Assignment of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase (ETF-QO) to Human Chromosome 4q33 by Fluorescence in Situ Hybridization and Somatic Cell Hybridization

Molecular Genetics and Metabolism 67, 364 –367 (1999) Article ID mgme.1999.2873, available online at http://www.idealibrary.com on

BRIEF COMMUNICATION Assignment of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase (ETF-QO) to Human Chromosome 4q33 by Fluorescence in Situ Hybridization and Somatic Cell Hybridization MATERIALS AND METHODS

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a nuclear-encoded protein located in the inner mitochondrial membrane. Inherited defects of ETF-QO cause glutaric acidemia type II. We here describe the localization of the ETF-QO gene to human chromosome 4q33 by somatic cell hybridization and fluorescence in situ hybridization. © 1999 Academic Press Key Words: electron transfer flavoprotein; gene localization; glutaric acidemia type II; GA II; human chromosome 4.

Localization of the ETF-QO gene to human chromosome 4 was accomplished by hybridizing a human 1.4-kb cDNA to EcoRI digested hamster- (BIOS Corp.) and mouse- (Dr. H. Willard) human somatic cell hybrid panels. Southern blot analysis was carried out using standard procedures. Fluorescence in situ hybridization (FISH) was performed using four genomic DNA clones (0.35- to 2.0-kb inserts in pBlueScript) and spanning approximately 10 kb of target DNA. A “cocktail” containing the biotin-16-dUTP-labeled clones (approximately 50 ng of each) was hybridized to metaphase chromosomes prepared from an individual with a normal human karyotype. Chromosomal slide preparations used in FISH experiments were R-banded according to a standard procedure (3). Metaphase preparations were stained with Hoechst 33258 (0.1 mg/ml in H 2O) for 20 min in the dark at room temperature and rinsed briefly in running tap water. One milliliter of 23 SSC (13 SSC is 0.1 NaCl, 0.015 M Na-citrate, pH 7.5) was applied under a coverslip and the slides were then irradiated with UV light (360 nm) for 30 min, rinsed in tap water, and allowed to air dry. Probe labeling, hybridization, and fluorescent detection of the probe were performed as described by Van Leuven et al. (4). For this experiment, the probe was labeled with digoxigenin-11-dUTP by nick translation for 2 h at 15°C. Slides were treated with RNase (100 mg/ml) for 1 h at 37°C and with pepsin (0.005% in 0.01 N HCl) for 30 min at 37°C. Hybridization was performed with the probe at a concentration of 2 ng/ml in 50% formamide in 23 SSC overnight in a moist

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) (EC 1.5.5.1) is a 64-kDa monomer located in the inner mitochondrial membrane. It serves as the electron acceptor for electron transfer flavoprotein (ETF) which oxidizes nine primary flavoprotein dehydrogenases. These dehydrogenases include the four chain-lengthspecific acyl-CoA dehydrogenases of fatty acid beta-oxidation and five dehydrogenases involved in amino acid catabolism. ETF-QO is reoxidized by ubiquinone. Deficiency of ETF-QO causes glutaric acidemia type II (GA II), an autosomal recessive inborn error of metabolism. Individuals with GA II accumulate and excrete substrates and/or metabolites of substrates of the primary dehydrogenases. Neonatal presentations of GA II are often fatal although milder late onset forms of the disorder have also been described (1). Complementary DNA (cDNA) to the human ETF-QO mature message was cloned (2) and was utilized to determine the chromosomal localization of the gene. 364 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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TABLE 1 Chromosomal Localization of ETF-QO Human chromosome Mouse hybrid

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

X

Y

Human Mouse A59-3A A11-4A A60-14 A50-2A T23-4C A71-1A A71-2B A49-5A A60-1A A60-8 A63-1A A68-2A A54-8A A23-1A A23-1A A60-10 A68-1A A60-2 A23-1A Discordant hybrids BIOS Discordant hybrids

2 2 1 2 1 2 2 2 2 1 2 1 2 2 2 2 2 1 2 1 2 6

2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 2 2 1 2 2 2 3

2 2 2 2 1 2 1 2 2 1 2 1 1 2 2 2 2 1 2 1 2 7

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0

2 2 2 2 1 2 2 2 2 2 1 1 1 2 2 2 2 1 2 2 2 5

2 2 2 2 1 2 2 2 2 2 2 1 1 2 2 2 2 1 2 1 2 5

2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 2

2 2 2 2 2 2 1 2 2 2 2 1 2 2 2 2 2 1 2 1 2 4

2 2 2 2 1 2 1 2 2 1 2 1 2 2 2 2 2 1 2 1 2 6

2 2 1 2 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 4

2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 1 2 1 2 3

2 2 2 2 1 2 1 2 2 2 2 1 2 2 2 2 2 2 2 1 2 4

2 2 1 2 2 2 1 2 2 1 2 1 2 2 2 2 2 1 2 1 2 6

2 2 2 2 2 2 2 2 2 1 1 2 1 2 2 2 2 1 2 1 2 5

2 2 1 2 2 2 1 2 2 1 2 1 1 2 2 2 2 1 2 1 2 7

2 2 2 2 1 2 1 2 2 2 2 1 2 2 2 2 2 1 2 1 2 5

2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2

2 2 1 2 2 2 2 2 2 1 2 2 2 2 2 2 2 1 2 1 2 4

2 2 2 2 1 2 2 2 2 2 2 1 1 2 2 2 2 1 2 1 2 5

2 2 1 2 1 2 2 2 2 2 1 1 1 2 2 2 2 1 2 1 2 7

2 2 2 2 2 2 2 2 2 2 1 1 2 2 2 2 2 1 2 2 2 3

2 2 2 2 2 2 1 2 2 1 1 1 1 2 2 2 2 2 2 2 2 5

2 2 1 2 2 2 2 2 2 2 1 2 1 2 2 2 2 2 2 2 2 3

2 2 2 2 1 2 1 2 2 1 2 1 2 2 2 2 2 1 2 1 2 5

4

2

5

1

21

5

3

4

4

4

2

5

7

8

5

4

3

5

8

4

8

3

2

5

Note. Discordant results are indicated by (1), concordant results are indicated by (2).

chamber at 37°C. Chemical detection of the probe and amplification of the signal were performed with successive 30-min incubations at 37°C with the following antibodies: mouse anti-digoxigenin (0.2 mg/ ml), sheep anti-mouse digoxigenin (20 mg/ml), and sheep anti-digoxigenen conjugated with rhodamine (20 mg/ml). Nonbinding antibodies were removed at each step. Finally, the slides were washed twice in PBS at room temperature, dehydrated, and airdried. DAPI/Antifade was applied before the slides were covered with a plastic coverslip. Slides were examined using a Zeiss fluorescence microscope. Nuclei were located using a DAPI filter and the rhodamine signal, indicating areas of hybridization of the ETF-QO probes, was visualized using a Zeiss fluorescence microscope with a Texas Red filter. RESULTS Hybridization of the 1.4-kb cDNA to the hamsterand mouse-human somatic cell hybrid panels iden-

tified a unique human signal at approximately 20 kb on Southern blots. The correlation of this signal with the human chromosomes found in the mouse-human somatic cell hybrid panel is presented in Table 1. One cell line (1049) in the BIOS panel gave discrepant results for this gene and for another gene, which was eventually localized to 19pter by in situ hybridization. This discrepancy may have been a result of submicroscopic rearrangements of DNA that occurred during formation of the hybrid cells. Results from the BIOS panel are summarized at the bottom of Table 1. The first two FISH procedures were performed using an ONCOR kit (5). Visualization of metaphases through a FITC filter revealed hybridization signals near the tip of the long arm of a B group-sized chromosome. Cohybridization with a cosmid probe to 4p (Wolf-Hirschhorn, Oncor, Inc.) confirmed chromosome 4 identity. R banding followed by FISH further localized the signal to band 4q33 (Fig. 1).

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FIG. 1. Fluorescence in situ hybridization of digoxigenen-labeled ETF-QO DNA to normal human metaphases from phytohemaglutinin-stimulated peripheral blood lymphocytes. Specific labeling was observed at 4q33. The images were obtained using a fluorescence imaging system from Oncor Image. The system consists of a MacIntosh Quadra 950 computer, a charge-coupled device (CCD) camera (Photometrics), and Ludl filter wheel.

DISCUSSION Somatic cell hybridization and fluorescence in situ hybridization were utilized to localize the ETF-QO gene to 4q33. Previous studies mapped the mouse ETF-QO (also referred to as ETFDH) gene (Etfdh) to

mouse chromosome 3 closely linked to the Si-s (sucrase-isomaltase structural gene) (6). This area of the mouse chromosome 3 shows homology to human chromosome 4q32-qter including the Glur-2 (glutamate receptor-2) locus whose human homolog maps to human chromosome band 4q32– q33 (7). There-

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L, De Strooper B, Fryns JP, Van den Berghe H. Cloning, characterization, and chromosomal localization to 4p16 of the human gene (LRPAP1) coding for the alpha 2-macroglobulin receptor-associated protein and structural comparison with the murine gene coding for the 44-dKa heparin-binding protein. Genomics 25:492–500, 1995.

fore, the ETF-QO data are consistent with the mouse localization of Etfdh. Examination of the human genome surrounding the human ETF-QO gene does not reveal any genes that might be related to the clinical presentation of GA II. 5.

OncorLight chromosome in situ system for single copy sequence detection in metaphase chromosomes or interphase nuclei: Fluorescence microscopy. Oncor Molecular Cytogenetics Instruction Manual, 1991.

6.

White RA, Dowler LL, Angeloni SV, Koeller DM. Assignment of Etfdh, Etfb, and Etfa to chromosomes 3, 7, and 13: The mouse homologs of genes responsible for glutaric acidemia type II in human. Genomics 33:131–134, 1996.

7.

Sun W, Ferrer-Montiel AV, Schinder AF, McPherson JP, Evans GA, Montal M. Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors. Proc Natl Acad Sci USA 89:1443–1447, 1992.

ACKNOWLEDGMENTS We acknowledge the generosity of Dr. Huntington Willard (Case Western Reserve, Cleveland, OH) in providing the mousehuman hybrid panel and Dr. Carl Hillaker (University of Colorado School of Medicine) for help with the FISH technology and imaging. This project was supported in part by NIH Grants HD04024 and HD08315.

REFERENCES 1.

Frerman FE, Goodman SI. Nuclear-encoded defects of the mitochondrial reparatory chain, including glutaric acidemia type II. In The Metabolic Basis of Inherited Disorders (Scriver C, Beaudet AL, Sly WS, Valle D, Eds.). New York: McGraw Hill, 1995, pp 1611–1629. 2. Goodman SI, Axtell KM, Bindoff LA, Beard SE, Gill RE, Frerman FE. Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase. Eur J Biochem 15:277–286, 1994. 3. Cherif D, Julier C, Delattre O, Derre J, Berger R. Simultaneous localization of cosmids and chromosome R-banding by fluorescence microscopy: Application to regional mapping of human chromosome 11. Proc Natl Acad Sci USA 87:6639 – 6645, 1990. 4. Van Leuven R, Hilliker C, Serneels L, Umans L, Overbergh

Elaine B. Spector* ,1 William K. Seltzer† Stephen I. Goodman* *Department of Pediatrics University of Colorado School of Medicine Denver, Colorado 80262 †Athena Diagnostics Worchester, Massachusetts 01605 Received April 28, 1999

1

To whom correspondence should be addressed at Campus Box C-233, 4200 E. Ninth Ave., Denver, CO 80262. Fax: (303) 3150349. E-mail: [email protected].