analysis of patients’ relatives docu- mented the existence of healthy individuals with very low activity of ARSA. Since these individuals lack symptoms but have a substantial ARSA deficiency, they have been designated as pseudodeficient. Pseudodefi- ciency (PD) is caused by homozygozity for the PD allele which includes two point mutations. One, PD(l), leads to the loss of an N-glycosylation site (A+G transition at the nucleotide 1788 in the genomic sequence), and the second mutation, PD(21, causes the loss of a polyadeny- lation signal (A+G transition at nucleotide 2723) [Gieselmann et al., 19891. Whereas PD( 1) explains the smaller size of the enzyme in pseudodeficiency, it does not affect its activity or stability. PD(2) causes the loss of 90% of cross reacting material and enzyme activity [Gieselmann et al., 19891. PD(2) has always been found to be associated with PD(l), while PD(1) has been found alone, and represents a rare polymorphism (allele fre- quency 4.5%) [Gieselmann et al., 1989; Nelson et al., 19911.

Reports suggesting a high frequency of a PD allele based on the ARSA activity in normal individuals have been confirmed by molecular studies. The PD allele fre-

Arylsulfatase A Pseudodeficiency 147

RESULTS Frequency of the PD Mutations

The frequency of the PD allele including both PD( 1) and PD(2) was 9.2% in the Arabs, 13.3% in the Ash- kenazim, and 12.2% in the non-Ashkenazi Jews (Table I). Among the Yemenite Jews, the PD allele frequency was found to be 22.7%.

PD(1) was found without PD(2) in all the communi- ties: the Ashkenazi (0.6%), non-Ashkenazi (3.6%), and Yemenite Jews (3.5%), as well as the Arabs (6.8%). PD(2) was never found alone in any of the 660 alleles screened.

quency has been found to be between 7-15% in the dif- ferent populations studied [Herz and Bach, 1984; Nel- son et al., 19911. In order to try to understand the cause and the origin of the high frequency of the PD polymor- phism, we analysed the ARSA haplotypes using three intragenic polymorphic sites in homozygotes and het- erozygotes for the PD allele.

METHODS The DNA analysed in the screening was obtained

from 204 nonrelated individuals living in Israel, origi- nating from various ethnic backgrounds (92 Ashkenazi, 51 non-Ashkenazi Jews, and 61 Christians or Muslim Arabs). We also analysed, as a separate group, DNA from 150 nonrelated Yemenite Jews, non-MLD carri- ers, who had been screened previously to determine the frequency of a mutation causing MLD.

We also examined DNA from 19 nonrelated PD ho- mozygotes, 5 from different Israeli ethnic groups and 14 which were referred from laboratories in Germany and England, originating mostly in those countries. These individuals were examined because of low ARSA activity and were diagnosed as PD homozygotes since they each carried the two PD mutations.

The ARSA gene was amplified in two fragments, A and C, as previously reported [Polten et al., 19911. The first fragment included a BgII polymorphic restriction site in exon 3. The second fragment included the two sites of the PD mutations (exon 6 and polyA tail), as well as a BsrI (exon 7) and a BamHI (intron 7) poly- morphic restriction site (Fig. 1). The BgII and BamHI polymorphisms were studied after digestion with the restriction enzyme according to the manufacturer's rec- ommendations. The BsrI polymorphism was studied af- ter an overnight digestion at 37" C. The digested frag- ments were run on 2% agarose gels. In each case we referred to the uncut polymorphism as (1).

PD(l), the A+G transition at nucleotide 1788, cre- ates a new BsrI site and this was used as a simple non- radioactive test for the detection of the mutation (Fig. 2). In addition, the same digestion allows detection of the BsrI polymorphism in exon 7. PD(2), the A+G tran- sition at nucleotide 2723, was detected by allele-specific hybridisation as described previously [Gieselmann et al., 19891.

Frequency of ARSA Intragenic Polymorphisms The respective frequencies of the polymorphisms in

non-PD alleles (Table 11) were in similar ranges in the different ethnic groups studied for BgII (l), from 8.6-lo%, and BamHI (l), from 76.8-79%. Some differ- ences between ethnic groups were found for the BsrI polymorphism: among Arabs the frequency of the allele BsrI (1) was 45.6% while it was between 53.2-56.3% among Jews.

Linkage Disequilibrium Between the PD Allele and a Haplotype Defined by ARSA Intragenic

Polymorphism We examined a total of 31 PD homozygotes: 19 dis-

covered because of low ARSA activity, and 12 found during screening. All 31 PD homozygotes were also ho- mozygous at the three polymorphic sites studied which defined the haplotype: BgII (21, BsrI (21, and BamHI (1). During the screening we found 72 additional PD carriers; the BsrI (2) polymorphism was observed in all of them. In 60 of these PD carriers we could determine the haplotype of the PD alleles, and they all had the BamHI (1) polymorphism (Table 111). Therefore, all of the 122 carriers of the PD allele, in whom the phase of the polymorphisms could be determined, had the hap- lotype [BsrI (21, BamHI (111. Additionally, in all of the PD carriers in which the BgII associated polymorphism could be determined, it was always BgII (2). When the distribution of each of the polymorphisms associated with the PD alelle was compared with that expected according t o the frequency of non-PD alleles, the dif-

3 Kb 0 1 2 I I I I

Bg ATG

Bs Ba t t

TGA

PD(1) PD(2) Fig. 1. The ARSA gene. The polymorphic restriction sites are marked as *Bg:BgII, Bs:BsrI, and

Ba:BamHI. The site of the two PD mutations are indicated as PD(1) for the mutation at the N-glycosyla- tion site and PD(2) for the mutation in the polyA tail.

148 Zlotogora et al.

I 1 I I I 2 I

822 bp

495 bp 327 bp

113bp 709 bp 1P I I

113bp 382 bp 327 bp I *P ' I

M1 1 2 3 4 5 M 2

1353 - 633 -

- 856 - 583

Fig 2 The BsrI polymorphism When fragment C is cut with BsrI, allele (1) may be obtained if the polymorphic site is absent (822 bp), and allele (2) if the polymorphic site is present (495 and 327 bp) If PD(1) is present, a new Bsr I site is created and, therefore, new fragments may be obtained allele (lp) if the polymorphic site is absent 1709 bp), and allele (2p) if the polymorphic site is present (382 and 327 Bpi On a 2 4 agarose gel, the markers used were M1 PhX174 RF DNA-Hae I11 digest (Biolabs, Beverly, MA) and M 2 Pucl9 Sau3A (Ambion, Austin, TX) (Lane 1, homozygote 1-1; lane 2, homozygote 2-2, lane 3, heterozygotc 1-2, lane 4, heterozygote lp-2p, lane 5, homozygote 2p-2p )

ferences were very significant for all three polymor- phisms. The frequencies of the haplotype LBgII (2), BsrI (21, BamHI (111 were significantly different among the carriers of the PD allele as compared to the non-PD carriers.

Among the 29 carriers of PD(l), the phase of the BamHI polymorphism was known in 18 and it was al- ways BamHI (1). In the 23 carriers in which the phase of the BsrI polymorphism could be determined. both al- leles were observed: BsrI (1) in 10 alleles and BsrI (2) in 13 alleles (Table 111). In all carriers of PD(1) in which the phase was known, the BgII polymorphism was always BgII ( 2 ) . The distribution of each the poly- morphisms in the carriers of the PD(1) mutation was not significantly different from that expected when compared to the non-PD alleles.

DISCUSSION In this study we have demonstrated a complete link-

age disequilibrium between the PD allele and the hap- lotype defined by the ARSA intragenic polymorphisms at the BgII, BsrI, and BamHI sites. There are two possible explanations for these observations. One is a common origin of the PD allele, a founder effect; the other is that the PD mutations occur mainly on one par- ticular haplotype.

In the Jewish Yemenite community the data support a common founder for the PD mutation. Jews have lived in Yemen for centuries, isolated from their neigh-

bows as well as from other Jewish communities. The community was relatively small, consanguineous mar- riages were frequent, and families were relatively large. These conditions may explain the very high fre- quency of the PD allele, observed today as a conse- quence of a founder effect with genetic drift. Another peculiarity of the Yemenite community is that late in- fantile MLD is relatively frequent because of a muta- tion which occurred in the PD allele [Zlotogora et al., submitted]. This mutation is found with a very high fre- quency as the result of a founder effect in a small Jew- ish isolate, i.e., among the Habbanite Jews who lived near Yemen [Zlotogora et al., 19801. As mentioned, the MLD mutation occurred in the PD allele, and all the Habbanites and Yemenite Jewish MLD carriers exam- ined also have the unique PD haplotype defined by the polymorphic sites.

TABLE I. Frequency of the Two PD Mutations Appearing Together (PD Allele) and Separately

P D ( l ) , PD(2)) in Various Communities

Origin PD allele (8) PD(1) (%) PD(2) (5%)

Jews Ashkenazi 13.3 0.6 0 Non-Ashkenazi 12.2 3.6 0 Yemenites 22.7 3.5 0 Arabs 9.2 6.8 0

Arylsulfatase A Pseudodeficiency 149

carrying the haplotype frequently associated with the fragile X mutation have a relatively high number of re- peats supported this possibility [Richards et al., 19921. In myotonic dystrophy, the mutatioo is in complete linkage disequilibrium with a two-allele insertion dele- tion polymorphism located 5 Kb from the CGT repeat. The study of this polymorphism and of a (CA), repeat strongly suggested that the myotonic dystrophy mutations are recurrent events which occur on the same haplotype [Imbert et al., 19931.

On the basis of our observations, since PD(1) was found alone as a rare polymorphism associated with different haplotypes, we propose that it occurred first as a recurrent event. Thereafter, one possibility is that PD(2) occurred in the ARSA allele including PD( 1) and the BgII (2), BsrI (2), and BamHI (1) polymorphisms, and, because of some advantage, this allele became fre- quent. This first PD allele, including both PD(1) and PD(2) mutations, must then have been very ancient since the same haplotype was found in all the very dif- ferent populations studied. However, in particular be- cause of the high frequency of the PD allele, one would have expected then to find PD(2) alone and/or the PD allele on other haplotypes because of recombinations, gene conversion, andlor mutations.

Another possibility is that PD(2) is a recurrent event which occurs only on one haplotype, including PD(1) and the three intragenic polymorphisms. The reasons for this association are unknown but it could, for in- stance, be that PD(2) causes the death of the cell when it occurs in other haplotypes. This may explain the complete linkage disequilibrium and the observation that PD(2) is never found alone.

ACKNOWLEDGMENTS This work was supported in part by a grant from the

German-Israeli Foundation (GIF). We thank CAPES (Brazil) for postgraduate scholarship to SG. We also thank Yosef Shiloh for the DNA of random Yemenite Jews, and Marcia Zeigler (Jerusalem) and Elisabeth Young (London) and colleagues for their cooperation.

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TABLE 11. Polymorphism in the ARSA Gene in the Non-PD Alleles

BsrI (1) BamHI (1) BgII (I) Origin (%I (%’) (%I Jews Ashkenazi 53.4 76.8 8.6 Non-Ashkenazi 53.2 ’77.1 Yemenite s 56.3 79 10 Arabs 45.6 78.6 9.4

As for the other populations, the possibility of a common founder may also explain the complete linkage disequilibrium observed between the PD allele and the haplotype defined by the BgII, BsrI, and BamHl poly- morphisms. The possibility that a common founder is at the origin of mutations frequent in large populations has been proposed in some genetic disorders. For in- stance, the PI*Z allele, which is the most common defi- ciency variant of alpha 1-antitrypsin, occurs on one unique haplotype. Thus, it was concluded that the PI*Z allele has a single origin, a hypothesis which was sup- ported by the data on its frequency and distribution [Cox et al., 19871. Similar studies of association of haplotypes with different mutations causing PKU in Europe demonstrated that there are five prevalent mutations, each strongly associated with one different haplotype defined by eight RFLP in or near the PAH gene. These data suggest that there were multiple geo- graphically and ethnically distinct origins of PKU within the European population, with each of the five major mutations arising from a single founding event [Eisensmith et al., 19921. In CF, a study of two poly- morphic sites flanking the gene demonstrated that in most European patients the major CF mutation AF508 occurred in the same haplotype, and a common founder is therefore suspected [European Working Group on CF Genetics, 19901. Similarly, the mutation G542X, which is frequent among CF patients from the Mediterranean area, is strongly associated with one haplotype, sug- gesting also a common founder for this mutation [Casals et al., 19931.

Linkage disequilibrium was also reported in two dis- eases which are caused by amplification of an unstable region of trinucleotide repeats: fragile X syndrome and myotonic dystrophy. While a founder effect was also suspected, the possibility that the frequent haplotype may be a haplotype which predisposes to amplification was proposed. The finding that normal individuals

TABLE 111. The Frequencies of the Three ARSA Intragenic Polymorphisms in PD and PD( 1) Alleles as Compared to

Those Found in Non-PD Alleles

BsrI (2) BamHI (1) BgII (2) (%) (%) (%I

P D homozygotes (31) 100 100 100 PD carriers (60) 100 100 PD(1) (23) 43.5 100 Non-PD alleles 45.6 74.9 90.4

150 Zlotogora et al.

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