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SHORT COMMUNICATION

Definition of the Locus Responsible for Systemic Carnitine


Deficiency within a 1.6-cM Region of Mouse Chromosome
11 by Detailed Linkage Analysis
KOHEI OKITA,*, TAKASHI TOKINO,* HIROYUKI NISHIMORI,* KOH MIURA,* HIROKO NIKAIDO,
JUN-ICHIRO HAYAKAWA, AKIRA ONO, MASAMICHI KUWAJIMA,
YUJI MATSUZAWA, AND YUSUKE NAKAMURA*,1
*Laboratory of Molecular Medicine, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo
108, Japan; Institute for Experimental Animals, School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920,
Japan; and Second Department of Internal Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan
Received October 13, 1995; accepted January 29, 1996

transport system (1). Carnitine deficiency results in


Carnitine is an essential cofactor for oxidation of failure of the mitochondria to oxidize long-chain fatty
mitochondrial fatty acids. Carnitine deficiency results acids for energy and in accumulation of triglycerides
in failure of energy production by mitochondria and in tissues, including muscles that depend most on oxi-
leads to metabolic encephalopathy, lipid-storage my- dation of long-chain fatty acids for the greater part of
opathy, and cardiomyopathy. The juvenile visceral the energy they need at rest and during exercise. Pa-
steatosis (JVS) mouse, an animal model of systemic tients with carnitine deficiency have been divided into
carnitine deficiency, inherits the JVS phenotype in au- two groups; patients with myopathic carnitine defi-
tosomal recessive fashion, through a mutant allele
ciency suffer from chronic muscle weakness (2, 5), and
mapped to mouse chromosome 11. As a step toward
those with systemic carnitine deficiency have recurrent
identifying the gene responsible for JVS by positional
cloning, we attempted to refine the jvs locus in the
episodes of hypoglycemia, hyperammonemia, hepatop-
mouse by detailed linkage analysis with 13 microsatel- athy, cardiomyopathy, and progressive muscle weak-
lite markers, using 190 backcross progeny. Among the ness (9, 12, 19).
13 loci tested, 5 (defined by markers D11Mit24, Since 1973, many patients with carnitine deficiency
D11Mit111, D11Nds9, D11Mit86, and D11Mit23) showed have been reported (18, 20), and a strain of mice
no recombination, with a maximum lod score of 52.38. (CH3.OH) exhibiting microvesicular fatty infiltration
Our results implied that the jvs gene can be sought on of viscera was reported in 1988 (10). This strain,
mouse chromosome 11 within a genetic distance no whose anomaly was termed juvenile visceral steatosis
greater than about 1.6 cM. q 1996 Academic Press, Inc. (JVS), suffered from hypoglycemia, hyperammo-
nemia, growth retardation, fatty liver, and cardiomy-
opathy; the JVS phenotype was inherited in an au-
Carnitine (b-hydroxy-g-trimethylaminobutyric acid) tosomal recessive mode (7). Later it was found that
is a multifunctional molecule that serves as an essen- JVS was caused by systemic carnitine deficiency in
tial cofactor for oxidation of mitochondrial fatty acids mice (11) and that the primary defect was probably
by transporting long-chain fatty acids as acylcarnitine an impaired system for reabsorption of carnitine in
esters across the mitochondrial inner membrane before the kidney (8). Hence, the JVS mouse is a useful ani-
b-oxidation (14, 16). It also modulates the ratio of coen- mal model for investigations of the pathophysiology
zyme A to acyl-CoA in mitochondria and can trap toxic and the molecular basis of systemic carnitine defi-
acyl-CoA metabolites. Dietary carnitine is present in ciency. Recently, the jvs locus was mapped on mouse
meat (especially red meat) and in milk, but it is also chromosome 11 by linkage analysis (13). Toward posi-
synthesized from lysine and methionine in the liver, tional cloning of the gene responsible for the JVS phe-
brain, and kidney. It cannot be synthesized in skeletal notype, we have constructed a detailed linkage map
or cardiac muscle; these tissues take up carnitine around the jvs locus. The jvs gene was introduced into
against a concentration gradient (the concentration in C57BL/6 mouse by a backcross breeding system to
plasma is about 10- to 50-fold less than that in tissues obtain JVS mice with good viability and fertility. The
such as skeletal and cardiac muscles) by an active homozygous mutant mouse (jvs/jvs) was mated to
Mus musculus molossinus mouse, and then F1 (jvs//)
1
To whom correspondence should be addressed. Telephone: 81- progeny were backcrossed to the homozygous mutant
35449-5372. Fax: 81-35449-5433. mouse (jvs/jvs). The 190 N2 offspring were examined

289 GENOMICS 33, 289291 (1996)


ARTICLE NO. 0194
0888-7543/96 $18.00
Copyright q 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

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290 SHORT COMMUNICATION

ics, Inc.) with the microsatellite markers used for link-


age analysis. Each positive YAC clone was grown from
a single colony and examined for size by pulsed-field
gel electrophoresis using the CHEF Mapper (Bio-Rad
Laboratories) (3, 17). We identified four YAC clones (Fig.
2): YAC25A1, with a 600-kb insert, contained
D11Mit154 and D11Mit87; YAC174B10, with an 850-kb
insert, contained D11Mit24, D11Mit111, and D11Nds9;
YAC92C5, with a 350-kb insert, contained D11Mit24,
D11Mit111, and D11Nds9; and YAC171D1, with a 950-
kb insert, contained D11Mit86, D11Mit23, D11Mit241,
and D11Mit207. Subsequently, we constructed cosmid
FIG. 1. A partial mouse chromosome 11 linkage map showing libraries of three YAC clones, YAC174B10, YAC92C5,
the location of jvs and haplotype analysis of 190 N2 progeny in the and YAC171D1, and isolated the end clones. To examine
backcross. The loci are listed to the right of the linkage map. Each chimerism of each YAC clone, we performed fluores-
column represents the chromosome identified in the backcross prog- cence in situ hybridization (FISH) on mouse interphase
eny that was inherited from the (jvs/jvs 1 Mus musculus molossinus)
F1 parent. Black squares represent the JVS allele. White squares
chromosomes using the end clones and several randomly
represent the M. m. molossinus allele. The number of N2 progeny selected cosmid clones as probes and confirmed that they
carrying each type of chromosome is listed at the bottom of each are not chimeric. We also performed Southern blot anal-
column. Genetic distances between the loci are shown to the left in ysis of four YAC DNAs using the end clones as well as
centimorgans. several randomly selected cosmid clones and found that
three YAC clones, 174B10, 92C5, and 171D1, overlap
for the phenotype, and then extraction of DNA from each other as shown in Fig. 2 and that there is a gap
appropriate tail samples was carried out according to between YAC25A1 and 174B10. On the basis of the link-
methods described previously (15). age analysis and this physical map of YAC clones, the
Oligonucleotide primer sequences defining 13 micro- order of the loci can be established as centromere
satellite marker loci (D11Mit153, D11Mit141, D11- D11Mit153(D11Mit141, D11Mit25, D11Mit154, D11-
Mit25, D11Mit154, D11Mit87, D11Mit24, D11Mit111, Mit87)(D11Mit24, D11Mit111, D11Nds9)(D11Mit86,
D11Nds9, D11Mit86, D11Mit23, D11Mit241, D11- D11Mit23)D11Mit241D11Mit207D11Mit131
Mit207, and D11Mit131) were identified through the da- telomere, unless some of these loci have been deleted
tabase on the e-mail server at the Whitehead Institute/ from YACs.
MIT Center for Genome Research. The 190 N2 progeny Secondary carnitine deficiency is caused by defects
were genotyped for 13 microsatellite marker loci, for such as deficiencies of enzymes required for fatty acid
which F1 progeny were confirmed as heterozygotes. The oxidation, defects in organic acid metabolism, or defi-
phenotype of each mouse was determined by examining ciencies of enzymes related to the mitochondrial respira-
its liver for fatty infiltration; of the 190 N2 progeny, 89 tory system (4, 6, 21). A tubular reabsorption defect that
were diagnosed as jvs/jvs affected, and 101 were jvs// causes renal loss of free and acylcarnitine, a defect in
unaffected. Linkage analyses detected no recombination carnitine biosynthesis, or a malabsorption of carnitine
events in these mice between the disease allele and in the intestine are possible causes of primary carnitine
D11Mit24, D11Mit111, D11Nds9, D11Mit86, or D11- deficiencies. The JVS mouse is thought to be an animal
Mit23; the maximum lod score was 52.38. One phenotypi- model of primary carnitine deficiency due to a defect in
cally unaffected mouse was recombinant for each of five the renal carnitine transport system (8), but the patho-
loci proximal to D11Mit24: D11Mit153, D11Mit141,
D11Mit25, D11Mit154, and D11Mit87 (Fig. 1). One other
mouse, with a JVS phenotype, was recombinant at the
most proximal locus only (D11Mit153). At loci distal to
the nonrecombinant cluster, two recombination events in
unaffected offspring were detected at D11Mit241, and
four (in one affected and three unaffected offspring) were
detected at the two most distal loci, D11Mit207 and
D11Mit131. The order of markers and the calculated ge-
netic distances between them suggested by this linkage
analysis is centromereD11Mit1530.52 { 0.52 cM
(D11Mit141, D11Mit25, D11Mit154, D11Mit87)0.52 {
0.52 cM(jvs, D11Mit24, D11Mit111, D11Nds9,
D11Mit86, D11Mit23)1.05 { 0.74 cMD11Mit241 FIG. 2. Physical map of the region containing the jvs gene on
mouse chromosome 11. Marker names and the 171D1 right end are
1.05 { 0.74 cM(D11Mit207, D11Mit131)telomere. shown above the map. Solid circles, microsatellite STSs; open circle,
To cover the region of interest, we screened mouse YAC end clone. Addresses of the YACs and the sizes of their inserts
yeast artificial chromosome DNA pools (Research Genet- are given below the map.

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SHORT COMMUNICATION 291

physiological mechanism causing systemic carnitine de- syndrome of systemic carnitine deficiency. Clinical, morphologi-
ficiency is still unclear. By detailed linkage analysis, we cal, biochemical and pathophysiologic features. Neurology 25:
1624.
have localized the jvs locus within a region of approxi- 10. Koizumi, T., Nikaido, H., Hayakawa, J., Nonomura, A., and
mately 1.6 cM on mouse chromosome 11, flanked by the Yoneda, T. (1988). Infantile disease with microvesicular fatty
loci defined by markers D11Mit141, D11Mit25, infiltration of viscera spontaneously occurring in the C3H-H-27
D11Mit154, or D11Mit87 proximally and D11Mit241 strain of mouse with similarities to Reyes syndrome. Lab.
distally. However, if in defining the jvs locus we base Anim. 22: 8387.
our calculations only on affected homozygote progeny on 11. Kuwajima, M., Kono, N., Horiuchi, M., Imamura, Y., Ono, A.,
Inui, Y., Kawata, S., Koizumi, T., Hayakawa, J., Saeki, T., and
the grounds that penetrance of the hereditary defect in Tarui, S. (1991). Animal model of systemic carnitine deficiency:
mice may not be 100%, the region containing the jvs Analysis in C3H-H-27 strain of mouse associated with juvenile
locus expands to a segment of approximately 3.2 cM visceral steatosis. Biochem. Biophys. Res. Commun. 174: 1090
between D11Mit153 and D11Mit207. In the former case, 1094.
we now cover the region of interest with YAC clones. In 12. McGarry, J. D., and Foster, D. W. (1980). Systemic carnitine
deficiency. N. Engl. J. Med. 303: 1413.
the latter case, we need to expand a physical contig
13. Nikaido, H., Horiuchi, M., Hashimoto, N., Saheki, T., and Haya-
map toward isolation of candidate genes in the mouse. kawa, J. (1995). Mapping of jvs (juvenile visceral steatosis)
Identification of the jvs gene will aid in further under- gene, which causes systemic carnitine deficiency in mice, on
standing fatty acid metabolism and diseases such as chromosome 11. Mamm. Genome 6: 369370.
cardiomyopathy. 14. Pande, S. V. (1975). A mitochondrial carnitine acylcarnitine
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15. Sato, T., Tanigami, A., Yamakawa, K., Akiyama, F., Kasumi,
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