- Email: [email protected]
Localization of the human dihydropteridine reductase gene to band p15.3 of chromosome 4 byin situ hybridization
GENOMICS
1,67-70
(1987)
Localization of the Human Dihydropteridine Reductase Gene to Band ~15.3 of Chromosome 4 by in Situ Hybridization R. M. Murdoch
Institute
BROWN’
AND H.-H.
May
6, 1987;
Press,
revised
Hospital, Melbourne,
Australia
June 15, 1987
Previous studies, using somatic cell hybrids, have shown that the DHPR gene is located on chromosome 4 (Kuhl et al., 1979). We have recently isolated and characterized a human DHPR cDNA clone (Dahl et al., 1987). Using this probe, we report the regional localization of the DHPR gene to band 4~15.3 by the technique of in situ hybridization.
We report the localization of the gene for dihydropteridine reductase (DHPR) to the human chromosome region 4~16.3 by in situ hybridization using a cDNA probe to the enzyme. The distal end of the short arm of chromosome 4 is of considerable interest because the gene responsible for Huntington’s disease is located in this region. Although this part of the chromosome is being extensively studied, DHPR is the first wellcharacterized gene to be assigned to the region. Restriction enzyme fragment length polymorphisms have been detected with a number of restriction endonucleases, including AuaII and MapI. These features may make the DHPR cDNA clone a useful probe not only for prenatal diagnosis of DHPR deficiency but also for linkage studies of Huntington’s disease. Academic
DAHL
for Research into Birth Defects, Royal Children’s Received
0 198’7
M.
MATERIALS
AND
METHODS
Chromosome Preparation
Lymphocytes from a normal, healthy male were stimulated with phytohemagglutinin (Wellcome) and cultured for 72 h at 37°C in medium 199 containing 15% fetal calf serum. Bromodeoxyuridine (200 rglml) was then added for a further 16 h (Zabel et al., 1983), after which the cells were washed twice in warm medium and incubated in fresh medium containing 10e5 M thymidine for a further 6.5 h. Chromosome preparations were made using standard techniques except that all procedures were carried out under safelights.
Inc.
INTRODUCTION
Dihydropteridine reductase (DHPR; EC 1.6.99.7) catalyzes the reduction of dihydropteridine to tetrahydrobiopterin, an essential cofactor in the reactions catalyzed by the three aromatic amino acid hydroxylases, phenylalanine, tyrosine, and tryptophan hydroxylase (Kaufman and Fisher, 1974). Lack of this cofactor leads to malignant hyperphenylalaninemia, a condition in which severe neurological disturbances persist in spite of dietary control of hyperphenylalaninemia and treatment with L-dopa and 5-hydroxytryptophan (Smith et al., 1985). DHPR deficiency was the first enzyme defect recognized as a cause of malignant hyperphenylalaninemia and it accounts for approximately 30% of all cases (Cotton, 1986). The disease is transmitted as an autosomal recessive disorder.
Preparation of Probe
The probe was a cDNA insert from hDHPR19 which was subcloned into plasmid pUC9 (Dahl et al., 1987). It covers the full coding region of human DHPR. The probe was labeled with either [lz51]dCTP or with three tritiated bases. Nick translation with [lz51]dCTP (Amersham International) resulted in a specific activity of a3 X 10’ dpm/pg. When tritiated compounds were used, the reaction mixture was 6 PM with respect to the three labeled nucleotides: [3H]dATP at 64 Ci/mmol, [3H]dCTP at 66 Ci/mmol, and [3H]TTP at 121 Ci/mmol (Amersham International); and 60 PM with respect to unlabeled dGTP. The specific activity of the tritiated probe was a2 X lo8 dpmlpg.
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. 503032. 1 To whom correspondence should be addressed at the Murdoch Institute, Royal Children’s Hospital, Parkville 3052, Victoria, Australia.
In Situ Hybridization
Hybridization of the probe to the chromosome preparations was carried out essentially as described 67
0868-7543187
$3.00
Copyright 8 1987 by Academic Preae, Inc. All righta of reproduction in any form reserved.
BROWN
AND
DAHL
6
-*&g
~tlmnhubRrjhm~~~ 13
FIG.
12
1.
Silver
grain
15
distribution
17
16
in 21 metaphases
after
19
in situ hybridization
by Buckle and Craig (1986). Slides were initially treated with 100 kg/ml RNAse in 2X SSC (20X SSC is 3 M NaCl, 0.3 A4 Na citrate, pH 7) at 37°C for 1 h. They were thoroughly rinsed with 2X SSC and dehydrated through an alcohol series. Chromosomal DNA was denatured by incubating at 65°C in 70% formamide, 0.1 mM EDTA, 2~ SSC for 4 min. Slides were rapidly cooled in cold 2~ SSC, rinsed in 2X SSC, and dehydrated. Approximately 100-200 ng of labeled probe was lyophilized and resuspended in 350 rl of hybridization fluid which contained 50% formamide, 5X Denhardt’s
A
18
m
of ‘%I-labeled
21
human
22
dihydropteridine
X
Y
reductase
probe.
solution (0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% Ficoll 400), 5X SSPE (0.9 M NaCl, 50 mA4 NaH2POI, 5 n&f EDTA, pH 7.2), 10% dextran sulfate, and 200 pg/ml salmon sperm DNA. The mixture was denatured by boiling for 5 min, followed by rapid cooling on ice. A 35-~1 aliquot of probe was placed on each of 10 slides, which were covered with a coverslip, sealed with rubber cement, and incubated overnight at 42°C. Coverslips were removed by rinsing in 5~ SSC and slides were washed in three changes of 2X SSC over 2
6
FIG. 2. (A) Silver grain distribution on chromosome cells after in situ hybridization with tritiated dihydropteridine ductase probe. (B) High-resolution analysis of chromosome cells.
4 in 40 re4 in 41
FIG. 3. Part of a human grains (arrows) on band ~15.3
metaphase spread of both chromosomes
showing 4.
silver
HUMAN
DIHYDROPTERIDINE
h. They were then usually washed in 0.5X SSC at 60°C for 30 min, followed by 1 h at the selected final stringency. This was either 0.5X SSC or 0.2X SSC, both at 6O”C, with a change of solution after 30 min. The slides were subsequently allowed to stand at room temperature for 1 h in 0.1X SSC before being dehydrated. Slides were dipped in L4 emulsion and exposed at 4°C for 5-12 days. They were developed at 20°C for 5 min in Kodak D19, diluted 1:l with water. They were then stained with Hoechst 33258 (15 pg/ml) for 30 min, exposed to black light (350 nm) in 2X SSC, and stained with 10% Giemsa for 25 min. Silver grains on or touching chromosomes were scored and related directly to chromosome bands. RESULTS
Analysis of the distribution of silver grains following in situ hybridization with a dihydropteridine reductase cDNA probe to chromosome preparations from male lymphocytes confirmed the assignment of the gene to chromosome 4. Initially, 21 cells that had been hybridized with ‘251-labeled probe were completely analyzed for silver grain distribution. The results show a clustering of grains in the region of 4~15.3 (Fig. 1). Slides from both 0.5X SSC and 0.2X SSC at 60°C stringency washes were included in the analysis. Grain distribution was similar at both stringencies, although a higher level of background was observed at 0.5~ SSC. In the subsequent experiments using a tritiated probe, a 0.2X SSC at 60°C stringency level was adopted. Figure 2 shows the results of an analysis of chromosome 4 alone after hybridization with a tritiated probe. Figure 2A shows a profile derived from the analysis of 40 cells and confirms the assignment of DHPR to band 4~15.3. In Fig. 2B, the results of analyzes of chromosomes where band ~15.3 was resolved into three subbands are shown. The higher resolution in these metaphases suggests a more precise regional localization to band ~15.31. DISCUSSION
The human gene for DHPR has previously been assigned to chromosome 4 using enzyme analysis of a panel of human-mouse somatic cell hybrids (Kuhl et al., 1979). Using a cDNA clone and the technique of in situ hybridization, we have confirmed this assignment and localized the gene to the chromosome region 4~15.3 (Fig. 3). The molecular genetics of chromosome 4 has attracted considerable interest and approximately 100 cloned DNA fragments have been assigned to this chromosome (Gusella et aZ., 1986; Gilliam et cd., 1987).
69
REDUCTASE
Fewer than 20 of these markers represent known genes and very few have been localized to the p arm. DHPR is the first well-characterized gene to be assigned to pl.5-pter. This region is of particular interest because the locus for Huntington’s disease is also found in this part of the chromosome. The isolation of the G8 marker (Gusella et al., 1983), which is linked to Huntington’s disease and is located in the region 4~16.1 + 16.3, has been a first step in the development of presymptomatic tests and in the isolation of the Huntington’s disease gene. However, additional polymorphic markers in the region need to be found. The preparation of a recombinant DNA library from a somatic cell hybrid containing only the p arm of a translocation chromosome derived from chromosomes 4 and 5 and containing the region 4p15.1-pter (Wasmuth, 1986) is one potential source for isolating such markers. The regional localization of DHPR to band 4~15.3, together with the paucity of either known genes or anonymous DNA sequences in this region, makes it an obvious candidate for linkage studies in Huntington’s disease. Several RFLPs have already been detected with our DHPR cDNA probe (Dahl et d., 1987). They include one MspI and two AuaII polymorphisms with allele sizes (kb) of 1.3/1.2, 9.0/7.0, and 5.7/4.3, respectively. These RFLPs may prove useful in the antenatal diagnosis of DHPR deficiency and also in linkage studies in Huntington’s disease. ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of E. Earle in these studies and thank Professor G. Brownlee and Dr. R. Cotton for their advice and helpful discussions. This work was partly supported by a Programme Grant from the National Health and Medical Research Council of Australia. REFERENCES 1. BUCKLE, V. J., AND CRAIG, I. W. (1986). In situ hybridisation. In “Human Genetic Diseases: A Practical Approach” (K. Davies, Ed.), pp. 85-100, IRL Press, Oxford. 2. COITON, R. G. H. (1986). Inborn errors of pterin metabolism. In “Folates and Pterins,” Vol. 3, “Nutritional, Pharmacological and Physiological Aspects” (R. L. Blakley, Ed.), Wiley, New York. 3. DAHL, H.-H. M., HUTCHISON, W., M&DAM, W., WAKE, S., MORGAN, F. J., AND COTTON, R. G. H. (1987). Human dihydropteridine reductase: Characterisation of a cDNA clone and its use in analysis of patients with dihydropteridine reductase deficiency. Nucleic Acids Res. 15: 1921-1936. 4. GILLIAM, T. C., HEALEY, S. T., MACDONALD, M. E., STEWART, G. D., WASMUTH, J. J., TANZI, R. E., ROY, J. C., AND GUSELLA, J. F. (1987). Isolation of polymorphic DNA fragments from human chromosome 4. Nucleic Acids Res. 15: 1445-1458. 5. GUSELLA, J. F., GILLIAM, T. C., MACDONALD, M. E., CHENG, S. V., AND TANZI, R. E. (1986). Molecular genetics of human chromosome 4. J. Med. Genet. 23: 193-199.
70
BROWN
AND
DAHL
6. GUSELLA,
J. F., WEXLER, N. S., CONNEALLY, P. M., et al. (1983). A polymorphic DNA marker genetically linked to Huntington’s disease. Nature (London) 308: 234-238.
phism localised hybridisation.
8. KAUFMAN,
S., HOLZMAN, N. A., MILSTEIN, AND KRUMHOLZ, A. (1975). Phenylketonuria ciency of dihydropteridine reductase. N.
S., BUTLER, I. J., due to a defi-
Engl. J. Med. 293:
P., OLEK, K., WARDENBACH, P., AND GRZESCHIK, H. (1979). Assignment of a gene for human quinoid-dihydropteridine reductase (QDPR, EC 1.6.5.1.) to chromosome
MAGENIS, R. E., GUSELLA, J., WELIKY, I-LUGHT, G., TOTH-FIDEL, S., AND SHEEHY, tington disease-liked restriction fragment
of chromosome
4 by in situ
12.
WANG, H. S., GREENBERG, C. R., HEWITT, J., KALOUSEK, D., AND HAYDEN, M. R. (1986). Subregional assignment of the linked marker G8 (D4SlO) for Huntington disease to chromosome 4~16.1-16.3. Amer. J. Hum. Genet. 39: 392-396. WASMUTH, J. J., CARLOCK, L. R., SMITH, B., AND IMMKEN, L. L. (1986). A cell hybrid and recombinant DNA library that facilitate identification of polymorphic loci in the vicinity of the Huntington disease gene. Amer. J. Hum. Genet. 39:
13.
K.
397-403. 4.
Hum. Genet. 53: 47-49. 10.
~16.1
SMITH, I., HYLAND, K., KENDALL, B., AND LEEMINC, R. (1985). Clinical role of pteridine therapy in tetrahydrobiopterin deficiency. J. Inherited Metab. Dis. S(Supp1. 1): 39-45.
785-790. 9. KUHL,
band
11.
7. KAUFMAN,
S., AND FISHER, D. B. (1974). Pterin-requiring aromatic amino acid hydroxylases. Irz “Molecular Mechanism of Oxygen Activation” (0. Hayaishi, Ed.), pp. 285-369, Academic Press, New York.
within
Amer. J. Hum. Genet. 39: 383-391.
K., OLSON, S., R. (1986). Hunlength polymor-
14.
ZABEL, B. U., NAYLOR, S. L., SAKAGUCHI, A. Y., BELL, G. I., AND SHOWS, T. B. (1983). High-resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin, and a DNA fragment (D3Sl) by in situ hybridisation. Proc. Natl. Acad. Sci. USA 80: 6932-6936.
1,67-70
(1987)
Localization of the Human Dihydropteridine Reductase Gene to Band ~15.3 of Chromosome 4 by in Situ Hybridization R. M. Murdoch
Institute
BROWN’
AND H.-H.
May
6, 1987;
Press,
revised
Hospital, Melbourne,
Australia
June 15, 1987
Previous studies, using somatic cell hybrids, have shown that the DHPR gene is located on chromosome 4 (Kuhl et al., 1979). We have recently isolated and characterized a human DHPR cDNA clone (Dahl et al., 1987). Using this probe, we report the regional localization of the DHPR gene to band 4~15.3 by the technique of in situ hybridization.
We report the localization of the gene for dihydropteridine reductase (DHPR) to the human chromosome region 4~16.3 by in situ hybridization using a cDNA probe to the enzyme. The distal end of the short arm of chromosome 4 is of considerable interest because the gene responsible for Huntington’s disease is located in this region. Although this part of the chromosome is being extensively studied, DHPR is the first wellcharacterized gene to be assigned to the region. Restriction enzyme fragment length polymorphisms have been detected with a number of restriction endonucleases, including AuaII and MapI. These features may make the DHPR cDNA clone a useful probe not only for prenatal diagnosis of DHPR deficiency but also for linkage studies of Huntington’s disease. Academic
DAHL
for Research into Birth Defects, Royal Children’s Received
0 198’7
M.
MATERIALS
AND
METHODS
Chromosome Preparation
Lymphocytes from a normal, healthy male were stimulated with phytohemagglutinin (Wellcome) and cultured for 72 h at 37°C in medium 199 containing 15% fetal calf serum. Bromodeoxyuridine (200 rglml) was then added for a further 16 h (Zabel et al., 1983), after which the cells were washed twice in warm medium and incubated in fresh medium containing 10e5 M thymidine for a further 6.5 h. Chromosome preparations were made using standard techniques except that all procedures were carried out under safelights.
Inc.
INTRODUCTION
Dihydropteridine reductase (DHPR; EC 1.6.99.7) catalyzes the reduction of dihydropteridine to tetrahydrobiopterin, an essential cofactor in the reactions catalyzed by the three aromatic amino acid hydroxylases, phenylalanine, tyrosine, and tryptophan hydroxylase (Kaufman and Fisher, 1974). Lack of this cofactor leads to malignant hyperphenylalaninemia, a condition in which severe neurological disturbances persist in spite of dietary control of hyperphenylalaninemia and treatment with L-dopa and 5-hydroxytryptophan (Smith et al., 1985). DHPR deficiency was the first enzyme defect recognized as a cause of malignant hyperphenylalaninemia and it accounts for approximately 30% of all cases (Cotton, 1986). The disease is transmitted as an autosomal recessive disorder.
Preparation of Probe
The probe was a cDNA insert from hDHPR19 which was subcloned into plasmid pUC9 (Dahl et al., 1987). It covers the full coding region of human DHPR. The probe was labeled with either [lz51]dCTP or with three tritiated bases. Nick translation with [lz51]dCTP (Amersham International) resulted in a specific activity of a3 X 10’ dpm/pg. When tritiated compounds were used, the reaction mixture was 6 PM with respect to the three labeled nucleotides: [3H]dATP at 64 Ci/mmol, [3H]dCTP at 66 Ci/mmol, and [3H]TTP at 121 Ci/mmol (Amersham International); and 60 PM with respect to unlabeled dGTP. The specific activity of the tritiated probe was a2 X lo8 dpmlpg.
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. 503032. 1 To whom correspondence should be addressed at the Murdoch Institute, Royal Children’s Hospital, Parkville 3052, Victoria, Australia.
In Situ Hybridization
Hybridization of the probe to the chromosome preparations was carried out essentially as described 67
0868-7543187
$3.00
Copyright 8 1987 by Academic Preae, Inc. All righta of reproduction in any form reserved.
BROWN
AND
DAHL
6
-*&g
~tlmnhubRrjhm~~~ 13
FIG.
12
1.
Silver
grain
15
distribution
17
16
in 21 metaphases
after
19
in situ hybridization
by Buckle and Craig (1986). Slides were initially treated with 100 kg/ml RNAse in 2X SSC (20X SSC is 3 M NaCl, 0.3 A4 Na citrate, pH 7) at 37°C for 1 h. They were thoroughly rinsed with 2X SSC and dehydrated through an alcohol series. Chromosomal DNA was denatured by incubating at 65°C in 70% formamide, 0.1 mM EDTA, 2~ SSC for 4 min. Slides were rapidly cooled in cold 2~ SSC, rinsed in 2X SSC, and dehydrated. Approximately 100-200 ng of labeled probe was lyophilized and resuspended in 350 rl of hybridization fluid which contained 50% formamide, 5X Denhardt’s
A
18
m
of ‘%I-labeled
21
human
22
dihydropteridine
X
Y
reductase
probe.
solution (0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% Ficoll 400), 5X SSPE (0.9 M NaCl, 50 mA4 NaH2POI, 5 n&f EDTA, pH 7.2), 10% dextran sulfate, and 200 pg/ml salmon sperm DNA. The mixture was denatured by boiling for 5 min, followed by rapid cooling on ice. A 35-~1 aliquot of probe was placed on each of 10 slides, which were covered with a coverslip, sealed with rubber cement, and incubated overnight at 42°C. Coverslips were removed by rinsing in 5~ SSC and slides were washed in three changes of 2X SSC over 2
6
FIG. 2. (A) Silver grain distribution on chromosome cells after in situ hybridization with tritiated dihydropteridine ductase probe. (B) High-resolution analysis of chromosome cells.
4 in 40 re4 in 41
FIG. 3. Part of a human grains (arrows) on band ~15.3
metaphase spread of both chromosomes
showing 4.
silver
HUMAN
DIHYDROPTERIDINE
h. They were then usually washed in 0.5X SSC at 60°C for 30 min, followed by 1 h at the selected final stringency. This was either 0.5X SSC or 0.2X SSC, both at 6O”C, with a change of solution after 30 min. The slides were subsequently allowed to stand at room temperature for 1 h in 0.1X SSC before being dehydrated. Slides were dipped in L4 emulsion and exposed at 4°C for 5-12 days. They were developed at 20°C for 5 min in Kodak D19, diluted 1:l with water. They were then stained with Hoechst 33258 (15 pg/ml) for 30 min, exposed to black light (350 nm) in 2X SSC, and stained with 10% Giemsa for 25 min. Silver grains on or touching chromosomes were scored and related directly to chromosome bands. RESULTS
Analysis of the distribution of silver grains following in situ hybridization with a dihydropteridine reductase cDNA probe to chromosome preparations from male lymphocytes confirmed the assignment of the gene to chromosome 4. Initially, 21 cells that had been hybridized with ‘251-labeled probe were completely analyzed for silver grain distribution. The results show a clustering of grains in the region of 4~15.3 (Fig. 1). Slides from both 0.5X SSC and 0.2X SSC at 60°C stringency washes were included in the analysis. Grain distribution was similar at both stringencies, although a higher level of background was observed at 0.5~ SSC. In the subsequent experiments using a tritiated probe, a 0.2X SSC at 60°C stringency level was adopted. Figure 2 shows the results of an analysis of chromosome 4 alone after hybridization with a tritiated probe. Figure 2A shows a profile derived from the analysis of 40 cells and confirms the assignment of DHPR to band 4~15.3. In Fig. 2B, the results of analyzes of chromosomes where band ~15.3 was resolved into three subbands are shown. The higher resolution in these metaphases suggests a more precise regional localization to band ~15.31. DISCUSSION
The human gene for DHPR has previously been assigned to chromosome 4 using enzyme analysis of a panel of human-mouse somatic cell hybrids (Kuhl et al., 1979). Using a cDNA clone and the technique of in situ hybridization, we have confirmed this assignment and localized the gene to the chromosome region 4~15.3 (Fig. 3). The molecular genetics of chromosome 4 has attracted considerable interest and approximately 100 cloned DNA fragments have been assigned to this chromosome (Gusella et aZ., 1986; Gilliam et cd., 1987).
69
REDUCTASE
Fewer than 20 of these markers represent known genes and very few have been localized to the p arm. DHPR is the first well-characterized gene to be assigned to pl.5-pter. This region is of particular interest because the locus for Huntington’s disease is also found in this part of the chromosome. The isolation of the G8 marker (Gusella et al., 1983), which is linked to Huntington’s disease and is located in the region 4~16.1 + 16.3, has been a first step in the development of presymptomatic tests and in the isolation of the Huntington’s disease gene. However, additional polymorphic markers in the region need to be found. The preparation of a recombinant DNA library from a somatic cell hybrid containing only the p arm of a translocation chromosome derived from chromosomes 4 and 5 and containing the region 4p15.1-pter (Wasmuth, 1986) is one potential source for isolating such markers. The regional localization of DHPR to band 4~15.3, together with the paucity of either known genes or anonymous DNA sequences in this region, makes it an obvious candidate for linkage studies in Huntington’s disease. Several RFLPs have already been detected with our DHPR cDNA probe (Dahl et d., 1987). They include one MspI and two AuaII polymorphisms with allele sizes (kb) of 1.3/1.2, 9.0/7.0, and 5.7/4.3, respectively. These RFLPs may prove useful in the antenatal diagnosis of DHPR deficiency and also in linkage studies in Huntington’s disease. ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of E. Earle in these studies and thank Professor G. Brownlee and Dr. R. Cotton for their advice and helpful discussions. This work was partly supported by a Programme Grant from the National Health and Medical Research Council of Australia. REFERENCES 1. BUCKLE, V. J., AND CRAIG, I. W. (1986). In situ hybridisation. In “Human Genetic Diseases: A Practical Approach” (K. Davies, Ed.), pp. 85-100, IRL Press, Oxford. 2. COITON, R. G. H. (1986). Inborn errors of pterin metabolism. In “Folates and Pterins,” Vol. 3, “Nutritional, Pharmacological and Physiological Aspects” (R. L. Blakley, Ed.), Wiley, New York. 3. DAHL, H.-H. M., HUTCHISON, W., M&DAM, W., WAKE, S., MORGAN, F. J., AND COTTON, R. G. H. (1987). Human dihydropteridine reductase: Characterisation of a cDNA clone and its use in analysis of patients with dihydropteridine reductase deficiency. Nucleic Acids Res. 15: 1921-1936. 4. GILLIAM, T. C., HEALEY, S. T., MACDONALD, M. E., STEWART, G. D., WASMUTH, J. J., TANZI, R. E., ROY, J. C., AND GUSELLA, J. F. (1987). Isolation of polymorphic DNA fragments from human chromosome 4. Nucleic Acids Res. 15: 1445-1458. 5. GUSELLA, J. F., GILLIAM, T. C., MACDONALD, M. E., CHENG, S. V., AND TANZI, R. E. (1986). Molecular genetics of human chromosome 4. J. Med. Genet. 23: 193-199.
70
BROWN
AND
DAHL
6. GUSELLA,
J. F., WEXLER, N. S., CONNEALLY, P. M., et al. (1983). A polymorphic DNA marker genetically linked to Huntington’s disease. Nature (London) 308: 234-238.
phism localised hybridisation.
8. KAUFMAN,
S., HOLZMAN, N. A., MILSTEIN, AND KRUMHOLZ, A. (1975). Phenylketonuria ciency of dihydropteridine reductase. N.
S., BUTLER, I. J., due to a defi-
Engl. J. Med. 293:
P., OLEK, K., WARDENBACH, P., AND GRZESCHIK, H. (1979). Assignment of a gene for human quinoid-dihydropteridine reductase (QDPR, EC 1.6.5.1.) to chromosome
MAGENIS, R. E., GUSELLA, J., WELIKY, I-LUGHT, G., TOTH-FIDEL, S., AND SHEEHY, tington disease-liked restriction fragment
of chromosome
4 by in situ
12.
WANG, H. S., GREENBERG, C. R., HEWITT, J., KALOUSEK, D., AND HAYDEN, M. R. (1986). Subregional assignment of the linked marker G8 (D4SlO) for Huntington disease to chromosome 4~16.1-16.3. Amer. J. Hum. Genet. 39: 392-396. WASMUTH, J. J., CARLOCK, L. R., SMITH, B., AND IMMKEN, L. L. (1986). A cell hybrid and recombinant DNA library that facilitate identification of polymorphic loci in the vicinity of the Huntington disease gene. Amer. J. Hum. Genet. 39:
13.
K.
397-403. 4.
Hum. Genet. 53: 47-49. 10.
~16.1
SMITH, I., HYLAND, K., KENDALL, B., AND LEEMINC, R. (1985). Clinical role of pteridine therapy in tetrahydrobiopterin deficiency. J. Inherited Metab. Dis. S(Supp1. 1): 39-45.
785-790. 9. KUHL,
band
11.
7. KAUFMAN,
S., AND FISHER, D. B. (1974). Pterin-requiring aromatic amino acid hydroxylases. Irz “Molecular Mechanism of Oxygen Activation” (0. Hayaishi, Ed.), pp. 285-369, Academic Press, New York.
within
Amer. J. Hum. Genet. 39: 383-391.
K., OLSON, S., R. (1986). Hunlength polymor-
14.
ZABEL, B. U., NAYLOR, S. L., SAKAGUCHI, A. Y., BELL, G. I., AND SHOWS, T. B. (1983). High-resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin, and a DNA fragment (D3Sl) by in situ hybridisation. Proc. Natl. Acad. Sci. USA 80: 6932-6936.