Am. J. Hum. Genet. 47:802-807, 1990

Chromosomal Localization of the Human a-L-iduronidase Gene (IDUA) to 4pl6.3 Hamish S. Scott,* Lesley J. Ashton,* Helen J. Eyret Elizabeth Baker,T Doug A. Brooks,* David F. Callen,t Grant R. Sutherlandt C. Phillip Morris,* and John J. Hopwood* Lysosomal Diseases Research Unit, Department of Chemical Pathology, Adelaide Children's Hospital, North Adelaide

and

tDepartment

of Cytogenetics and Molecular

Genetics,

Summary The lysosomal hydrolase a-L-iduronidase (IDUA) is one of the enzymes in the metabolic pathway responsible for the degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. In humans a deficiency of IDUA leads to the accumulation of glycosaminoglycans, resulting in the lysosomal storage disorder mucopolysaccharidosis type I. A genomic subclone and a cDNA clone encoding human IDUA were used to localize IDUA to chromosome 4p16.3 by in situ hybridization and this was confirmed by Southern blot analysis. This localization is different from that of a previous report mapping IDUA to chromosome 22 and places the gene for IDUA in the same region of chromosome 4 as the Huntington disease gene. Measurement of expressed human IDUA activity in human-mouse hybrid cell lines confirmed that IDUA is on chromosome 4.

Introduction

a-L-Iduronidase (a-L-iduronide iduronohydrolase E.C.3.2.1.76; IDUA) is a lysosomal hydrolase required for the breakdown of the glycosaminoglycans heparan sulfate and dermatan sulfate (Hopwood 1989; Neufeld and Muenzer 1989). Lysosomal enzymes undergo a series ofprocessing and maturation events for which IDUA has served as a model (Shapiro et al. 1976; Rome et al. 1978; Myerowitz and Neufeld 1981; Schuchman et al. 1984b; Clements et al. 1985, 1989). In humans a deficiency of IDUA results in the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). MPS I is inherited as an autosomal recessive disease and has a wide variation of clinical presentation. MPS I has been clinically divided into three syndromes: Hurler syndrome (severe), Scheie syndrome (mild), and Hurler/ Scheie syndrome (intermediate). Patients that are severely affected with MPS I generally have mental retardaReceived March 22, 1990; revision received March 10, 1990. Address for correspondence and reprints: Dr. John J. Hopwood, Lysosomal Diseases Research Unit, Department of Chemical Pathology, Adelaide Children's Hospital, 72 King William Road, North Adelaide, S. A. 5006, Australia. i 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4705-0006$02.00

802

tion, severe skeletal deformations including dwarfism, stiff joints, hepatosplenomegaly, corneal clouding, and a life span of less than 10 years. Patients with mild MPS I have a normal life span and normal intelligence, but usually have stiff joints, corneal clouding, and mild somatic changes. MPS I patients are diagnosed by determination of a deficiency of IDUA activity in leukocytes and fibroblasts. A large number of different mutant alleles at the IDUA locus are thought to be responsible for the continuous spectrum of clinical phenotypes (Neufeld and Muenzer 1989; Hopwood and Morris, in press), but biochemical characterization of the residual IDUA activity has enabled discrimination only between the extremes of clinical phenotype (Hopwood and Muller 1979; Muller and Hopwood 1984). In order to provide a DNA probe for molecular analysis of MPS I, amino acid sequence data from purified human liver IDUA (Clements et al. 1989) has been used to isolate both a genomic clone and a cDNA clone for IDUA. The genomic and cDNA clones both contain nucleotide sequences which are colinear with peptide sequences from purified IDUA (H. S. Scott, unpublished

data). In this paper we report the localization of the gene for IDUA to human chromosome 4pl6.3 by in situ hy-

Localization of IDUA

803

bridization to human metaphase chromosomes and Southern blot analysis of human-mouse cell hybrids. This report differs from a previous localization of the IDUA gene to chromosome 22 (Schuchman et al. 1984a). In order to resolve whether IDUA is on chromosome 4 or 22, the localization was confirmed by detecting human IDUA activity in human-mouse cell hybrids by use of a monoclonal antibody specific to human IDUA. Material and Methods DNA Probes

Amino acid sequence data from purified IDUA (Cleal. 1989) was used to isolate IDUA genomic and cDNA clones. In brief, oligonucleotides were used to isolate a genomic clone which contained 12 tryptic peptides and four amino terminal sequences totaling 243 amino acid matches. A genomic fragment was used to isolate cDNA clones encoding the full sequence of mature IDUA. The genomic probe used was a 1.5-kb PstI subclone of which over half is coding sequence. The cDNA probe was a 700-bp clone containing only coding sequence. ments et

In Situ Chromosomal Hybridization

Metaphase chromosomes were prepared from lymphocyte cultures of two normal males and in situ hybridization carried out according to a method described elsewhere (Sutherland et al. 1988). Briefly, the IDUA probes were nick-translated to a specific activity of approximately 1 x 108 cpm/gg with three tritiated nucleotides and hybridized to the metaphases from the

two normal males -the cDNA probe at a concentration of 0.5-0.1 pg/ml for 14-18 d and the genomic probe at 0.01 gg/ml for 13-20 d. All individual silver grains touching chromosomes were counted to determine the pattern of hybridization.

Hybrid Cell Lines Wegroth-D2 is described by Geurts van Kessel et al. (1983). WAIVA is described by Deisseroth et al. (1977). Construction of the human-mouse cell lines designated CY is described by Callen (1986). CY12, CY13, and CY14 are described by Callen et al. (1989). CY120 and CY123 were made from fibroblast lines and are previously unpublished. All CY cell lines contain a chromosome background of mouse A9 cells. The human chromosome content of all cell hybrids is shown in table 1. Southern Blot Analysis

High-molecular-weight DNA (8-10 gg per track) from A9 mouse cells, normal human cells, and the human-mouse hybrid cell lines was digested with EcoRI, electrophoresed through 0.8% agarose, and then transferred to Genescreen Plus nylon membrane (Dupont). The IDUA cDNA probe was labeled with a-32P-dCTP using a random primer extension kit (Amersham) according to the manufacturer's instructions. Unincorporated nucleotides were separated using a 1-ml Sephadex G-50 column, and hybridization was carried out in 50O formamide, 10% wt/vol dextran sulfate (Mr = 500,000), 1 M NaCl, 1% wt/vol SDS with sonicated herring-sperm DNA (100 gg/ml) at 420C according to the manufacturer's instructions (Dupont). The hybridized filter was washed once in 2 x SSC, 0.1%

Table I Human Chromosome Content of Human-Mouse Cell Hybrids HUMAN CHROMOSOME NUMBER

HYBRID WAIVA

........

Wegroth-D2

.....

1 -

+

2 3 --

--

4

5

7 8 11

12

-

14 -

-

-

16

17 20 21 22 Ex So

+

-

-

-

-

+

-

-

--

CY12 .......... ++ + - +; q24-qter + --pll.2-qter CY13 .......... q44-qter + - -+ + + - +p13.11-qterr+ CY123 ......... - +; q35.2-qter - - - pl2.2-qter. CY120 ......... -pter-q25 q22-qter - - - - + + CY14 .......... - - +; q31.1-qter + - - + + + pl3.3-qter - + + - + + NOTE. -The + and - symbols signify the presence or absence, respectively, of an entire human chromosome in the cell hybrids. All CY cell hybrids have a translocation chromosome containing the portions of the autosomes indicated. Expression of human IDUA in a cell hybrid (Ex) and the presence of a human specific band in Southern blots (So) is indicated by the + and - signs in the two columns at the extreme right-hand side of the table.

Scott et al.

804

wt/vol SDS for 30 min and then once in 0.2 x SSC, 0.1% wt/vol SDS at 650C for 20 min.

at 200C. The blocking solution was then decanted, and extracts from the skin fibroblasts, leukocytes, and cell hybrids (30 id) were applied and incubated overnight at 40C. The wells were washed three times with 1% wt/vol ovalbumin solution and incubated with 30 gl of 4-methylumbelliferyl-a-L-iduronide as described elsewhere (Clements et al. 1985) for 4 h at 37°C. Relative fluorescence values were converted to pmol/min/mg protein. Protein was determined by the method of Markwell et al. (1978).

Immunocapture of a-L-Iduronidose Cells were grown to confluent monolayers in 75-cm2 flasks in Eagle basal medium (modified) (Flow Laboratories) containing 10% vol/vol FCS (Gibco). Cells were harvested and washed twice by centrifugation in 20 ml of PBS at 500 g at room temperature and finally resuspended in 100 gd of 0.02 M Tris-HCl pH 7.0, 0.5 M NaCl. Leukocytes were harvested from 10 ml of whole blood as previously described by Kampine et al. (1966) and resuspended in 100 gd of 0.02 M Tris-HCl

Results

pH 7.0, 0.5 M NaCl. All cells were lysed by freezing and thawing the cell preparations five times. The cell debris was removed by centrifugation in a microfuge twice at 12,000 g for 15 min. Aliquots of the suspension were removed for determination of total IDUA activity as described by Clements et al. (1985). The monoclonal antibody designated IdlA has been described elsewhere (Clements et al. 1985, 1989). It was immunopurified from culture supernatants on a sheep anti-mouse immunoglobulin affinity column. One hundred microlitres of IdlA (approximately 10 gg of antibody) was applied to the wells of a polyvinyl ELISA plate (Costar) and incubated overnight at 40C. Residual reactive sites on the plates were blocked by incubation with a blocking solution containing 0.5 M NaCl, 0.02 M Tris-HCI, and 1% wt/vol ovalbumin for 2 h

To define the chromosomal localization ofthe IDUA gene, cDNA and genomic clones were used for in situ hybridization to metaphase chromosomes from normal males. -The distribution of grains obtained using the genomic DNA probe on 52 metaphases is shown in figure 1. Of 158 grains, 25 (16%) were over the short arm of chromosome 4. A similar distribution was obtained when the cDNA probe was used with 15% of grains localizing over the short arm of chromosome 4 in 25 metaphases (data not shown). For the cDNA probe, scoring was then undertaken on 30 additional metaphases with chromosomes at the 600-1,000-band level of resolution which had grains over the short arm of chromosome 4. The combined data (fig. 2) show a localization of the IDUA cDNA probe to the short arm of chromosome 4 with most

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Chromosomes Figure I Ideograms of G-banded human chromosomes showing the distribution of 158 silver grains from 52 metaphases after hybridization to the 3H-labeled a-L-iduronidase genomic DNA. Note 16% of the silver grains localize over chromosome 4p.

Localization of IDUA I6 3 16 2

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Southern blot analysis of EcoRI-digested DNA from normal human male (lane H), mouse A9 cells (lane M) and the human-mouse cell hybrids CY14, CY120, CY123, CY12, CY13, Wegroth, and WAVIA in lanes 1-7, respectively. The size (in kilobases) of the human and mouse EcoRI fragments is indicated on the left. Note that only cell lines containing some of or all of human chromosome 4, CY14 and CY120 (table 1), have the human-specific band for IDUA. For convenience lane 2 has been photographically inserted from another position on the same gel. a

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An ideogram of G-banded chromosome 4 illustratFigure 2 ing the distribution of silver grains in 30 metaphases with chromosomes at the 600-1,000-band level of resolution after hybridization to the a-L-iduronidase cDNA probe. Note that most of the silver grains are distributed over bands 4p15.3 to 4p16.3 with a peak at 4pl6.3.

grains distributed over 4pl5.3-4pter with a peak at 4p16.3. This indicates that the most likely localization of the human a-L-iduronidase gene is 4pl6.3. In situ hybridization of the genomic clone showed an almost identical result from 31 high-resolution metaphases with signal on 4p. Hybridization of both probes to metaphase spreads from another unrelated normal male showed an identical localization (data not shown). The localization of the IDUA gene to chromosome 4 was confirmed by performing Southern blot analysis on the hybrid cell lines shown in table 1 with the cDNA clone. The gene probe hybridizes to a single 8-kb EcoRI band in mouse DNA and to a single 23-kb EcoRI band in human DNA. The 23-kb EcoRI human-specific band

is present only in the cell lines CY120 and CY14 (fig. 3). CY14 contains a translocation chromosome, der(16) t(4;16)(q31.1;p13.3), an entire chromosome 4, and five other human chromosomes. CY120 contains the translocation chromosome der(4)t(4;16)(q25;q22) as the only human chromosome present. The other cell lines in the panel contain human chromosomes that allow exclusion of the possibility that the presence of a human IDUA-specific band is due to any of the human chromosomes present in CY14 other than chromosome 4 (see table 1). Thus human IDUA must be on human chromosome 4 and, in particular, in the region included in the CY120 translocation chromosome, 4pter-4q25. Wegroth-D2, WAIVA, and CY13 contain chromosome 22 but do not produce human-specific bands. The localization of IDUA to chromosome 4 was directly demonstrated by the immunocapture of human IDUA with the monoclonal antibody IdlA (Clements et al. 1989) on human-mouse hybrids (table 1). When used against mixed leukocytes, IdlA captures 44% of the IDUA enzyme activity present. In mouse A9 cells IdlA captures less than 1% of activity, showing that IdlA is specific for human IDUA. Significant human IDUA activity was detected only in the hybrid cell lines CY14 and CY120 (table 2). Thus, human IDUA activity was expressed from human chromosome 4, in particular from the region included in CY120, 4pter-4q25, and no discordant chromosomes were recorded. This is consistent with the in situ hybridization and Southern blot results using the IDUA genomic and cDNA clones and confirms that IDUA is on chromosome 4. Wegroth-D2, WAIVA, and CY13 all contain chromosome 22 and were all negative for expression of human IDUA (table 2).

Scott et al.

806 Table 2 Expression of Human a-L-iduronidase

a-L-IDURONIDASE ACTIVITY (pmol/min/mg protein) WAIVA

Wegroth D-2

CY12

CY13

CY123

CY120

CY14

A9

Human Leukocytes

Human Fibroblasts

504

152

224

191

980

966

823

222

135

2441

.17

.46

.43

.22

.50

11.3

19.1

.21

58.8

318

Total IDUA activity in extract Immuno-

........

captured IDUA activity

NOTE.-For experimental details see Material and Methods.

Discussion

The assignment of the structural gene for human IDUA to chromosome 4 was based on the demonstration that both a cDNA and a genomic clone for IDUA were localized to chromosome 4p15.3-4p16.3 by in situ hybridization to human metaphase spreads, and this was confirmed by Southern blot analysis of humanmouse hybrids. As this result conflicted with published data assigning IDUA to chromosome 22 (Schuchman et al. 1984a), the chromosome 4 localization was confirmed by direct measurement of expressed IDUA activity using an enzyme-specific substrate assay. A monoclonal antibody specific to human IDUA was used in order to distinguish human IDUA from mouse IDUA. The use of this human-specific enzyme assay maps IDUA to human chromosome 4pter-4q25. The only cell lines that expressed detectable human IDUA activity were CY14 and CY120. While the quantities expressed were 10-20-fold lower than the values for normal human fibroblasts, they are 10-fold higher than the A9 mouse control and are comparable to values obtained from human leukocytes. These lower values probably reflect a lower level of expression of human IDUA in the human-mouse cell hybrids than in human fibroblasts. It can be seen from the leukocyte IDUA activities recorded in table 2 that the immunocapture assay is detecting 44% of the human IDUA activity present in the cell extracts. Thus it can be said that human IDUA accounts for only 2%-4% of the total IDUA activity present in CY120 and CY14, respectively, which demonstrates the sensitivity of the assay. The assay captured only 13% of activity present in the human fibroblasts, but this reflects the loading capacity of the assay, not the sensitivity. That is, that the level of IDUA activity detected in a normal human fibroblast control

extract (318 pmol/min/mg protein) reflects the maximum binding capacity of the assay. In a previous report, Schuchman et al. (1984a) used a polyclonal antibody against purified human IDUA to localize the gene for human IDUA to chromosome 22pter-22qll using human-mouse cell hybrids. Our in situ hybridization, Southern blot analysis, and immunocapture results disagree with that report. It is significant that there was no specific signal over chromosome 22 (fig. 1) and hybrid cell lines containing human chromosome 22 (Wegroth-D2, WAIVA, and CY13) show no human-specific bands in Southern blot analysis with EcoRI (fig. 3) or using three other restriction enzymes (data not shown). More significantly, we show that hybrid cell lines containing human chromosome 22 do not express detectable human IDUA (tables 1, 2). Schuchman et al. (1984b) purified two forms of human IDUA, a high- and low-uptake form, as defined by their uptake into IDUA-deficient (MPS I) fibroblasts. The antibody used in the localization study (Schuchman et al. 1984a) was produced against the low-uptake form of IDUA (Schuchman et al. 1984c). This antiserum may be detecting a contaminating polypeptide present in Schuchman et al.'s enzyme preparation. This is supported by the fact that the antiserum detects immunologically cross-reactive protein, in a range of 38% to 105% of normal, in MPS I patient fibroblasts (Schuchman and Desnick 1988). This is in contrast to results obtained by other workers (Myerowitz and Neufeld 1981) and ourselves (Ashton et al. 1990). In a study of 20 different MPS I cell lines, using two different monoclonal antibodies, all but one cell line contained less than 5% of the IDUA protein found in normal controls. Two of the patient fibroblast lines used in the Schuchman and Desnick (1988) study were shown to

Localization of IDUA contain less than 1% of normal IDUA protein using the monoclonal antibody detection system of Ashton et al. (1990). This highlights the potential danger of relying exclusively on antibodies to detect a low-abundance protein in mixtures that may contain other crossreactive materials. The gene for Huntington disease (or Huntington chorea) is also localized to human chromosome 4pl6.3 (Cox et al. 1989). Further studies will be performed to determine how closely IDUA and the Huntington disease gene are linked.

Acknowledgments This study was supported by the Australian National Health and Medical Research Council and the Adelaide Children's Hospital Research Foundation. We thank A. B. Deisseroth for the cell line WAIVA and A. Geurts van Kessel for WegrothD2. Dr. Peter R. Clements is acknowledged for valuable discussions, and we thank Sarah Moore and Stuart Purvis-Smith for the parent cell lines used to make CY123 and CY120, respectively. We would also like to acknowledge Dr. Liang Z. Chen for some of the DNA samples and preliminary Southern blot analysis.

References Ashton L, Brooks DA, McCourt PAG, Clements PR, Hopwood JJ (1990) Immunoquantification and enzyme kinetics of a-L-iduronidase in cultured fibroblasts for mucopolysaccharidosis type I patients (submitted) Callen DF (1986) A mouse-human hybrid cell panel for mapping human chromosome 16. Ann Genet 29:235-239 Callen DF, Hyland VL, Baker EG, Fratini A, Gedeon AK, MulleyJC, Fernandez KEW, et al (1989) Mapping the short arm of human chromosome 16. Genomics 4:348-354 Clements PR, Brooks DA, McCourt PAG, HopwoodJJ (1989) Immunopurification and characterization of human a-Liduronidase with the use of monoclonal antibodies. Biochem J 259:199-208 Clements PR, Brooks DA, Saccone GTP, HopwoodJJ (1985) Human a-L-iduronidase. I. Purification, monoclonal antibody production, native and subunit molecular mass. Eur J Biochem 152:21-28 Cox DR, Murray JC, Buetow KH (1989) Report of the committee on the genetic constitution of chromosome 4. Cytogenet Cell Genet 51:121-136 Deisseroth A, Nienhuis A, Turner P, Velez R, Anderson WF, Ruddie F, Lawrence J, et al (1977) Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay. Cell 12:205-218 Geurts van Kessel AHM, Tetteroo PAT, von dem Bourne AEGKr, Hajemeijer A, Bootsma D (1983) Expression of human myeloid-associated surface antigens in human-

807 mouse myeloid cell hybrids. Proc Natl Acad Sci USA 80: 3748-3752 HopwoodJJ (1989) Enzymes that degrade heparin and heparan sulphate. In: Lane DW, Lindahl U (eds) Heparin: chemical and biological properties, clinical applications. Arnold, London, pp 190-229 Hopwood JJ, Morris CP. The mucopolysaccharidoses: diagnosis, molecular genetics and treatment. Mol Biol Med (in press) Hopwood JJ, Muller V (1979) Biochemical discrimination of Hurler and Scheie syndromes. Clin Sci 57:265-272 KampineJP, Brady RO, KanferJN, Feld M, Shapiro D (1966) Diagnosis of Gaucher's disease and Niemann-Pick disease with small samples of venous blood. Science 155:86-88 Markwell MK, Haas SM, Bilber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:211-222 Muller V, Hopwood JJ (1984) a-L-Iduronidase deficiency in mucopolysaccharidosis type I against a radiolabeled sulfated disaccharide substrate derived from dermatan sulfate. Clin Genet 26:414-421 Myerowitz R. Neufeld EF (1981) Maturation of a-L-Iduronidase in cultured human fibroblasts. J Biol Chem 256:3044-3048 Neufeld EF, Muenzer J (1989) The mucopolysaccharidoses. In: Scriver CR, Beaudet ML, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th ed. McGraw-Hill, New York, pp 1565-1587 Rome LH, Garvin AJ, Neufeld EF (1978) Human kidney a-L-iduronidase: purification and characterization. Arch Biochem Biophys 189:344-353 Schuchman EH, Astrin KH, Aula P, Desnick RJ (1984a) Regional assignment of the structural gene for human a-Lidurc dase. Proc Natl Acad Sci USA 81:1169-1173 Schuchman EH, Desnick RJ (1988) Mucopolysaccharidosis type I subtypes: presence of immunologically cross reactive material and in vitro enhancement of the residual a-Liduronidase activities. J Clin Invest 81:98-105 Schuchman EH, Guzman NA, Desnick RJ (1984b) Human a-L-iduronidase. I. Purification and characterization of the high uptake (higher molecular weight) and low uptake (processed) forms. J Biol Chem 259:3132-3140 Schuchman EH, Guzman NA, Takada G, Desnick RJ (1984c) Human a-L-iduronidase. II. Comparative biochemical and immunologic properties of the purified low and high uptake forms. Enzyme 31:166-175 Shapiro LJ, Hall CW, Leder IJ, Neufeld EF (1976) The relationship of a-L-iduronidase and Hurler corrective factor. Arch Biochem Biophys 172:156-161 Sutherland GR, Baker E, Callen DF, Hyland VJ, May BK, Bawden MJ, Healy HM, Borthwick IA (1988) 5-Aminolevulinate synthase is at 3p2l and thus not the primary defect in X-linked sideroblastic anemia. Am J Hum Genet 43:331-335