Hum Genet (1991) 87 : 28-32
9 Springer-Verlag1991
A novel missense mutation in exon 8 of the ornithine transcarbamylase gene in two unrelated male patients with mild ornithine transcarbamylase deficiency Akira Hata 1'2, Toshinobu Matsuura 1'2, Chiaki Setoyama 1, Kazuniro Shimada 1, Tohru Yokoi 3, Izumi Akaboshi 2, and Ichiro Matsuda 2 1Department of Biochemistry, Kumamoto University Medical School, 2-2-1 Honjo, Kumamoto, 860 Japan 2Department of Pediatrics, Kumamoto University Medical School, 1-1-1 Honjo, Kumamoto, 860 Japan 3Department of Pediatrics, Kanazawa University Medical School, 13-1 Takaramachi, Ishikawa, 920 Japan Received April 17, 1990 / Revised September 26, 1990
Summary. We studied two unrelated male probands with mild ornithine transcarbamylase (OTC) (E.C.2.1.3.3) deficiency presenting a similar clinical course. Previous analyses of their liver OTCs also revealed similar properties. To identify the underlying molecular defects, we first cloned the entire coding region of the OTC gene from one proband and found a single base-substitution (C to T) leading to the substitution of tryptophan for arginine at amino acid position 277. Using a genomic amplification technique followed by allele specific oligonucleotide hybridization, we identified the same point mutation in the O T C gene of the other proband. We observed the presence of the mutation among family members in at least three generations, and in one asymptomatic hemizygous sibling in each family.
Introduction Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) deficiency, the most common inborn error of the urea cycle (Brunsilow and Horwich 1989), exhibits X-linked inheritance with frequent new mutations. In male patients presenting with the classical form, OTC activity is often undetectable, and the patients usually die during the neonatal period. Several male patients with mild clinical symptoms have been reported, and biochemical studies of these patients have revealed a variety of abnormal kinetic properties of OTC (Briand et al. 1982; Matsuda et al. 1984). Therefore, O T C deficiency is a heterogeneous entity caused by a variety of defects in the OTC gene, and at present, the molecular properties of most of the defects are poorly understood. To search for a possible correlation between the clinical course and molecular defects in OTC deficiency, cloning and characterization of the normal human OTC Offprint requests to: I. Matsuda
gene was required. We reported the structural organization of the human OTC gene and the complete nucleotide sequence of its coding region (Hata et al. 1986, 1988a, b). The gene spans 73 kb and contains 10 exons encoding a polypeptide that consists of 354 amino acids, including a leader sequence of 32 amino acids. Although exons 3 and 9 are known to encode the carbamyl phosphate binding site and the ornithine binding site, respectively (Hata et al. 1988a), other functional epitopes of OTC are less well understood. The availability of cloned human OTC c D N A and genomic D N A has made feasible the characterization of the molecular defects in patients with OTC deficiency. In several patients, deletions of part or all of the OTC gene have been identified (Old et al. 1985; Rozen et al. 1985; 1986, McClead et al. 1986; Maddalena et al. 1988a). However, the majority of patients with O T C deficiency do not have this deletion, and point mutations or small structural changes in the OTC gene are presumably involved. Recently, several point mutations in the OTC gene have been reported (Maddalena et al. 1988b; Hata et al. 1989; Grompe et al. 1989). We describe here a novel missense mutation in exon 8 of the OTC gene in two unrelated male patients with mild OTC deficiency. Using in vitro genomic amplification followed by allele-specific oligonucleotide (ASO) hybridization, we observed the presence of this mutation among family members in at least three generations.
Materials and methods Case histories
Data on probands Y and M, two Japanese male subjects with mild OTC deficiency, have been reported (previously designated patients i and 2) (Yokoi et al. 1981). Their families were non-consanguineous and of disticnt last names. Proband Y developed normally until 13 months of age when an episode of vomiting and lethargy lasting for three days occurred.
29 Reye syndrome was diagnosed on the basis of hyperammonemia and the increased levels of GOT (glutamic oxaloacetic transarninase), GPT (glutamic pyruvic transaminase) and CPK (creatine phosphokinase). At 15 months of age, OTC deficiency was suspected from a history of repeated attacks of vomiting, lethargy, hyperammonemia, serum amino acids profile and a high excretion of orotic acids. Hepatic OTC activity was 19% of control values and the Km for ornithine was 8.3mM at pH 7.5, approximately ten-fold higher than the control. Although normal OTC activity is maximal at about pH 7.7-7.8, this optimum was shifted to pH 8.5 and over in proband Y. After the diagnosis of OTC deficiency was established, proband Y and his siblings followed a low protein diet. The IQ of proband Y was normal at age 9 years. Proband M developed normally until 13 months of age when he experienced an episode of vomiting and lethargy. At 14 months of age, OTC deficiency was suspected from clinical findings and laboratory data similar to those of proband Y. The OTC of this proband also showed a decrease in enzyme activity (16% of the control values), an abnormal Km for ornithine (9.8mM at pH 7.5), and a shift of pH optimum to the alkaline side. Dietary care similar to that of family Y was initiated in the proband and family members, including two brothers; none of them have had any clinical symptoms. The IQ of proband M was normal at 8 years.
Slot blots and ASO hybridization For slot blot analysis, 5 ~tlarnplifed genomic DNA were denatured with 0.3 N NaOH in a final volume of 50 ~tl and heated for 30 min at 65~ The samples were cooled to room temperature, neutralized by adding 50 ~tl 2 M NH4OAc, and loaded into the wells of a Schleieher and Schuell Minifold II Slot Blotter (FRG) containing a nitrocellulose filter pre-wetted in 1M NH4OAc. Two replicate filters were prepared. One was hybridized with the wild type 19-met oligonucleotide probe end-labeled with 32p, the other with the mutant 19mer oligonucleotide probe. Hybridization was carried out in 5 • SSPE (1 x SSPE = 150 mM NaC1, 10 mM NaHzPO4, l m M EDTA, pH 7.4), 5 x Denhardt's solution (1 • Denhardt's solution = 0.002% bovine serum albumird0.02% polyvinylpyrrolidone/0.02% Fieoll), and 0.5% sodium dodecyl sulfate (SDS) for 6h at 37~ The blots were washed three times in 6 • SSC(1 • SSC = 150mM NaCY 15mM sodium citrate, pH 7.0)/0.1% SDS at room temperature and once for 30min at 57~ for the wild type probe and 55~ for the mutant probe hybridization, in the same buffer. These conditions were determined from the melting temperatures calculated for the homo-duplexes generated between each oligonucleotide and its complementary genomic sequence (Thein and Wallace 1986).
A test of heterozygosity for OTC deficiency Genomic DNA extraction Genomic DNAs were prepared either from Epstein-Barr virus transformed lymphoid cell lines (Hata et al. 1986), or from 3-4 ml of whole peripheral blood ceils (Old 1986).
Two female family members were tested for heterozygosity for the mutant OTC allele by allopurinol treatment (Brusilow and Valle 1987). After an initial 6-h urine collection, each woman took a single 300 mg allopurinol tablet and collected a second 6-h urine sample. Urinary orotic acid was measured by the procedure described by Adachi et al. (1963).
Cloning and sequencing Two independently constructed human genomic libraries were screened for the clones carrying the OTC gene. The first was constructed from EcoRI partial digests of high molecular weight lymphoblastoid DNA derived from proband Y, using EMBL4 DNA (Stratagene, USA) as the vector. Approximately 9 x 10s phages from the unamplified DNA library were screened with the cDNA probe EC5, which covers all exons of the human OTC gene (Hata et al. 1988a). All except exon 10 were cloned from this library. To clone the genomic fragment containing exon 10, genomic DNA was completely digested by EcoRI and a DNA fraction containing 3-5 kb fragments was cloned into the EcoRI sites of lambda gt 10 (Stratagene). Approximately 5 x 105 phages were screened with the cDNA probe EC7, spanning exon 7 to exon 10 (Hata et al. 1988a). DNA sequencing was performed as described elsewhere (Hate et al. 1988a).
Oligonucleotide synthesis Two 25-mer oligonucleotide primers and two 19-mer probes were synthesized on a ZEON GENET A-III (Nippon Zeon, Japan), using the methoxyphosphoramidite method; they were purified by high-pressure liquid chromatography on a C 18column.
Amplification of genomic D NA Genomic DNAs prepared from the patients and their family members were amplified in vitro by the polymerase chain reaction (PCR) (Saiki et al. 1985, 1988), using Taq DNA polymerase (Perkin Elmer Cetus, USA) and two synthetic primers (5'-AATGGTACCAAGC TGTTGCTGACAA-3' and 5'-CTTCATTGTAACCTGGTAAC CTTGG-3'). One of the two primers corresponds to the 5' end sequence of exon 8, and the other, to the 3' end anti-sense strand sequence of exon 8 (Hata et al. 1988a). PCR was carried out on a Program Temp Control System PC-500 (Astec, Japan). Genomic DNA (1 gg) was subjected to 30 cycles of amplification, (60 s denaturation at 94~ 60 s reannealing at 50~ and 120 s polymerization at 72~ The amplified DNA was examined by electrophoresis on a 4% NuSieve GTG agarose gel (FMC, USA).
Secondary structure and hydrophobicity profile prediction Secondary structures of the normal and mutant OTCs were predicted from their amino acid sequences by the method of Chou and Fasman (1978); hydrophobicity profiles were predicted by the method of Rose and Roy (1980) using the PRINAS program obtained from Mitsui Knowledge Industry (Japan).
Results
Cloning and sequencing of the entire coding region of the OTC gene of proband Y W e s c r e e n e d a p p r o x i m a t e l y 9 • 105 p h a g e clones carrying g e n o m i c D N A d e r i v e d f r o m p r o b a n d Y, using as a p r o b e the h u m a n O T C c D N A E C 5 a n d c o v e r i n g all the exons of the h u m a n n o r m a l O T C gene ( H a t a et al. 1988a). W e isolated 12 i n d e p e n d e n t clones carrying the O T C gene. S o u t h e r n blot analysis and D N A sequencing showed that a l t o g e t h e r they c o v e r e d e x o n 1 to e x o n 9, b u t n o t e x o n 10. A s o u r p r e v i o u s studies showed that e x o n 10 of the h u m a n O T C g e n e is p r e s e n t o n a 3.9-kb EcoR! fragm e n t ( H a t a et al. 1988a), we c o n s t r u c t e d a n e w l i b r a r y after fractionating the EcoRI c o m p l e t e digests of g e n o m i c D N A f r o m p r o b a n d Y a n d collecting the f r a c t i o n that c o n t a i n e d 3 - 5 kb D N A f r a g m e n t s . A p p r o x i m a t e l y 5 • 105 plaques of this library were screened, using as a p r o b e the h u m a n O T C c D N A E C 7 , which covers e x o n 7 to e x o n 10 ( H a t a et al. 1988a). O f two positive clones, o n e carried the g e n o m i c D N A s e q u e n c e c o r r e s p o n d i n g to e x o n 10 of the h u m a n O T C gene. W e d e t e r m i n e d the n u c l e o t i d e s e q u e n c e of the e n t i r e coding r e g i o n a n d e x o n / i n t r o n b o u n d a r i e s of the O T C
30
Fig. 1. Sequencing gels around the site of a point mutation in exon 8 from wild type and proband Y (patient). A thymine has replaced the normal cytosine in codon 277 (arrows), causing a change from arginine to tryptophan at amino acid 245 of the mature protein
gene of p r o b a n d Y, and c o m p a r e d them with those of the previously determined h u m a n normal O T C gene ( H a t a et al. 1988a). We found that the sequence of the coding region of the O T C gene of p r o b a n d Y could be aligned perfectly with that of the h u m a n normal O T C gene, except for a single-base substitution present in exon 8. This change was a C to T transition in codon 277 and resulted in the replacement of an arginine by a tryptophan residue (Fig. 1).
Identification of the mutation in proband M with synthetic oligonucleotide probes As the clinical symptoms and properties of hepatic OTCs of probands Y and M were similar, we presumed that they carried the same O T C mutation. For confirmation, we analyzed exon 8 of the O T C gene of proband M. Using total genomic D N A prepared from peripheral leukocytes of p r o b a n d M, the entire D N A sequence of exon 8 was amplified by the P C R and analyzed by A S O hybridization. For this purpose, we synthesized two different 19m e r oligonucleotide as probes, one corresponding to exon 8 of the normal O T C gene from codon 274 to the first base of codon 280 ( 5 ' - A A G A A A A A G C G G C T C C A G G - 3 ' ) ( H a t a et al. 1988a) (Fig. 1 wild type), the other corresponding to the same part of the O T C gene of proband Y ( 5 ' - A A G A A A A A G T G G C T C C A G G - 3 ' ) (Fig. 1 patient). As shown in Fig. 2, genomic D N A s from probands Y amd M hybridized strongly to the mutant probe, but not to the normal one. These results confirm that the probands Y and M carry the same point mutation in their O T C genes.
Genotypic analysis of familis Y and M As O T C deficiency is an X-linked disorder, a mutation present in the O T C gene of a male patient can be trans-
Fig. 2A-D. Segregation of the mutant allele in the two families. A Pedigree of family Y. Squares represent males and circles females. Open symbols represent the normal allele and solid symbols the mutant allele. Stippled square denotes asymptomatic sibling of hemizygote with mutant allele. An arrow indicates the proband. B ASO hybridization analysis of family Y. Total genomic DNAs were isolated from each individual, and the DNA region containing exon 8 of the OTC gene was amplified by the PCR method. Amplified DNA samples were spotted on a nitrocellulose filter, using a slot blot template and hybridized with 32p-labeled synthetic oligonucleotide probes. Wild-type probe corresponds to the normal sequence of exon 8, and mutant probe contains the substitution of T to C of the normal sequence. C Pedigree of family M. Symbols are the same as shown in A. D ASO hybridization analysis of family M. Experimental procedures are as described in B
mitted from his mother or can arise de novo in the germ line during early devleopment. In family Y, the proband is the only m e m b e r of the family clinically afflicted with disease (Fig. 2A). However, the pattern of A S O hybridization revealed that his brother is also hemizygous for the mutant allele. His sister, mother and aunt were all heterozygous for the O T C gene (Fig. 2B). Thus, the grandmother of proband Y carried this mutation.
31 Table 1. Urinary orotic acid level (mg/g creatinine)
Attack Pedigree Y I-2 I-3 II-1 II-2 II-3
Allopurinol(-) Allopurinol(+) a wild type mutant
750
2.8 52.4 4.8 14.4 6.8
24.4 74.6
Glu Lys Lys LyslArg I Leu Gin Ala Phe e e e e p e e e e hh hh hh hhT~re
A
2.0
I
t
1.5-
Pedigree M II-2 III-1 III-2
Controls b
1070
3.2 7.0 3.3
x
~" 7
gm 0.5
3.0
a During the first 6 h after an oral administration of allopurinol (300 rag) b n = 12. Standard error was _+0.3 and the range of urinary orotic acid levels for normal subjects was between 1.5 and 4.8 In family M, of the two phenotypically normal brothers of proband M, one was hemizygous for the mutant allele (Fig. 2D). The mother of proband M and his grandmother were also heterozygous for the OTC gene and had the same mutation in one of their alleles. Thus, the C to T mutation in the OTC gene has been stably inherited through at least three generations in these two unrelated families. Urinary orotic acid levels in the pedigree of Y and M After allopurinol loading, urinary orotic acid levels increased from 2.8 mg/g creatinine to 24.4mg/g creatinine in the mother (pedigree Y, I-2) and from 52.4 mg/g creatinine to 74.6 mg/g creatinine in the aunt (pedigree Y, I-3) (Table 1, Fig. 2A), indicating a carrier state of O T C deficiency (Brusilow and Valle 1987). Two siblings (pedigree Y, II-2 and II-3) and proband M excreted slightly but significantly higher amounts of orotic acid compared with controls without allopurinol load (Table 1, Fig. 3A). During hyperammonemic attack both probands Y and M excreted extremely high amounts of orotic acid in urine (Table 1).
Discussion A novel point mutation in the OTC gene, a C to T transition that leads to the replacement of the arginine 277 by tryptophan, was detected in two unrelated male patients with mild O T C deficiency. Based on the following supporting evidence, we conclude that this mutation is the direct cause of the O T C deficiency. This nucleotide substitution has not been observed in more than 100 different human X chromosomes derived from either healthy persons or patients with O T C deficiency (A. Hata, K. Shimada, and I. Matsuda, unpublished observations), and is not likely to represent one neutral polymorphism. We expected to see mutation(s) in the coding region of the OTC gene of these two unrelated male probands, because enzymatic characteristics of their OTCs showed abnormalities in kinetic or physicochemical properties, such as increased Km values for ornithine and a shift of p H opti-
1.o
0.0 -0.5
!
250 B
3;0 Residue number
Fig. 3A, B. Effects of the mutation on the structure of OTC. A Effects of the mutation on the secondary structure, from amino acid residues 273 to 281. B Effects of the mutation on the hydrophobic-
ity profile. In A, h and e denote alpha-helix and beta-sheet, respectively. In B, solid line shows the profile of the wild type protein and broken line the mutant OTC mum to the alkaline side. We found no other base substitution resulting in amino acid change in the entire coding region of the O T C gene of proband Y. The arginine 277 is completey conserved in all the OTCs of human, rat, mouse, E. coli and Aspergillus (Huygen et al. 1987; Veres et al. 1987). This observation strongly suggests that arginine 277 is essential for the full activity of OTCs. The arginine to tryptophan substitution is a change from a basic amino acid residue to a neutral large one. When we compared the secondary structures of normal and mutant sequences as predicted by the method of Chou and Fasman (1978), we found that the amino acid residue at position 277 was located at the boundary between a-helix and [3-sheet structures and that the substitution from arginine to tryptophan resulted in a shift of that boundary (Fig. 3A). A comparison of hydrophobicity profiles also revealed that this change induces a significant increase in hydrophobicity (Fig. 3B). These predictions support the conclusion that this mutation causes a significant conformational change of the protein, either altering the affinity for substrates, or affecting the stability of the protein. Two individuals heterozygous for this mutation (Table 1, pedigree Y, I-2 and I-3) showed abnormally increases orotic aciduria after allopurinol loading, a response not observed in normal control individuals (Brusilow and Valle 1987). These results eliminate the possiblity that the C to T mutation reflects a neutral polymorphism. The difference in response to the drug between the heterozygous females is considered to reflect both differences in protein intake and the effect of random X inactivation in hepatic cells. The almost normal or slightly elevated urinary orotic acid level in both subjects without allopurinol loading is consistent with the moderate phenotypic expression of this mutation in hemizygous males.
32 W e f o u n d n o g e n e t i c link b e t w e e n families Y a n d M in t e r m s o f f a m i l y h i s t o r y . F u r t h e r m o r e , as we discussed in d e t a i l in t h e p r e v i o u s r e p o r t o f O T C deficiency ( H a t a et al. 1989), t h e c y t o s i n e at C p G is a h o t s p o t for m u t a tion in h u m a n s , a n d is k n o w n to b e m e t h y l a t e d ; s p o n t a n e o u s d e a m i n a t i o n to t h y m i n e results in a C to T transition. W e initially p r e s u m e d t h a t t h e s e two m u t a t i o n s occ u r r e d i n d e p e n d e n t l y . F o r t h e p u r p o s e o f p r o v i d i n g evid e n c e for i n d e p e n d e n t r e c u r r e n t m u t a t i o n e v e n t s , the two probands were haplotyped by restriction fragment l e n g t h p o l y m o r p h i s m s ( R F L P s ) in t h e O T C g e n e using MspI, TaqI a n d BamHI. H o w e v e r , as b o t h p r o b a n d s s h o w e d t h e s a m e h a p l o t y p e (MspI: 6.2 a n d 5.1 k b ; TaqI: 3.7 kb; BamHI: 18.0 k b ) , w e c o u l d n o t e x c l u d e the possibility o f this m u t a t i o n o r i g i n a t i n g f r o m the s a m e ancestry. T o o u r s u r p r i s e , p e d i g r e e a n a l y s e s using the A S O hyb r i d i z a t i o n t e c h n i q u e r e v e a l e d t h a t e a c h p o r b a n d has ano t h e r m a l e sibling c a r r y i n g t h e s a m e m u t a t i o n , a l t h o u g h t h e r e a r e as y e t n o clinical s y m p t o m s , p r e s u m a b l y b e cause t h e i r m o t h e r s h a v e t h e b o y s o n a p r o t e i n restrict i o n diet; t h e p o s s i b i l i t y o f a n u n i d e n t i f i e d g e n e t i c f a c t o r r e l a t e d to t h e a p p e a r a n c e o f s y m p t o m s awaits clarification. S e v e r a l m a l e p a t i e n t s with O T C d e f i c i e n c y of l a t e o n s e t a r e k n o w n to r e a c t well to a p r o t e i n r e s t r i c t e d diet ( M a t s u d a et al. 1991); o u r p a t i e n t s a n d t h e i r b r o t h e r s can be i n c l u d e d in this g r o u p .
Acknowledgements. We thank Dr. Sumio Tanase for pertinent discussion on the protein conformation of OTC and Dr. Jean-Marc Lalouel for helpful comment. Editorial assistance by Mariko Ohara is also gratefully acknowledged. This work was supported by grants from the Ministry of Education, Science and Culture (62304042, 63571086), a grant for Pediatric Research (63A-01) from the Ministry of Health and Welfare of Japan and grant-in-aid from the Japan Medical Association (I.M.).
References
Adachi T, Tanimura A, Asahina M (1963) A colorimetric determination of orotic acid. J Vitaminol 9 : 217-226 Briand P, Francois B, Rabier D, Cathelineau L (1982) Ornithine transcarbamylase deficiencies in human males; kinetic and immunochemical classification. Biochem Biophys Acta 704 : 100106 Brusilow SW, Horwich AL (1989) Urea cycle enzymes. In: Scriber CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGraw-Hill, NewYork, pp 629-663 Brusilow S, Valle D (1987) Allopurinol (AP) induced orotidinuria (ODNU): a test of heterozygosity for ornithine transcarbamylase (OTC) deficiency. Pediatr Res 21 : 289A Chou PY, Fasman GD (1978) Prediction of secondary structure of proteins from their amino acid sequence. Adv Enzymo147 : 45148 Finkelstein JE, Francomano CA, Brusilow SW, Traystman MD (1990) Use of denaturing gradient gel electrophoresis for detection of mutation and prospective diagnosis in late onset ornithine transcarbamylase deficiency. Genomics 7 : 167-172 Grompe M, Muzny DM, Caskey CT (1989) Scanning detection of mutations in human ornithine transcarbamylase by chemical mismatch cleavage. Proc Natl Acad Sci USA 86 : 5888-5892 Hata A, Tsuzuki T, Shimada K, Takiguchi M, Mori M, Matsuda I (1986) Isolation and characterization of the human ornithine transcarbamylase gene: structure of the 5'-end region. J Biochem 100 : 717-725
Hata A, Tsuzuki T, Shimada K, Takiguchi M, Mori M, Matsuda I (1988a) Structure of the human ornithine transcarbamylase gene. J Biochem 103 : 302-308 Hata A, Tsuzuki T, Shimada K, Takiguchi M, Mori M, Matsuda I (1988b) Structural organization of the human ornithine transcarbamylase gene. J Inherited Metab Dis 11:337-340 Hata A, Setoyama C, Shimada K, Takeda E, Kuroda Y, Akaboshi I, Matsuda I (1989) Ornithine transcarbamylase deficiency resulting from a C-to-T substitution in exon 5 of the ornithine transcarbamylase gene. Am J Hum Genet 45 : 123-127 Huygen R, Crabeel M, Glansdorff N (1987) Nucleotide sequence of the ARG3 gene of the yeast Saccharomyces cerevisiae encoding ornithine carbamoyltransferase. Comparison with other carbamoyltransferases. Eur J Biochem 166: 371-377 Maddalena A, Sosnoski DM, Berry GT, Nussbaum RL (1988a) Mosaicism for an intragenic deletion in a boy with mild ornithine transcarbamylase deficiency. N Engl J Med 319:999-1003 Maddalena A, Spence JE, O'Brien WE, Nussbaum RL (1988b) Characterization of point mutations in the same arginine codon in three unrelated patients with ornithine transcarbamylase deficiency. J Clin Invest 82 : 1353-1358 Matsuda I, Nagata N, Oyanagi K, Tsuchiyama A, Yamamoto H, Hase Y, Kodama H, Kai H (1984) Biochemical heterogeneity of ornithine carbamyl transferase (OTC) in patients with OTC deficiency. Jpn J Hum Genet 29 : 327-333 Matsuda I, Nagata N, Matsuura T, Oyanagi K, Tada K, Narisawa K, Kitagawa T, Sakiyama T, Yamashita F, Yoshino M (1991) Retrospective survey of urea cycle disorders. I. Clinical and laboratory observations of thirty-two Japanese male patients with ornithine transcarbamylase (OTC) deficiency. Am J Med Genet 38 : 85-89 McClead RE Jr, Rozen R, Fox J, Rosenberg L, Menke J, Bickers R, Morrow G l I I (1986) Clinical application of DNA analysis in a family with OTC deficiency. Am J Med Gcnet 25:513-518 Old JM, Briand PL, Purvis-Smith S, Howard N J, Wilcken B, Hammond J, Pearson P, Cathelineau L, Williamson R, Davies KE (1985) Prenatal exclusion of ornithine transcarbamylase deficiency by direct gene analysis. Lancet I : 73-75 Old JM (1986) Fetal DNA analysis. In: Davies KE (ed) Human genetic diseases: a practical approach. IRL, Oxford Washington, DC, pp 1-17 Rose GD, Roy S (1980) Hydrophobic basis of packing in globular proteins. Proc Natl Acad Sci USA 77 : 4643-4647 Rozen R, Fox J, Fenton WA, Horwich AL, Rosenberg LE (1985) Gene deletion and restriction fragment length polymorphisms at the human ornithine transcarbamylase locus. Nature 313: 815-817 Rozen R, Fox JE, Hack AM, Fenton WA, Horwich AL, Rosenberg LE (1986) DNA analysis for ornithine transcarbamylase deficiency. J Inherited Metab Dis 9 [Suppl 1] : 49-57 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of ~-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 : 487-491 Thein SL, Wallace RB (1986) The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders. In: Davies KE (ed) Human genetic diseases: a practical approach. IRL, Oxford Washington, DC, pp 33-50 Veres G, Gibbs RA, Sherer SE, Caskey CT (1987) The molecular basis of the sparse for mouse mutation. Science 237 : 415-417 Yokoi T, Honke K, Funabashi T, Hayashi R, Suzuki Y, Taniguchi N, Hosoya M, Saheki T (1981) Partial ornithine transcarbamylase deficiency simulating Reye syndrome. J Pediatr 99:929-931 Note added in proof. After this paper had been submitted, a report appeared dealing with the characterization of the same point mutation (Finkelstein et al. 1990).
9 Springer-Verlag1991
A novel missense mutation in exon 8 of the ornithine transcarbamylase gene in two unrelated male patients with mild ornithine transcarbamylase deficiency Akira Hata 1'2, Toshinobu Matsuura 1'2, Chiaki Setoyama 1, Kazuniro Shimada 1, Tohru Yokoi 3, Izumi Akaboshi 2, and Ichiro Matsuda 2 1Department of Biochemistry, Kumamoto University Medical School, 2-2-1 Honjo, Kumamoto, 860 Japan 2Department of Pediatrics, Kumamoto University Medical School, 1-1-1 Honjo, Kumamoto, 860 Japan 3Department of Pediatrics, Kanazawa University Medical School, 13-1 Takaramachi, Ishikawa, 920 Japan Received April 17, 1990 / Revised September 26, 1990
Summary. We studied two unrelated male probands with mild ornithine transcarbamylase (OTC) (E.C.2.1.3.3) deficiency presenting a similar clinical course. Previous analyses of their liver OTCs also revealed similar properties. To identify the underlying molecular defects, we first cloned the entire coding region of the OTC gene from one proband and found a single base-substitution (C to T) leading to the substitution of tryptophan for arginine at amino acid position 277. Using a genomic amplification technique followed by allele specific oligonucleotide hybridization, we identified the same point mutation in the O T C gene of the other proband. We observed the presence of the mutation among family members in at least three generations, and in one asymptomatic hemizygous sibling in each family.
Introduction Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) deficiency, the most common inborn error of the urea cycle (Brunsilow and Horwich 1989), exhibits X-linked inheritance with frequent new mutations. In male patients presenting with the classical form, OTC activity is often undetectable, and the patients usually die during the neonatal period. Several male patients with mild clinical symptoms have been reported, and biochemical studies of these patients have revealed a variety of abnormal kinetic properties of OTC (Briand et al. 1982; Matsuda et al. 1984). Therefore, O T C deficiency is a heterogeneous entity caused by a variety of defects in the OTC gene, and at present, the molecular properties of most of the defects are poorly understood. To search for a possible correlation between the clinical course and molecular defects in OTC deficiency, cloning and characterization of the normal human OTC Offprint requests to: I. Matsuda
gene was required. We reported the structural organization of the human OTC gene and the complete nucleotide sequence of its coding region (Hata et al. 1986, 1988a, b). The gene spans 73 kb and contains 10 exons encoding a polypeptide that consists of 354 amino acids, including a leader sequence of 32 amino acids. Although exons 3 and 9 are known to encode the carbamyl phosphate binding site and the ornithine binding site, respectively (Hata et al. 1988a), other functional epitopes of OTC are less well understood. The availability of cloned human OTC c D N A and genomic D N A has made feasible the characterization of the molecular defects in patients with OTC deficiency. In several patients, deletions of part or all of the OTC gene have been identified (Old et al. 1985; Rozen et al. 1985; 1986, McClead et al. 1986; Maddalena et al. 1988a). However, the majority of patients with O T C deficiency do not have this deletion, and point mutations or small structural changes in the OTC gene are presumably involved. Recently, several point mutations in the OTC gene have been reported (Maddalena et al. 1988b; Hata et al. 1989; Grompe et al. 1989). We describe here a novel missense mutation in exon 8 of the OTC gene in two unrelated male patients with mild OTC deficiency. Using in vitro genomic amplification followed by allele-specific oligonucleotide (ASO) hybridization, we observed the presence of this mutation among family members in at least three generations.
Materials and methods Case histories
Data on probands Y and M, two Japanese male subjects with mild OTC deficiency, have been reported (previously designated patients i and 2) (Yokoi et al. 1981). Their families were non-consanguineous and of disticnt last names. Proband Y developed normally until 13 months of age when an episode of vomiting and lethargy lasting for three days occurred.
29 Reye syndrome was diagnosed on the basis of hyperammonemia and the increased levels of GOT (glutamic oxaloacetic transarninase), GPT (glutamic pyruvic transaminase) and CPK (creatine phosphokinase). At 15 months of age, OTC deficiency was suspected from a history of repeated attacks of vomiting, lethargy, hyperammonemia, serum amino acids profile and a high excretion of orotic acids. Hepatic OTC activity was 19% of control values and the Km for ornithine was 8.3mM at pH 7.5, approximately ten-fold higher than the control. Although normal OTC activity is maximal at about pH 7.7-7.8, this optimum was shifted to pH 8.5 and over in proband Y. After the diagnosis of OTC deficiency was established, proband Y and his siblings followed a low protein diet. The IQ of proband Y was normal at age 9 years. Proband M developed normally until 13 months of age when he experienced an episode of vomiting and lethargy. At 14 months of age, OTC deficiency was suspected from clinical findings and laboratory data similar to those of proband Y. The OTC of this proband also showed a decrease in enzyme activity (16% of the control values), an abnormal Km for ornithine (9.8mM at pH 7.5), and a shift of pH optimum to the alkaline side. Dietary care similar to that of family Y was initiated in the proband and family members, including two brothers; none of them have had any clinical symptoms. The IQ of proband M was normal at 8 years.
Slot blots and ASO hybridization For slot blot analysis, 5 ~tlarnplifed genomic DNA were denatured with 0.3 N NaOH in a final volume of 50 ~tl and heated for 30 min at 65~ The samples were cooled to room temperature, neutralized by adding 50 ~tl 2 M NH4OAc, and loaded into the wells of a Schleieher and Schuell Minifold II Slot Blotter (FRG) containing a nitrocellulose filter pre-wetted in 1M NH4OAc. Two replicate filters were prepared. One was hybridized with the wild type 19-met oligonucleotide probe end-labeled with 32p, the other with the mutant 19mer oligonucleotide probe. Hybridization was carried out in 5 • SSPE (1 x SSPE = 150 mM NaC1, 10 mM NaHzPO4, l m M EDTA, pH 7.4), 5 x Denhardt's solution (1 • Denhardt's solution = 0.002% bovine serum albumird0.02% polyvinylpyrrolidone/0.02% Fieoll), and 0.5% sodium dodecyl sulfate (SDS) for 6h at 37~ The blots were washed three times in 6 • SSC(1 • SSC = 150mM NaCY 15mM sodium citrate, pH 7.0)/0.1% SDS at room temperature and once for 30min at 57~ for the wild type probe and 55~ for the mutant probe hybridization, in the same buffer. These conditions were determined from the melting temperatures calculated for the homo-duplexes generated between each oligonucleotide and its complementary genomic sequence (Thein and Wallace 1986).
A test of heterozygosity for OTC deficiency Genomic DNA extraction Genomic DNAs were prepared either from Epstein-Barr virus transformed lymphoid cell lines (Hata et al. 1986), or from 3-4 ml of whole peripheral blood ceils (Old 1986).
Two female family members were tested for heterozygosity for the mutant OTC allele by allopurinol treatment (Brusilow and Valle 1987). After an initial 6-h urine collection, each woman took a single 300 mg allopurinol tablet and collected a second 6-h urine sample. Urinary orotic acid was measured by the procedure described by Adachi et al. (1963).
Cloning and sequencing Two independently constructed human genomic libraries were screened for the clones carrying the OTC gene. The first was constructed from EcoRI partial digests of high molecular weight lymphoblastoid DNA derived from proband Y, using EMBL4 DNA (Stratagene, USA) as the vector. Approximately 9 x 10s phages from the unamplified DNA library were screened with the cDNA probe EC5, which covers all exons of the human OTC gene (Hata et al. 1988a). All except exon 10 were cloned from this library. To clone the genomic fragment containing exon 10, genomic DNA was completely digested by EcoRI and a DNA fraction containing 3-5 kb fragments was cloned into the EcoRI sites of lambda gt 10 (Stratagene). Approximately 5 x 105 phages were screened with the cDNA probe EC7, spanning exon 7 to exon 10 (Hata et al. 1988a). DNA sequencing was performed as described elsewhere (Hate et al. 1988a).
Oligonucleotide synthesis Two 25-mer oligonucleotide primers and two 19-mer probes were synthesized on a ZEON GENET A-III (Nippon Zeon, Japan), using the methoxyphosphoramidite method; they were purified by high-pressure liquid chromatography on a C 18column.
Amplification of genomic D NA Genomic DNAs prepared from the patients and their family members were amplified in vitro by the polymerase chain reaction (PCR) (Saiki et al. 1985, 1988), using Taq DNA polymerase (Perkin Elmer Cetus, USA) and two synthetic primers (5'-AATGGTACCAAGC TGTTGCTGACAA-3' and 5'-CTTCATTGTAACCTGGTAAC CTTGG-3'). One of the two primers corresponds to the 5' end sequence of exon 8, and the other, to the 3' end anti-sense strand sequence of exon 8 (Hata et al. 1988a). PCR was carried out on a Program Temp Control System PC-500 (Astec, Japan). Genomic DNA (1 gg) was subjected to 30 cycles of amplification, (60 s denaturation at 94~ 60 s reannealing at 50~ and 120 s polymerization at 72~ The amplified DNA was examined by electrophoresis on a 4% NuSieve GTG agarose gel (FMC, USA).
Secondary structure and hydrophobicity profile prediction Secondary structures of the normal and mutant OTCs were predicted from their amino acid sequences by the method of Chou and Fasman (1978); hydrophobicity profiles were predicted by the method of Rose and Roy (1980) using the PRINAS program obtained from Mitsui Knowledge Industry (Japan).
Results
Cloning and sequencing of the entire coding region of the OTC gene of proband Y W e s c r e e n e d a p p r o x i m a t e l y 9 • 105 p h a g e clones carrying g e n o m i c D N A d e r i v e d f r o m p r o b a n d Y, using as a p r o b e the h u m a n O T C c D N A E C 5 a n d c o v e r i n g all the exons of the h u m a n n o r m a l O T C gene ( H a t a et al. 1988a). W e isolated 12 i n d e p e n d e n t clones carrying the O T C gene. S o u t h e r n blot analysis and D N A sequencing showed that a l t o g e t h e r they c o v e r e d e x o n 1 to e x o n 9, b u t n o t e x o n 10. A s o u r p r e v i o u s studies showed that e x o n 10 of the h u m a n O T C g e n e is p r e s e n t o n a 3.9-kb EcoR! fragm e n t ( H a t a et al. 1988a), we c o n s t r u c t e d a n e w l i b r a r y after fractionating the EcoRI c o m p l e t e digests of g e n o m i c D N A f r o m p r o b a n d Y a n d collecting the f r a c t i o n that c o n t a i n e d 3 - 5 kb D N A f r a g m e n t s . A p p r o x i m a t e l y 5 • 105 plaques of this library were screened, using as a p r o b e the h u m a n O T C c D N A E C 7 , which covers e x o n 7 to e x o n 10 ( H a t a et al. 1988a). O f two positive clones, o n e carried the g e n o m i c D N A s e q u e n c e c o r r e s p o n d i n g to e x o n 10 of the h u m a n O T C gene. W e d e t e r m i n e d the n u c l e o t i d e s e q u e n c e of the e n t i r e coding r e g i o n a n d e x o n / i n t r o n b o u n d a r i e s of the O T C
30
Fig. 1. Sequencing gels around the site of a point mutation in exon 8 from wild type and proband Y (patient). A thymine has replaced the normal cytosine in codon 277 (arrows), causing a change from arginine to tryptophan at amino acid 245 of the mature protein
gene of p r o b a n d Y, and c o m p a r e d them with those of the previously determined h u m a n normal O T C gene ( H a t a et al. 1988a). We found that the sequence of the coding region of the O T C gene of p r o b a n d Y could be aligned perfectly with that of the h u m a n normal O T C gene, except for a single-base substitution present in exon 8. This change was a C to T transition in codon 277 and resulted in the replacement of an arginine by a tryptophan residue (Fig. 1).
Identification of the mutation in proband M with synthetic oligonucleotide probes As the clinical symptoms and properties of hepatic OTCs of probands Y and M were similar, we presumed that they carried the same O T C mutation. For confirmation, we analyzed exon 8 of the O T C gene of proband M. Using total genomic D N A prepared from peripheral leukocytes of p r o b a n d M, the entire D N A sequence of exon 8 was amplified by the P C R and analyzed by A S O hybridization. For this purpose, we synthesized two different 19m e r oligonucleotide as probes, one corresponding to exon 8 of the normal O T C gene from codon 274 to the first base of codon 280 ( 5 ' - A A G A A A A A G C G G C T C C A G G - 3 ' ) ( H a t a et al. 1988a) (Fig. 1 wild type), the other corresponding to the same part of the O T C gene of proband Y ( 5 ' - A A G A A A A A G T G G C T C C A G G - 3 ' ) (Fig. 1 patient). As shown in Fig. 2, genomic D N A s from probands Y amd M hybridized strongly to the mutant probe, but not to the normal one. These results confirm that the probands Y and M carry the same point mutation in their O T C genes.
Genotypic analysis of familis Y and M As O T C deficiency is an X-linked disorder, a mutation present in the O T C gene of a male patient can be trans-
Fig. 2A-D. Segregation of the mutant allele in the two families. A Pedigree of family Y. Squares represent males and circles females. Open symbols represent the normal allele and solid symbols the mutant allele. Stippled square denotes asymptomatic sibling of hemizygote with mutant allele. An arrow indicates the proband. B ASO hybridization analysis of family Y. Total genomic DNAs were isolated from each individual, and the DNA region containing exon 8 of the OTC gene was amplified by the PCR method. Amplified DNA samples were spotted on a nitrocellulose filter, using a slot blot template and hybridized with 32p-labeled synthetic oligonucleotide probes. Wild-type probe corresponds to the normal sequence of exon 8, and mutant probe contains the substitution of T to C of the normal sequence. C Pedigree of family M. Symbols are the same as shown in A. D ASO hybridization analysis of family M. Experimental procedures are as described in B
mitted from his mother or can arise de novo in the germ line during early devleopment. In family Y, the proband is the only m e m b e r of the family clinically afflicted with disease (Fig. 2A). However, the pattern of A S O hybridization revealed that his brother is also hemizygous for the mutant allele. His sister, mother and aunt were all heterozygous for the O T C gene (Fig. 2B). Thus, the grandmother of proband Y carried this mutation.
31 Table 1. Urinary orotic acid level (mg/g creatinine)
Attack Pedigree Y I-2 I-3 II-1 II-2 II-3
Allopurinol(-) Allopurinol(+) a wild type mutant
750
2.8 52.4 4.8 14.4 6.8
24.4 74.6
Glu Lys Lys LyslArg I Leu Gin Ala Phe e e e e p e e e e hh hh hh hhT~re
A
2.0
I
t
1.5-
Pedigree M II-2 III-1 III-2
Controls b
1070
3.2 7.0 3.3
x
~" 7
gm 0.5
3.0
a During the first 6 h after an oral administration of allopurinol (300 rag) b n = 12. Standard error was _+0.3 and the range of urinary orotic acid levels for normal subjects was between 1.5 and 4.8 In family M, of the two phenotypically normal brothers of proband M, one was hemizygous for the mutant allele (Fig. 2D). The mother of proband M and his grandmother were also heterozygous for the OTC gene and had the same mutation in one of their alleles. Thus, the C to T mutation in the OTC gene has been stably inherited through at least three generations in these two unrelated families. Urinary orotic acid levels in the pedigree of Y and M After allopurinol loading, urinary orotic acid levels increased from 2.8 mg/g creatinine to 24.4mg/g creatinine in the mother (pedigree Y, I-2) and from 52.4 mg/g creatinine to 74.6 mg/g creatinine in the aunt (pedigree Y, I-3) (Table 1, Fig. 2A), indicating a carrier state of O T C deficiency (Brusilow and Valle 1987). Two siblings (pedigree Y, II-2 and II-3) and proband M excreted slightly but significantly higher amounts of orotic acid compared with controls without allopurinol load (Table 1, Fig. 3A). During hyperammonemic attack both probands Y and M excreted extremely high amounts of orotic acid in urine (Table 1).
Discussion A novel point mutation in the OTC gene, a C to T transition that leads to the replacement of the arginine 277 by tryptophan, was detected in two unrelated male patients with mild O T C deficiency. Based on the following supporting evidence, we conclude that this mutation is the direct cause of the O T C deficiency. This nucleotide substitution has not been observed in more than 100 different human X chromosomes derived from either healthy persons or patients with O T C deficiency (A. Hata, K. Shimada, and I. Matsuda, unpublished observations), and is not likely to represent one neutral polymorphism. We expected to see mutation(s) in the coding region of the OTC gene of these two unrelated male probands, because enzymatic characteristics of their OTCs showed abnormalities in kinetic or physicochemical properties, such as increased Km values for ornithine and a shift of p H opti-
1.o
0.0 -0.5
!
250 B
3;0 Residue number
Fig. 3A, B. Effects of the mutation on the structure of OTC. A Effects of the mutation on the secondary structure, from amino acid residues 273 to 281. B Effects of the mutation on the hydrophobic-
ity profile. In A, h and e denote alpha-helix and beta-sheet, respectively. In B, solid line shows the profile of the wild type protein and broken line the mutant OTC mum to the alkaline side. We found no other base substitution resulting in amino acid change in the entire coding region of the O T C gene of proband Y. The arginine 277 is completey conserved in all the OTCs of human, rat, mouse, E. coli and Aspergillus (Huygen et al. 1987; Veres et al. 1987). This observation strongly suggests that arginine 277 is essential for the full activity of OTCs. The arginine to tryptophan substitution is a change from a basic amino acid residue to a neutral large one. When we compared the secondary structures of normal and mutant sequences as predicted by the method of Chou and Fasman (1978), we found that the amino acid residue at position 277 was located at the boundary between a-helix and [3-sheet structures and that the substitution from arginine to tryptophan resulted in a shift of that boundary (Fig. 3A). A comparison of hydrophobicity profiles also revealed that this change induces a significant increase in hydrophobicity (Fig. 3B). These predictions support the conclusion that this mutation causes a significant conformational change of the protein, either altering the affinity for substrates, or affecting the stability of the protein. Two individuals heterozygous for this mutation (Table 1, pedigree Y, I-2 and I-3) showed abnormally increases orotic aciduria after allopurinol loading, a response not observed in normal control individuals (Brusilow and Valle 1987). These results eliminate the possiblity that the C to T mutation reflects a neutral polymorphism. The difference in response to the drug between the heterozygous females is considered to reflect both differences in protein intake and the effect of random X inactivation in hepatic cells. The almost normal or slightly elevated urinary orotic acid level in both subjects without allopurinol loading is consistent with the moderate phenotypic expression of this mutation in hemizygous males.
32 W e f o u n d n o g e n e t i c link b e t w e e n families Y a n d M in t e r m s o f f a m i l y h i s t o r y . F u r t h e r m o r e , as we discussed in d e t a i l in t h e p r e v i o u s r e p o r t o f O T C deficiency ( H a t a et al. 1989), t h e c y t o s i n e at C p G is a h o t s p o t for m u t a tion in h u m a n s , a n d is k n o w n to b e m e t h y l a t e d ; s p o n t a n e o u s d e a m i n a t i o n to t h y m i n e results in a C to T transition. W e initially p r e s u m e d t h a t t h e s e two m u t a t i o n s occ u r r e d i n d e p e n d e n t l y . F o r t h e p u r p o s e o f p r o v i d i n g evid e n c e for i n d e p e n d e n t r e c u r r e n t m u t a t i o n e v e n t s , the two probands were haplotyped by restriction fragment l e n g t h p o l y m o r p h i s m s ( R F L P s ) in t h e O T C g e n e using MspI, TaqI a n d BamHI. H o w e v e r , as b o t h p r o b a n d s s h o w e d t h e s a m e h a p l o t y p e (MspI: 6.2 a n d 5.1 k b ; TaqI: 3.7 kb; BamHI: 18.0 k b ) , w e c o u l d n o t e x c l u d e the possibility o f this m u t a t i o n o r i g i n a t i n g f r o m the s a m e ancestry. T o o u r s u r p r i s e , p e d i g r e e a n a l y s e s using the A S O hyb r i d i z a t i o n t e c h n i q u e r e v e a l e d t h a t e a c h p o r b a n d has ano t h e r m a l e sibling c a r r y i n g t h e s a m e m u t a t i o n , a l t h o u g h t h e r e a r e as y e t n o clinical s y m p t o m s , p r e s u m a b l y b e cause t h e i r m o t h e r s h a v e t h e b o y s o n a p r o t e i n restrict i o n diet; t h e p o s s i b i l i t y o f a n u n i d e n t i f i e d g e n e t i c f a c t o r r e l a t e d to t h e a p p e a r a n c e o f s y m p t o m s awaits clarification. S e v e r a l m a l e p a t i e n t s with O T C d e f i c i e n c y of l a t e o n s e t a r e k n o w n to r e a c t well to a p r o t e i n r e s t r i c t e d diet ( M a t s u d a et al. 1991); o u r p a t i e n t s a n d t h e i r b r o t h e r s can be i n c l u d e d in this g r o u p .
Acknowledgements. We thank Dr. Sumio Tanase for pertinent discussion on the protein conformation of OTC and Dr. Jean-Marc Lalouel for helpful comment. Editorial assistance by Mariko Ohara is also gratefully acknowledged. This work was supported by grants from the Ministry of Education, Science and Culture (62304042, 63571086), a grant for Pediatric Research (63A-01) from the Ministry of Health and Welfare of Japan and grant-in-aid from the Japan Medical Association (I.M.).
References
Adachi T, Tanimura A, Asahina M (1963) A colorimetric determination of orotic acid. J Vitaminol 9 : 217-226 Briand P, Francois B, Rabier D, Cathelineau L (1982) Ornithine transcarbamylase deficiencies in human males; kinetic and immunochemical classification. Biochem Biophys Acta 704 : 100106 Brusilow SW, Horwich AL (1989) Urea cycle enzymes. In: Scriber CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGraw-Hill, NewYork, pp 629-663 Brusilow S, Valle D (1987) Allopurinol (AP) induced orotidinuria (ODNU): a test of heterozygosity for ornithine transcarbamylase (OTC) deficiency. Pediatr Res 21 : 289A Chou PY, Fasman GD (1978) Prediction of secondary structure of proteins from their amino acid sequence. Adv Enzymo147 : 45148 Finkelstein JE, Francomano CA, Brusilow SW, Traystman MD (1990) Use of denaturing gradient gel electrophoresis for detection of mutation and prospective diagnosis in late onset ornithine transcarbamylase deficiency. Genomics 7 : 167-172 Grompe M, Muzny DM, Caskey CT (1989) Scanning detection of mutations in human ornithine transcarbamylase by chemical mismatch cleavage. Proc Natl Acad Sci USA 86 : 5888-5892 Hata A, Tsuzuki T, Shimada K, Takiguchi M, Mori M, Matsuda I (1986) Isolation and characterization of the human ornithine transcarbamylase gene: structure of the 5'-end region. J Biochem 100 : 717-725
Hata A, Tsuzuki T, Shimada K, Takiguchi M, Mori M, Matsuda I (1988a) Structure of the human ornithine transcarbamylase gene. J Biochem 103 : 302-308 Hata A, Tsuzuki T, Shimada K, Takiguchi M, Mori M, Matsuda I (1988b) Structural organization of the human ornithine transcarbamylase gene. J Inherited Metab Dis 11:337-340 Hata A, Setoyama C, Shimada K, Takeda E, Kuroda Y, Akaboshi I, Matsuda I (1989) Ornithine transcarbamylase deficiency resulting from a C-to-T substitution in exon 5 of the ornithine transcarbamylase gene. Am J Hum Genet 45 : 123-127 Huygen R, Crabeel M, Glansdorff N (1987) Nucleotide sequence of the ARG3 gene of the yeast Saccharomyces cerevisiae encoding ornithine carbamoyltransferase. Comparison with other carbamoyltransferases. Eur J Biochem 166: 371-377 Maddalena A, Sosnoski DM, Berry GT, Nussbaum RL (1988a) Mosaicism for an intragenic deletion in a boy with mild ornithine transcarbamylase deficiency. N Engl J Med 319:999-1003 Maddalena A, Spence JE, O'Brien WE, Nussbaum RL (1988b) Characterization of point mutations in the same arginine codon in three unrelated patients with ornithine transcarbamylase deficiency. J Clin Invest 82 : 1353-1358 Matsuda I, Nagata N, Oyanagi K, Tsuchiyama A, Yamamoto H, Hase Y, Kodama H, Kai H (1984) Biochemical heterogeneity of ornithine carbamyl transferase (OTC) in patients with OTC deficiency. Jpn J Hum Genet 29 : 327-333 Matsuda I, Nagata N, Matsuura T, Oyanagi K, Tada K, Narisawa K, Kitagawa T, Sakiyama T, Yamashita F, Yoshino M (1991) Retrospective survey of urea cycle disorders. I. Clinical and laboratory observations of thirty-two Japanese male patients with ornithine transcarbamylase (OTC) deficiency. Am J Med Genet 38 : 85-89 McClead RE Jr, Rozen R, Fox J, Rosenberg L, Menke J, Bickers R, Morrow G l I I (1986) Clinical application of DNA analysis in a family with OTC deficiency. Am J Med Gcnet 25:513-518 Old JM, Briand PL, Purvis-Smith S, Howard N J, Wilcken B, Hammond J, Pearson P, Cathelineau L, Williamson R, Davies KE (1985) Prenatal exclusion of ornithine transcarbamylase deficiency by direct gene analysis. Lancet I : 73-75 Old JM (1986) Fetal DNA analysis. In: Davies KE (ed) Human genetic diseases: a practical approach. IRL, Oxford Washington, DC, pp 1-17 Rose GD, Roy S (1980) Hydrophobic basis of packing in globular proteins. Proc Natl Acad Sci USA 77 : 4643-4647 Rozen R, Fox J, Fenton WA, Horwich AL, Rosenberg LE (1985) Gene deletion and restriction fragment length polymorphisms at the human ornithine transcarbamylase locus. Nature 313: 815-817 Rozen R, Fox JE, Hack AM, Fenton WA, Horwich AL, Rosenberg LE (1986) DNA analysis for ornithine transcarbamylase deficiency. J Inherited Metab Dis 9 [Suppl 1] : 49-57 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of ~-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 : 487-491 Thein SL, Wallace RB (1986) The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders. In: Davies KE (ed) Human genetic diseases: a practical approach. IRL, Oxford Washington, DC, pp 33-50 Veres G, Gibbs RA, Sherer SE, Caskey CT (1987) The molecular basis of the sparse for mouse mutation. Science 237 : 415-417 Yokoi T, Honke K, Funabashi T, Hayashi R, Suzuki Y, Taniguchi N, Hosoya M, Saheki T (1981) Partial ornithine transcarbamylase deficiency simulating Reye syndrome. J Pediatr 99:929-931 Note added in proof. After this paper had been submitted, a report appeared dealing with the characterization of the same point mutation (Finkelstein et al. 1990).
