Human monoamine oxidase gene (MAOA): Chromosome position (Xp21-p11) and DNA polymorphism

GENOMICS

3,53-58

(1988)

Human Monoamine Oxidase Gene (MAOA): Chromosome (Xp21 -pl 1) and DNA Polymorphism

Position

LAURIE Ozmus,*~t YUN-PUNG P. Hsu,*,lT GAIL BRUNS,* JOHN F. POWELL,~ SHIUAN CHEN,~~ WALTER WEYLER,# MARGOT UTTERBACK,*~~ DEBORAH Zucm, t JONATHAN HAINES,** JAMES A. TROFATTER,** P. MICHAEL CONNEALLY,** JAMES F. GusELm,taB’tt AND XANDRA

0.

BREAKEFIELD*‘t’T

*Molecular Neurogenetics, E. K. Shriver Center, Waltham, Massachusetts 02254; tLaboratory of Neurogenetics, Massachusetts General Hospital, Boston, Massachusetts 027 14; *Genetics Division, Children’s Hospital, Boston, Massachusetts 02115; §Endocrinology Research Group, Clinical Research Center, Harrow, England; “Beckman Research Institute, City of Hope, Duarte, California 9 10 70; #Molecular Biology Division, VA Medical Center, San Francisco, California 94 12 1; llNeuroscience Program (Neurology) and ttDepartment of Genetics, Harvard Medical School, Boston, Massachusetts 02115; and **Department of Medical Genetics, Indiana University Medical Center, Indianapolis, Indiana 46223 Received

May

20, 1988

MAO-B (60,660) (Cawthon et aZ., 1981; Weyler and Salach, 1985). The flavin-bearing subunit of MAO-A can be distinguished from that of MAO-B by partial proteolysis and peptide mapping of [3H]pargyline-labeled peptides (Cawthon and Breakefield, 1979). Antibodies that can distinguish each form of the enzyme have been described (Pintar et aZ., 1983; Russell et aZ., 1979; Denney et al., 1982a, b). Comparison of amino acid sequences suggests that MAO-A and MAO-B are encoded by separate genes (Powell et aZ., manuscript submitted; Bach et aZ., 1988). Previously, a gene for MAO-A was mapped to the human X chromosome by resolution on SDS-polyacrylamide gels of human and rodent enzymes using somatic cell hybrids whose only human chromosomal material was the X chromosome (Pintar et al., 1981). A gene for MAO-B was also assigned to the human X chromosome using hybrids and immunological distinction of human and rodent forms of the enzyme (Kochersperger et aZ., 1986). Recently, a 2.0-kb cDNA clone from human liver, HMll, containing essentially a complete coding sequence for MAO-A, was identified on the basis of sequence match (97% or 156 of 161 amino acids compared) with several peptides of human MAO-A (Hsu et aZ., manuscript submitted). This cDNA clone was used here to assign the position of the corresponding gene locus on the X chromosome by Southern blot analysis of DNA from a panel of human-rodent somatic cell hybrids. A restriction fragment length polymorphism (RFLP) for this locus was also found and used to evaluate linkage distances between this MAOA locus and several other loci on Xp.

An essentially full-length cDNA clone for the human enzyme monoamine oxidase type A (MAO-A) has been used to determine the chromosomal location of a gene encoding it. This enzyme is important in the degradative metabolism of biogenic amines throughout the body and is located in the outer mitochondrial membrane of many cell types. Southern blot analysis of PstI-digested human DNA revealed multiple fragments that hybridized to this probe. Using rodenthuman somatic cell hybrids containing all or part of the human X chromosome, we have mapped these fragments to the region Xp21-pll. A restriction fragment length polymorphism (RFLP) for this MAOA gene was identified and used to evaluate linkage distances between this locus and several other loci on Xp. The MAOA locus lies between DXSl4 and OTC, about 29 CM from the former. Q 1988 Acedemic Prsu, Inc.

Monoamine oxidase (MAO), an enzyme located in the outer mitochondrial membrane of most cells, is primarily responsible for the degradation of biogenic amines (for review see Murphy, 1978; Pintar and Breakefield, 1982; Glover and Sandler, 1986). Two forms of this enzyme, MAO-A and MAO-B, have been distinguished on the basis of biochemical properties, including substrate affinity and inhibitor sensitivity. These forms are thought to consist of one or two subunits, each of which bears a covalently bound flavin cofactor (Minamura and Yasunobu 1978; Walker et al., 1971; Nagy and Salach, 1981; Weyler, manuscript submitted). The subunits of MAO-A appear to be slightly larger (apparent M, 63,660) than those for 53

oSS&7543/SS $3.00 Copyright Q 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

54

OZELIUS METHODS

ET AL. kb

DNA Clones

10.24

The human cDNA clone of MAO-A, HMll, contains a 2.0-kb fragment inserted in the EcoRI site of Xgtll (Hsu et al. manuscript submitted). Other cloned DNA probes used in this study were cDNAs or genomic sequences of human origin. Table 1 shows the chromosomal location of these probes and includes references for the RFLPs used. Probe pH1 was kindly provided by Dr. A. L. Horwich, c7 and ~55.5 by Dr. L. Kunkel, and ~58.1 by G.B.

8.0

4

5.64

4.13.gp

2.34

Genomic DNA

Genomic DNA was obtained from lymphoblasts by extraction with phenol/chloroform (Gusella et al., 1979), as described elsewhere (Breakefield et al., 1986). DNA was isolated from the nuclei of human-

-kb 10.2 8.0

5.8

2.3 1.9 1.8

1.3

1234587

FIG. 1. Dosage analysis of human MAOA gene. Ten-microgram aliquots of DNA from a human-hamster cell hybrid G95, hamster parent line (E36), or human lymphoblast lines were digested with I?&. Electrophoresis in 0.8% agarose gels was carried out at 70 V for 16 h. Ethidium bromide staining and uv visualization confirmed essentially equal loading and digestion of DNA in lanes 2-7. A Southern blot of the gel was hybridized to HMll. Lane 1, size markers, XDNA cut with HindHI; lane 2, E36 (hamster parent); lane 3, G96 (Xpter-qter); lane 4, GUS4413 (control male); lane 5, GUS1392 (control male); lane 6, GUS2202 (control female); lane 7, GM1202 (4X male). Arrows (right) indicate human bands.

1

2

3

4

5

6

7

8

9

,o

FIG. 2. Regional localization of human MAOA gene. Ten-microgram aliquots of DNA from human-rodent somatic cell hybrids, rodent parental lines (hamster, E36; mouse, A9), or human lymphoblast lines (control male, GUS1392) were digested with P&I and analyzed aa described in the legend to Fig. 1. Lane 1, E36 (hamster); lane 2, A9 (mouse); lane 3, GUS1392 (human). The following lines are hybrids, with the portion of the human X chromosome retained indicated in parentheses: lane 4, G89E5 (Xpter-qt.&; lane 5, 749 (Xql2-qter); lane 6, 31-l/5 (Xcen-qter); lane 7, 836 (Xpter-ten); lane 8, 422 (Xpll-qter); lane 9, 617 (Xp2l-qter); lane 10,40-9121 (Xp22.3-qter) (Mohandas et al., 1979; Rrune et aL, 1982; Wieacker et al., 1984). Arrows (left) indicate human fragments.

rodent somatic cell hybrids and deletion lines as described (Kunkel et al., 1982). Hybrid lines were generously provided by Dr. T. Mohandas (Mohandas et aZ., 1979) and Dr. H. H. Ropers (Wieacker et al., 1984). Ten micrograms of DNA was digested with restriction endonucleases, fractionated by agarose gel electrophoresis, and transferred to nylon filters as described (Breakefield et al, 1986). The filters were hybridized to a gel-purified EcoRI fragment of cDNA clone HMll which had been labeled by random oligonucleotide priming (Feinberg and Vogelstein, 1984). Labeling, hybridization, and autoradiography were performed as described (Breakefield et oz., 1986). Linkage Analysis

Two-point analyses were carried out using the computer program LIPED (Ott, 1976). Multipoint analyses were performed using the LINKMAP program of the LINKAGE package (Lathrop et aZ., 1984).

HUMAN

1

2

3

FIG. 3. RFLP for MAOA gene. DNA from three unrelated individuals (lanes l-3) was digested with EcoRV. Electrophoresis is 0.8% agarose gels was carried out at 90 V for 16 h. Southern blots were hybridized to HMll. Arrows (left) indicate polymorphic fragments.

Data were entered into the computer using the data management program LIPIN (Trofatter et aZ., 1986). RESULTS

The cDNA clone, HMll, containing the entire coding sequence for human MAO-A, revealed nine bands in a Southern blot of total human genomic DNA cut with P&I (Fig. 1). Five of these bands, 8.0,5.6,4.1,3.9, and 2.3 kB, gave strong signals; the other four, 10.2, 1.9, 1.8, and 1.3 kb, were faint and not always visible. This dosage blot using DNA from an X only hybrid, two normal males, a normal female, and a 4X female demonstrates that all nine bands map to the human X chromosome. To further prove that all these bands corresponded to sequences encoded in the human X chromosome, the HMll probe was hybridized to a panel of human-rodent somatic cell hybrids which contained a full complement of human chromosomes. The only human bands seen were in a cell line that retained the X chromosome (data not shown). Regional localization of the MAOA locus was determined using a panel of human-rodent somatic cell hybrids containing the human X chromosome or portions of it (Fig. 2). Hybrid line 422 (lane 8, Fig. 2), which retains Xpll-qter, was missing all human bands, whereas hybrid line 617 (lane 9, Fig. 2), which retains Xp21-qter, was positive for all bands. An RFLP for the MAOA locus was sought in order to determine its linkage relationship with other loci in this chromosomal region. HMll was hybridized to a set of screening gels which contained DNA from five unrelated individuals; each DNA was cut with 35 different restriction enzymes. One of these enzymes, EcoRV, detected a polymorphic variation having two

55

GENE

MAOA

alleles, Al (10.2 kb) and A2 (6.5 and 3.7 kb) (Fig. 3). Two constant bands with sizes of 5.8 and 5.4 kb were also seen. Subsequent hybridization to filters containing DNA from Caucasian, unrelated North American individuals (19 females and 13 males) revealed a frequency of 65% for allele Al and 35% for allele A2. The distribution of genotypes inferred from the observed phenotypes did not deviate significantly from that expected for Hardy-Weinberg equilibrium. Approximately 42% of females are heterozygous for this trait. The polymorphism was traced through three nuclear families (27 individuals) and found to be inherited in an X-linked Mendelian fashion. To establish the linkage position of the MAOA locus recognized by HMll, alleles distinguished by the EcoRV polymorphism were traced through a large Venezuelan kindred established for use as a reference pedigree (Tanzi et al., 1987). Four loci-ornithine transcarbamylase (OTC; pHl), DXS14 (p58.1), DXS28 (c7), and ~55.5 (see Table 1)-known to be in the vicinity of this MAO-A locus and typed previously in the Venezuelan pedigree (Zucker, unpublished data) were analyzed relative to the MAOA locus using the computer program LIPED (Ott, 1976). Lod scores and the maximum likelihood estimates of recombination fractions are given in Table 2. Data from the Venezuelan pedigree were also used to provide map distances separating these four marker loci for use in multipoint analysis to establish the relative position of MAOA. DXS14, which is closer to the centromere than the other loci used (Goodfellow et al., 1985), was made the reference point and arbitrarily assigned a map position of 0.0. On the basis of multipoint linkage analysis of the data from the Venezuelan reference family, OTC and ~55.5 had been placed previously at 43 CM from DXS14, and DXS28 at 67.2 CM from DXS14 (Haines et al., unpublished data). Using LINKMAP, location scores were calculated for MAOA at various distances along this map to determine its most likely position. Figure 4 shows that the peak Log10 likelihood ratio for MAOA is approximately +5.5. It is about 4 units higher than any other peak, indicating odds of about 10,OOO:l favoring this position between DXS14 and OTC/p55.5 (Fig. 5). This peak places MAOA at about 29 CM from DXS14. TABLE

1

DNA Probes Used for Linkage Analysis Probe pH1 Cl

~58.1 p55.5

Locus OTC DXS28 DXSl4

-

RE

Location

Ref.

Tap1 EcoRV

xp21.1 Xp21.3 Xpll-cen xp21.1

(18,35, 10) (3,lO) (5,U (22a)

MspI XmnI

56

OZELIUS

ET

AL.

TABLE 2 Lod Score at Recombination Fractions e Probe

0.00

p58.1 PHI p55.5 c7

--cc) -W -W -W

0.01 -9.8 -1.7 +0.5 -14.8

0.05

0.10

0.15

0.20

0.30

0.40

8

-3.97 +0.11 +4.10 -6.75

-1.78 +0.75 +5.02 -3.58

-0.74 +0.99 +5.13 -1.92

-0.18 +1.07 +4.88 -0.91

+0.20 +0.95 +3.74 +0.11

+0.11 +0.58 +2.02 -0.34

0.32 0.21 0.14 0.39

DISCUSSION

By using somatic cell hybrid panels and genetic linkage, we have shown that a gene coding for a MAO-A is located on the human X chromosome in the region Xpll-p21 between loci OTC and DXSld We have also identified an PULP that will allow the investigation of the role of this MAOA locus in Xlinked genetic diseases. It is interesting to note that for any particular restriction enzyme used, the probe HMll detects several bands of varying intensities in total human DNA (Fig. 1). There are two possible 1

IOTC/S& YAOA I

GENETIC

0 LOCATION

OF YAO-A

I

I

50

100

LOCUS

0.21 1.07 5.15 0.35

explanations for this variation. First, this MAOA gene maybe large with many exons and some of these exons may be small or contain sites for P&I within them. If this is the case, the cDNA probe HMll may hybridize only partially to these short exon stretches, resulting in bands of lighter intensity. Second, it is possible that HMll cross-hybridizes with other MAO loci in this chromosomal region that are partially homologous to the MAOA locus mapped here. The deduced amino acid sequence from a bovine cDNA for MAO-A showed about 68% homology to bovine MAO-B peptides from purified enzyme over 214 amino acids compared (Powell et aZ., manuscript submitted). Since the mismatches are interspersed in most of the peptides, two separate genes rather than a complex alternative splicing mechanism seem likely. In addition, some of the human bands could correspond to genes for as yet uncharacterized forms of MAO or for pseudogenes.

a DXS28

I -50

m

DXS14

I

IcMl

FIG. 4. The ratio of log, likelihood at a stated position relative to the logic likelihood at -10 (approximating the value of the unlinked state). The LINKMAP program of the LINKAGE package was used to determine the location of the MAOA locus in relationship to four other loci on Xp. Locations are determined relative to DXS14.

X FIG. 5. Human X chromosome showing position of MAOA locus relative to other loci.

HUMAN

Selective drug inhibition of MAO activity in adult rodents and humans leads to accumulation of biogenie amines and alteration of neuronal activity (Campbell et al, 1979; Murphy et aZ., 1983). This indicates that there are neurophysiologic consequences to variations in MAO activity. In quail embryos the gross morphological development of the nervous system and overall viability were not affected when MAO-A activity was inhibited essentially completely with clorgyline (Pintar et al., 1983), indicating that a deficiency in MAO is compatible with life. It seems possible then that a defect in a human MAO gene may cause an inherited neurologic and/or psychiatric disease. With the detection of an RFLP for an MAOA locus, linkage analysis can now be used to determine possible roles for this gene in inherited X-linked diseases. ACKNOWLEDGMENTS The authors thank Suzanne Eschenbach and Timothy Corey for help with these experiments; Janice Poisson for skilled preparation of this manuscript; Vijaya Ramesh for helpful discussions; Drs. T. Mohandas and H. Ropers for providing hybrid cell lines; and Dr. A. L. Horwich for pH1 and Dr. L. Kunkel for c7 and 65.5. This work was supported by the Scottish Rite Schizophrenia Research Program to X.O.B. and Y-P.P.H. and NIH Grant NS21921 (Senator Jacob Javits Neuroscience Investigator Award) to X.O.B.; Grants NS26912, NS18637, and NS22031 to J.F.G.; Grants MH42462 and NS25786 to S.C.; and Grant HD18658 to G.B. Computing facilities were provided by IUPUI computing services. REFERENCES 1. ALDRIDGE, J., KUNKEL, L., BRUNS, G., TANTRAVAHI, U., LALANDE, M., BREWSTER, T., MOREAU, E., WILSON, M., BROMLEY, W., RODERICK, T., AND LATT, S. (1984). A strategy to reveal high-frequency RFLPs along the human X chromosome. Amer. J. Hum. Genet. 36: 548-584. 2. BACH, A. W. J., LAN, N. C., BURKE, D. J., ABELL, C. W., BEMBENER, M. E., KWAN, S-W., SEEBURG, P. H., AND SHIH, J. C. (1988). Molecular cloning of human monoamine oxidase A and B. FEBS, Abstract 8351. 3. BAKKER, E., HOFKER, M. H., GOOR, N., MANDEL, J-L., WROGEMANN, K., DAVIES, K. E., KUNKEL, L. M., WILLARD, H. F., FENTON, W. A., SANDKUYL, L., MAJOOR-KRAKAUER, D., ESSEN, A. J. V., JAHODA, M., SACHS, E., VAN OMMEN, G., AND PEARSON, P. (1985). Prenatal diagnosis and carrier detection of Duchenne muscular dystrophy with closely linked RFLPs. Lancet1: 655-658. 4. BREAKEFIELD, X. O., BRESSMAN, S., KRAMER, P. L., OZELIUS, L., MOSKOWITZ, C., TANZI, R., BRIN, M. F., HOBBS, W., KAUFMAN, D., TOBIN, A., KIDD, K. K., FAHN, S., AND GuSELLA, J. F. (1986). Linkage analysis in a family with dominantly inherited torsion dystonia: Exclusion of the pro-opiomelanocortin and glutamic acid decarboxylase genes and other chromoeomal regions using DNA polymorphisms. J. Neurogenet. 3: 159-175. 5. BRUNS, G., GUSELLA, J., KEYS, C., LEARY, A., HOUSMAN, D., AND GERALD, P. (1982). Isolation of X chromosome DNA sequences. Adv. Exp. Med. Biol 154: 60-72.

MAOA

GENE

57

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31.

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