EVIEWS

T h e mitochondrial matrix enzyme ornithine transcarbamylase (OTC; EC 2.1.3.3) catalyses the second step of the urea cycle, the conversion of ornithine and carbamyl phosphate to citrulline (Fig. 1). The OTC structural gene is located on the short arm of the X chromosome at p21.1. Deficiency of this enzyme is a common cause of a urea cycle disorder in humans and leads to the accumulation of ammonia, causing severe neurological disturbances 1. In cases of severe deficiency, this manifests itself as lethargy, vomiting and coma, starting soon after birth. Typically, affected male infants are well for a short interval after birth ranging from one to several days, but then they rapidly deteriorate, becoming comatose with blood ammonia levels usually exceeding 1000 ~ti. Plasma glutamine levels are also elevated. Additional biochemical markers of the disorder help to distinguish it from other urea cycle enzyme deficiencies. For example, plasma citrulline levels are very low in OTC-deficient patients, often below the limit of detection by amir.o acid chromatography. In addition, these patients excrete large amounts of orotic acid and orotidine in their urine. This orotic aciduria is due to the intramitochondrial accumulation of carbamyl phosphate, which diffuses into the cytoplasm and is converted to orotic acid by the pyrimidine synthesis pathway. Thus, the triad of hyperammonemia, low plasma citrulline, and orotic aciduria is the biochemical hallmark of this disorder. Final confirmation of the diagnosis is obtained by measuring enzyme activity in a liver specimen. Although most patients suffer from the typical neonatal hyperammonemic crisis, males with milder enzyme defects have been described, probably representing heterogeneity of the disorder at the molecular level. These patients become ill later in life and

Molecular detection and correction of 0rnithine transcarbamylase deficiency MARKUS GROMPE,STEPHENN. JONES AND C. THOMASCASKEY

The application of new diagnostic techniques has led to improvement in carrier deteaion and prenatal diagnosis in ornithine transcarbamylase deficiency. Progress has also been made towards somatic gene therapy. sometimes have atypical symptoms, such as bizarre behavior and other psychiatric disorders 2. It is not uncommon for heterozygous females to express some aspects of the disease and to have symptoms ranging from ..... d protein intolerance to hyperarnmonemit_ coma in the newborn period& Recently, it has been recognized that adult OTC carrier females are at particular risk for becoming symptomatic after child birth't. The prognosis for patients with severe OTC deficiency is grim. Despite improved dietary and pharmacologic therapy (see below), many patients die eady in life and the incidence of mental retardation among survivors is very high. Because of the devastating nature of the disorder there is strong demand for prenatal and carrier diagnosis in families of patients with OTC deficiency.

F/GiI

Mammalian urea cycle. CPS l, mitochondrial carbamyl phosphate synthetase; NAGS, N-acetylglutamate synthetase; OTC, omithine transcarbamylase; AS, argininosuccinate synthetase; AL, argininosuccinate lyase; ARG, arginase; OT, mitochondriai omithine transporter; CPS II, cytoplasmic carbamyl phosphate synthetase. The cytoplasmic conversion of carbamyi phosphate to orotic acid via the pyrimidine synthesis pathway is shown to the left without the enzymes responsible for the reactions. AUopurinol metabolites inhibit thu synthesis of uridine 5'-phosphate, thus augmenting orotic acid and orotidine levels.

Mitochondrial Matrix

Glutamlne

i:O~:H~:: ATP

* C02" ATP

c--- LL Ph°sinate

~

OTC Cttrulllne

CarDamyl

Jr

~1 I OT

Aspait;~te DlhyOroorotate

Cltrulllne

Ornlthlne

1

Urea

~

ARG

Orotldlne S'-phosphate

Orot1Olne

l e

t UrlOtne 5"-phosDhate

TIG OCTOBER1990 VOL.6 NO. 10 © lt~)O Elsevier S c i e n c e P u b l i s h e r s Ltd (UK) 0 1 6 8 - 9 4 7 9 / 9 0 / 5 0 2 . 0 0

AL

~'glnosucctnate

Fumarate

Inhibition Dy AIIopurlnol metaboiltes

I

Aspartate

ArIP* PPl"~A5

Orotlc ACld Arglnlne

f

Acetyl-CoA * Glutamate NAGS N-Acetylglutamate

Cytoplasm

IEVIEWS

T~mt 1. RFLPs detected with full-length human

OTC eDNA Fnzyme BamHI

mspl mspI Taql

PolymorpMc

Constant

bands Odb)

bands Od~)

18, 5.2 6.6, 6.2 5.1, 4.4 3.7, 3.6

20, 17.5, 11, 7 14, 5.4, 3.3, 2.3, 1.9 14, 5.4, 3.3, 2.3, 1.9 4.6, 2.6, 1.8, 1.5

The OTC gene Early mapping studies placed the OTC gene in the vicinity of the Duchenne muscular dystrophy locus at Xp21.1 on the short arm of the X chromosome s. In 1983, a rat OTC cDNA was isolated by the use of OTC antibodies 6, and the following year a human OTC cDNA was cloned by Horwich et al. 7. The mature OTC message is approximately 1.6 kb long, with a coding region of 1062 nucleotides accounting for a protein of 354 amino acids. The amino terminus contains a 32 amino acid mitochondrial leader sequence, allowing A

D

t ,, I,,

4456p

7

¢5

bp

ATG I

J J M B

RC

t 5Z

5

I

6 3 0 bp 975 bp

i

Diagnosis of 0TC deficiency

i

,I 025 bp

'

1

2

1196-- m,,-~ 1025 -975--

transport of the protein into mitochondria after synthesis. There, the leader peptide is cleaved and the enzyme assumes its active homotrimeric conformation. In 1988, Hata et al. described the structure of the human OTC gene 8. It spans 73 kb and contains ten exons and nine introns. The structures of the mouse 9 and raO0 0 T C geues have also been determined and are very similar to that of the human. The OTC gene is expressed in a tissue-specific manner, with highest levels detected in liver and lesser amounts in small bowel11. This tissue-specific expression is mediated by DNA sequence elements in the 5' flanking region of the gene. A DNA fragment containing 750 bp of sequence 5' to the translation initiation codon in the mouse OTC gene has been found to confer proper tissue specificity of expression in vitro 12. This promoter element has also been linked to both the chloramphenicol acetyltransferase (CAT) reporter gene and human OTC cDNA, resulting in high levels of expression in small intestinal mucosa and low levels of expression in the liver of transgenic micen3. Since OTC levels are normally highest in the liver in vivo, this indicates that additional sequences are required to obtain high levels of transcription in this organ. An OTC enhancer element capable of increasing transcription 20-fold in hepatoma cells has been recently identified for the rat OTC geneS4; the rat OTC enhancer resides 11.1 kb 5' to the transcriptional start site, contains a C/EBP recognition sequence and functions in a tissue-specific manner.

3

4

5 .

.

6 .

.

.

.

.

7

8

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630 - -

445 - -

~TGta Results of chemical mismatch cleavage on five patients with OTC deficiency. The top part of the figure gives a schematic representation of the OTC cDNA with the flanking PCR amplification primers A and D. The sites of the mutations in patients M, B, RC, SZ and S are marked by triangles. The expected chemical cl,.avage products are shown as barred lines. In the lower part of the ~'igurean autoradiogram with the respective chemical cleavage fragments is shown. Lane i corresponds to patient RC, lanes 2 and 3 to S, lanes 5 and 6 to SZ, lane 7 to B and lane 3 to M. Lane ~, is a wild-type control.

The advances in the understanding of the molecular biology of the OTC gene described above have also been very useful in clinical applications. After the initial cloning of the human cDNA, it was used as a probe in Southern blot analysis, and four diagnostically useful intragenic restriction iragment length polymorphisms (RFLPs) have been identified 1~ (Table 1). Once biochemical tests indicate that a patient has OTC deficiency, it becomes important to determine whether the mother carries the disease allele. If the mother can unambiguously be identified as a carrier, DNA-based diagnosis becomes feasible using the above-mentioned RFLPs. A positive family history can be used to determine carrier status, but many of the OTC-deficient patients are isolated cases in a family and represent new mutations, as is the case for other X-linked lethal conditions 16. In a recent study, biochemical carrier testing was used to show that one-third of the mothers of isolated male patients and two-thirds of the mothers of female cases from monoplex families were non-carriers 17, suggesting that the mutation rate is higher in male gametes than in female gametes. Biochemical carrier testing can be very helpful. The at-risk female is subjected to a metabolic challenge by administering either a protein load or allopurinol (Fig. 1), after which orotic acid or orotidine levels in the urine are measured, with elevated amounts indicating carrier status. Measurements of on~tidine are more sensitive than determination of orotic acid levels and detect obligate carriers in about 90% of cases17. Positive results are highly reliable (close to

TIC-OCTOBER1990 VOL.6 NO. 10

~3~

[k~EVIEWS 100%), but a negative challenge cannot rule out the heterozygous state; the remaining risk is approximately 5-15%. This difficulty, typical for X-linked disorders, is due to random X chromosome inactivation-in OTC-expressing cells, which can lead to both highly symptomatic carder females as well as women who appear metabolically normal even though they carry the disease gene. A different carder test involves antibody staining of OTC-producing enterocytes in small bowel biopsies TM, but no data are yet available on its reliability in larger numbers of patients. Even families in which the heterozygous females can be accurately diagnosed can sometimes not be studied by linkage-based DNA diagnosis because there are no informative polymorphisms in that particular family. Dii'ect detection of the disease-causing mutation can solve these diagnostic problems in uninformative families and when the carrier status cannot be clarified by biochemical means. This has been achieved in the past in cases of major deletions or when a point mutation altered a restriction sitem20. Direct detection of mutations would also be useful for prenatal diagnosis in cases of gonadal mosaicism, although no such cases have been reported for OTC deficiency to date. Since OTC deficiency is a disease with a high incidence of new mutations, it is likely that the disease-causing alteration is different in each patient. Any strategy for mutation detection in this disorder must therefore be able to scan large regions of the gene for the presence of sequence alterations.

Direct detection of mutations in the OTC gene Several recently developed methods have been used in the molecular analysis of OTC deficiency. One strategy being pursued by Finkelstein et al. involves amplification of individual exons, followed by denaturing gel gradient electrophoresis or direct sequencing in order to identify the mutation 21. Denaturing gel gradient electrophoresis is based on the effect of single base alterations on the melting behavior of a doublestranded DNA molecule, such that mutant and wildtype DNA can be separated in a denaturing gradient gel. This approach has the advantage of not being dependent on an mRNA source, but involves the laborious analysis of ten individual exons. In contrast, another approach uses RNA from an OTC-expressing tissue instead of genomic DNA, so that the entire peptide-coding region can easily be studied. Our laboratory has adapted the newly developed technique of chemical mismatch cleavage zz to scan for and directly identify mutations in PCRamplified OTC cDNA, isolated from liver specimens. In this method, heteroduplexes are formed between radiolabeled wild-type DNA and mutant DNA, followed by chemical modification and cleavage at the site of the mutation. Several patients with OTC deficiency have now been studied in this fashion (Fig. 2), and in each case a putative mutation was identified, with the sequence alteration differing between individuals, as expected23. This approach is limited by the invasive procedures that are necessary to obtain the material needed for the analysis, unless an autopsy liver specimen from an affected family member is available. Such

i

m

m

m

u

X

~C

u

cu

c

ucu b~

180 112 68

FIG[] Direct detection of a Taql site mutation in OTC exon 9. In a family with OTC deficiency, exon 9 of the OTC gene was amplified by PCR, using primers from the flanking introns, and the 180 bp product was subsequently digested with Taql restriction enzyme. Lanes with undigested PCR product are marked with a U, and lanes with digested PCR product by a C. The PCR product was not cut in the OTC-deficient patient (shaded square) (lots of Taql site), whereas complete digestion occurred in the unaffected brother and an unrelated control (112 and 68 bp). The patient's mother showed all three bands, indicating that she is a carrier. post-mortem samples can be extremely useful for diagnosis in OTC deficiency families and should be obtained whenever possible.

Taql site mutations and deletions Base changes altering TaqI restriction sites in the OTC cDNA make up an important subgroup of mutations and are found in 10-15% of patients. They are easy to detect, because fragments of altered size will be seen on Southern blot analysis after Taql digestion. There are four Taql sites in the OTC cDNA, one each in exons 1, Y, 5 and 9. Until recently only mutations in the TaqI site in exon 5 had been described for OTC deficiency 2°, but now additional mutations affecting the TaqI sites in exons 1, 3 and 9 have been identified and sequenced (M. Grompe et al., unpublished). Prenatal and carrier diagnosis by direct detection of mutations in families with such alterations can be greatly simplified by amplification of the respective exon by the polymerase chain reaction followed by a TaqI digest (Fig. 3). The simultaneous

TIC,ocroBra !.090 VOL.6 NO. 10

EVIEWS

OTC d e f i c i e n c y

l~OSltlve family l~Istory

negatlve family hlstory

ot)taln DNA for mrormatlve

.

t:)1oct~ernlcal carrier testlng

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amt)Iguous resu]t

I citrec~:,~'i~uta:t':i°n :clet'ec~°n :] : by :cDNAanaiysis o~:stucly :::::,I

virtually all the survivors are mentally handicapped. Additional illnesses often precipitate hyperammonemic crisis to which the patient eventually succumbs. Liver transplantation is at present the only fo~'m of therapy that offers any hope for an improved !.outcome. However, the morbklity and mortality of this procedure are significant and donor livers are not easily available. These factors make OTC deficiency a candidate disorder for somatic gene replacement therapy.

Animal models of OTC deficiency

Research into gene therapy for OTC d;eficiency is greatly facilitated by the availability /qGm of two murine models of OTC Current diagnostic procedure in OTC deficiency. deficiency: sparse fur (spf) amplification of several OTC exons has also been used and sparse fur-abnormal skin aad hair (sp..(-ash). The to map the extent of deletions of the gene. molecular defect in both mutant strains has been idenIn summary, the mainstay of diagnosis in OTC de- tified: in the spfmouse, a single base pair mutation ficiency is RFLP-based DNA diagnosis, after biochemi- replaces a histidine residue with an asparagine residue cal clarification of the mother's carrier status by at amino acid position 117 of OTC, altering the pH allopurinol loading. However, this approach is unsuc- optimum of the enzyme and leading to a decrease in cessful in some families and direct detection of the hepatic OTC activity to 15% of wild-type levels24; in mutation responsible for the disease is now feasible in spf-asb mice, a point mutation in the last base pair of most of the uninformative families. The techniques exon 4 alters a guanosine to a.n adenosine, thus interneeded for such direct mutation detection are labor- fering with efficient splicing at this site 2~. Normal OTC and cost-intensive and therefore not useful as primary message levels are greatly reduced and hepatic OTC diagnostic tools. Because of the heavy burden enzyme activity is approximately 5% of normal levels. imposed by the disease, however, their application is Both strains of mice exhibit many of the classic sympjustified in those families in which the routine strategy toms of human OTC deficiency, including elevated has failed. A schematic representation of the current blood ammonia levels, orotic aciduria, and retarded diagnostic approach to OTC deficiency is shown in Fig. 4.

Treatment of OTCdeficiency Despite the marked progress in our diagnostic capabilities in OTC deficiency, only limited progress has been made in treatment]. Current therapy consists of dietary and pharmacologic manipulation. The patient's protein intake is severely restricted and citrulline is supplemented in order to avoid arginine depletion (Fig. 1). In addition, sodium benzoate and sodium phenylacetate or sodium phenylbutyrate are used. Benzoate binds glycine, and phenylacetate and butyrate bind glutamine, forming compounds that can be excreted in urine, thereby reducing the nitrogen load. Although these therapies have improved the outcome in mildly affected males and heterozygous females, many patients with severe OTC deficiency die and

FIGI~ Germ-line correction of OTC deficiency in spfmice. Photograph of 9 day old spf littermates. Top mouse expresses the OTC transgene containing a 750 bp fragment of the mouse OTC promoter at high levels in the mucosal layer of the small bowel, and is phenotypicallyand metabolicallycorrected for OTC deficiency. Bottom mouse failed to mcorporate the transgene and retains the spfphenotype. 116 OCTOBER1990 VOL.6 NO. 10

m

~EVIEWS growth. In additiopj the fur of both mutants is abnormally sparse. As a first step io somatic gene therapy for OTC deficiency, we and others have used these OTCdeficient mice to determine whether gene transfer could correct the inborn error. Cavard and coworkers26 created transgenic spf-ash mice by microinjecting rat OTC cDNA coupled to the SV40 large T antigen promoter region into fertilized ~r-ash oocytes. One line of transgenic mice was reported.to be phe~otypically and metabolically corrected for the spf-ashinduced defect. These mice were found to express the transgene solely in the liver, indicating that OTC gene transfer and expression in the liver could correct OTC deficiency in this mouse model. Recently, we have created several lines of transgenic spfmice that were found to be similarly corrected for OTC deficiency13. Fertilized spf oocytes were microinjected with human OTC cDNA placed under transcriptional control of a 750 bp fragment of the mouse OTC promoter region described above. The resulting transgenic mice were phenotypically and biochemically corrected for the inborn error (Fig. 5). Northern analysis and OTC enzyme assays indicated that gene transfer and expression of OTC in the small bowel is sufficient to correct OTC deficiency- in the spfmice. This ectopic correction of the inborn error in these mice augurs well for somatic gene therapy of OTC deficiency by intestine-targeted gene transfer. Somatic g e n e therapy f o r OTC deficiency

In order to achieve biochemical correction of OTC deficiency, the OTC gene must be targeted to a tissue in which carbamyl phosphate synthetase is expressed, since carbamyl phosphate is a substrate in the conversion of ornithine to citrulline by OTC. This limits the target tissues for OTC gene transfer to liver and small intestine. Since all components of the urea cycle reside in the liver, this tissue has received the most attention. Unfortunately, liver tissue is not amenable to the standard methods of gene transfer used in other tissues, such as retrovirus-mediated gene transfer into bone marrow cells z7,28, since it is not possible to reimplant hepatocytes into host animals after they have been removed and transfected. Retrovirus-mediated gene transfer of several genes encoding liver-specific functions into hepatocytes in vitro has been demonstrated; for example, Woo and co-workers recently reported transduction of the human phenylalanine hydroxylase gene into primary cultures of mouse hepatocytes z9. Wilson et aL3O used retroviral vectors to corred, in vitro, the genetic defect in hepatocytes isolated from Watanabe rabbits with heritable hyperlipidemia. However, little information is available regarding the long-term viability of such hepatocytes when reintroduced into recipients. Although some progress has been made recently in delivering genes to the liver in vivo31, truly efficient methods for such in vivo transduction have yet to be found. Nor is it known whether recombinant retroviruses will be able to transduce genes into intestinal epithelium either in vitro or in vivo. However, we are hopeful that these obstacles will be overcome eventually, pe,,TniRing somatic gene therapy for OTC deficiency.

Acknowledgements M.G. is an Association of Medical School Pediatric Department Chairmen, Inc., Pediatric Scientist Training Program Fellow supported by NIH grant no. HD00850. S.N.J. was supported by a National Research Service Award from the PHS, no. DK 08254. C.T.C is an Investigator of the Howard Hughes Medical institute.

References 1 Brusilow, S. and Horwich A.L. (1989) in The Metabolic ~asis oflnherited Disease, 6th edn (Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D., eds), pp. 629-663, McGraw-Hill 2 Rowe, P.C., Newman, S.L. and Brusilow, S.W. (1986) New Engl. J. Med. 314, 541-547 3 Girgis, N. et al. (1987)J. Inherit. Metab. Dis. 10, 274-275 4 Hawks-Am, P. et al. New Engl. J. Med. (in press) 5 Lindgren, V. et al. (1984) Science 226, 698-701 6 Horwich, A.L. et al. (1983) Proc. NatlAcad. Sci. USA 80, 4258-4263 7 Horwich, A.L. et al. (1984) Science 224, 1068--1074 8 Hata, A. et al. (1987) J. Biochem. 103, 302-308 9 Scherer, S.E., Veres, G. and Caskey, C.T. (1988) Nucleic AcidsRes. 16, 1593-1601 10 Takiguchi, M., Murakami, T., Miura, S. and Mori, M. (1987) Proc. Natl Acad. ScL USA 84, 6136--6140 11 Hoogenraad, N.J. et al. (1985) Am.J. Physiol. 249, G792--G799 12 Veres, G., Craigen, w.J. and Caskey, C.T. (1986) J. Biol. Chem. 261, 7588-7591 13 Jones, S.N. et aL J. Biol. Chem. (in press) 14 Murakami, T., Nishiyori, A., Takiguchi, M. and Mori, M. (1990) Mol. Cell. Biol. 10, 1180-1191 15 Fox, J.E., Hack, A.M., Fenton, W.A. and Rosenberg, L.E. (1986) Am.J. Hum. Genet. 38, 841-847 16 Haldane, J.B.S. (1935)J. Genet. 31, 317-326 1 7 Hauser, R.H., Finkelstein, j.E., Valle, D. and Brusilow, S.W. New Engl. J. Med. (in press) 18 Hamano, Y. et al. (1988) New Engl.J. Med. 318, 1521-1523 19 Rozen, R. etal. (1985) Nature313, 815--817 20 Maddalena, A., Spence, J.E., O'Brien, W.E. and Nussbaum, R.L. (1988)J. Clin. Invest. 82, 1353-1358 21 Finkeistein, J.E. et al. (1990) Genomics 7, 167-173 22 Cotton, R.G.H., Rodrigues, N.R. and Campbell, R.D. (1988) Proc. Natl Acad. Sci. USA 85, 4397--4401 23 Grompe, M., Muzny, D.M. and Caskey, C.T. (1989) Proc. Nail Acad. Sci. USA 86, 5888-5892 24 Veres, G., Gibbs, R.A., Scherer, S.E. and Caskey, C.T. (1987) Science 237, 415--417 25 Hodges, P.E. and Rosenberg, L.E. (1989) Proc. Natl Acad. Sci. USA 86, 4142-4146 26 Cavard, C. et al. (1988) Nucleic Acids Res. 16, 2099-2110 2 7 Belmont, J.W. et al. (1988) Mol. Cell. Biol. 8, 5116--5125 28 Bender, M.A., Miller, A.D. and Gelinas, R.E. (1989) Mol. Cell. Biol. 9, 1426-1434 29 Peng, H. et al. (1988) Proc. Natl Acad. Sci. USA 85, 8146--8150 30 Wilson, J.M., Johnson, D.E., Jefferson, D.M. and Mulligan, R.C. (1988) Proc. Natl Acad. Sci. USA 85, 4421--4425 31 Wu, C.H., Wilson, J.M. and Wu, G.Y. (1989) J. Biol. Chem. 264, 16985-16987

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