A Review of Ornithine Transcarbamylase Deficiency (OTCD)

Ⅵ JMA Medical Awards

Hyperammonemia in Pediatric Clinics: A review of ornithine transcarbamylase deﬁciency (OTCD) based on our case studies

JMAJ 47(4): 160–165, 2004

Ichiro MATSUDA

Professor Emeritus, Kumamoto University

Abstract: Ornithine transcarbamylase, which is the enzyme to synthesize citrulline from carbamyl phosphate and ornithine, is located on the X chromosome. Male patients with OTCD present a wide clinical picture, as shown in the neonatal type (usually ending in death within one year) and the late-onset type (appearing between 2 and 56 years and characterized by long-term survival). OTC activity in the neonatal type is essentially undetectable, whereas those with the late-onset of the control level. Mutations of male patients with the 6.3%עtype have 8.1 -include base insertion/deletion, exon skipping, and non (23סneonatal type (n sense and missense mutations. Mutations may lead to unstable mRNA or trun- cated protein, or involve the active site or cord domain of the enzyme, leading to are only (25סstructural changes. Mutations associated with the late-onset type (n of the missense type, with most occurring on the surface of the enzyme. We performed prenatal monitoring for OTCD in 21 cases, among which one male fetus with Arg129His and two male fetuses with Arg40His (both belonging to the late- onset type) were diagnosed on the basis of gene analysis of amniotic cells. Treat- ment was initiated immediately after birth, enabling them to enjoy a normal school life. Two fetuses diagnosed as having neonatal-type mutations were terminated. Gene analysis provides the most reliable information about the future conse- quences of OTCD, especially in male patients. Key words: Hyperammonemia; Urea cycle disorder; Ornithine transcarbamylase deﬁciency (OTCD); Genotype-phenotype correlation

Introduction ditions including liver diseases accompanied with liver cirrhosis (e.g., congenital biliary atre- Hyperammonemia in children is observed in sia) and hereditary diseases such as urea-cycle association with a variety of diseases and con- enzyme defects, defective transport of ornithine

This article is a revised English version of a paper originally published in the Journal of the Japan Medical Association (Vol. 129, No. 1, 2003, pages 60–64). This research was awarded the Medical Prize of the Japan Medical Association for 2002 in Clinical Medicine.

160 JMAJ, April 2004—Vol. 47, No. 4 ORNITHINE TRANSCARBAMYLASE DEFICIENCY

into mitochondria, and certain organic acide- with central nervous system (CNS) symptoms mias. In recent years, gene analysis of certain such as vomiting, spasm, coma, and lethargy, hereditary diseases has revealed the relation- within 30 days after birth. In most cases, it ends ship between genotype and phenotype (the in patient death within the first several months pathologic condition), and the results of analy- of life. Even if the patient survives, severe neu- sis have come to be applied in clinical practice. rological disorders will remain. OTC activity in This paper describes ornithine transcarba- the liver is below the limit of detection.7) mylase deficiency (OTCD), one of the most frequent of such hereditary diseases in Japan 2. Late-onset type and the subject of our research since we Late-onset disease occurs in patients of vari- reported the first case of this disease in Japan ous ages, ranging from those in infancy, puberty, in 1971.1) Our group was the first to succeed and adolescence, to middle age or even later. in elucidating the structure of the OTC gene One patient had been known to be asympto- in 1988.2) In addition, identification of the matic until the age of 65 years. Patients com- carbamyl phosphate synthetase and arginase monly lead a normal life until the onset of genes and elucidation of their mutant genes disease. However, some patients have been were also initially carried out by our group.3–6) reported to have mild symptoms (vomiting and OTC, a urea cycle enzyme localized in mito- mild neural confusion) particularly when they chondria of the liver, is involved in synthesizing have fever. Hepatic OTC activity in patients citrulline from carbamyl phosphate and orni- with normal IQ and normal electrocardiographic -of the nor 5.5%עthine. This enzyme is first synthesized as a pre- findings corresponds to 16.6 cursor protein with a molecular mass of about mal level.7) 40,000 daltons in the cytosol, then transported to the mitochondrial matrix, where it is pro- 3. Female patients cessed to the mature enzyme of about 36,000 Signs and symptoms vary widely among fe- daltons. It becomes active after being formed male patients (heterozygotes). Some are asymp- into a trimer. The OTC gene maps to Xp21.1 tomatic, while others eventually die after onset. and is 73 kb long with 10 exons.1) OTC defi- Differences in the rate of inactivation of the X ciency is an X-linked semidominant urea cycle chromosome owing to lyonization are involved disorder, and has the highest incidence (1 out in how the disease manifests.7) of 50,000 people) among the various urea cycle disorders. Clinically, the age of onset is wide- Relation between Disease Type ranging, involving infants to adults. The reason (Phenotype) and Mutant Gene (Fig. 1) for such variety remained unclear until gene analysis provided relevant information. Mutation of the OTC gene is basically indi- vidual, varying among those who are affected.8) Classification of Disease Types 1. Gene mutation of neonatal-onset OTCD About half of male patients (hemizygotes) About half the patients with neonatal-onset have a neonatal onset, with the disease occur- disease have nonsense mutations (mutations ring within 1 month after birth; the other half with a stop codon present in the sequence), experience late-onset disease. Most female pa- base insertion, and base deletion. In this case, tients (heterozygotes) have late-onset disease.7) enzyme protein is always greatly decreased, and the activity level is virtually nil. The other 1. Neonatal-onset type half of patients have missense mutations (muta- OTCD in neonates is severe and manifests tions accompanied with substitution of amino

JMAJ, April 2004—Vol. 47, No. 4 161 I. MATSUDA

Exon

Mutation 1 R40H Y55D 2 3

D126G 4 R129H Age at first episode R141Q I172M 5 Age at detection of mutation on genetic screening in the absence Q180H of symptoms S192R D196V L201R 6 S207R H214Y L221L (AAGǞAAA) 7 T264A T264I M268T 8 R277W

L304F 9 V337L 10 Ͻ1 month 1 year 10 years 50 years

Fig. 1 Relationship between patient age at the ﬁrst episode of OTCD and missense mutant genes Mutations in patients who died within one month after birth (neonatal-onset disease) were located at the active site of the enzyme protein or at the site indispensable for composition of a trimer. Mutations in patients with late-onset disease were located on the surface of the enzyme protein. (From Matsuda, I.: Iden 54; 2000: 48Ð54)

acids: e.g., D126G, R141Q, I172M, S192R, at the ﬁrst examination and had never experi- D196V, or L201R). All these mutations are enced episodes of hyperammonemia, as well as missense mutations at sites important for en- male patients who developed the disease at the zyme activity, such as the site responsible for ages of 9, 15, 17, and 48 years. The 65-year-old trimer formation and the active site of the patient was found to have a daily protein intake enzyme.8) In any case, OTC activity as exam- of less than 65 g. Interestingly, the disease ined in gene expression studies was below the manifested in all these patients in the 1980s, limit of detection.8,9) when the protein intake among Japanese adult men reached about 80 g/day.10) 2. Gene mutation of late-onset OTCD In late-onset cases, all mutations are mis- 3. Gene mutation of OTCD in females sense mutations, and most of them are located In female patients, nonsense mutation, base on the surface of the enzyme protein. The OTC insertion, base deletion, exon skipping, and activity corresponds to 10–15% of the normal missense mutation have been observed. How- level. Unlike neonatal-onset disease, identical ever, mutations in female patients are basically gene mutations (R40H, R277W, R129H, M268T) of the same nature as those in male patients are found in about 30% of the families of with neonatal-onset disease.8) patients. The R4OH mutation, in particular, has been found in 6 families. In these families, 4. Patient gender and carrier diagnosis there was a male patient who was 65 years old It is known empirically that the percentage

162 JMAJ, April 2004—Vol. 47, No. 4 ORNITHINE TRANSCARBAMYLASE DEFICIENCY

Table 1 Prenatal Diagnosis of OTCD

Site of mutation Case Family Specimen Sex targeted for diagnosis Mutation Restriction Result Consequence enzyme Exon/Intron AǞC) Rsa IPatient Aborted .4ם) Villi Male Intron 1 11 21 Villi Male Normal Continued 31 Villi Female Normal Continued 42 Villi Male Exon 2 CGTǞCAT Arg 40 His Nla III, Mae II Patient Continued 53 Villi Male Exon 2 CGTǞCAT Arg 40 His Nla III, Mae II Patient Continued 64 Villi Male Exon 4 GACǞGGC Arg 126 Gly Sdu IPatient Aborted 75Amniotic Male Exon 4 CGTǞCAT Arg 129 His Msp IPatient Continued fluid 86 Villi Female Exon 5 CGAǞTGA Arg 141 Ter Taq I Carrier Continued 96Amniotic Female Carrier Continued fluid 10 7 Villi Female Exon 5 CGAǞCAA Arg 141 Gln Taq I Carrier Continued 11 7 Villi Male Normal Continued 12 8* Amniotic Male Exon 6 AGCǞAGG Ser 192 Arg Pvu II Unevaluable Continued fluid (patient) 13 8 Villi Male Patient Aborted 14 9 Amniotic Female Exon 9 TGGǞTGA Ser 332 Ter Base sequence Carrier Continued fluid analysis GǞA) Msp I, RFLP Normal Continued .1ם) Amniotic Male Intron 8 10 15 fluid 16 11 Amniotic Female Msp I, RFLP Normal Continued fluid 17 12** Amniotic Unknown Unevaluable Continued fluid (patient) 18 13 Villi Male SSCP (Normal) Continued (normal) *Maternal blood contamination **Bacterial contamination SSCP: single-strand conformation polymorphism, RFLP: restriction fragment length polymorphism (From Matsuda, I.: Ethical issues around prenatal diagnosis: From the standpoint of molecular biology. Shusanki Igaku 1998; 28: 999Ð1003. (in Japanese))

of carriers among mothers of male patients is or R129H mutant gene. In these cases, treat- not the same as that among mothers of female ment was begun just after delivery without patients. Whereas 92% of mothers of male pa- abortion. The patients are currently in elemen- tients are carriers, fresh mutation was common tary school and are showing healthy growth. in female patients, with only 20% of mothers of Three of the 18 cases underwent abortion after female patients being carriers.11) prenatal diagnosis.12)

Prenatal Diagnosis of OTCD Treatment and Prognosis

The author has been involved in prenatal Administration of sodium benzoate or sodium monitoring for OTCD in 18 patients from 13 phenylacetate is employed as pharmacotherapy, families (Table 1). The male-female ratio was and a low protein diet (protein 1.0–1.5 g/kg/ 11:7. Four of the 7 females were carriers, and 6 day plus essential amino acids at the required of the 11 males had a mutant gene. Three of level) is used as nutritional therapy. However, these 6 had late-onset OTCD, showing an R40H since arginine is essential in this disease, in

JMAJ, April 2004—Vol. 47, No. 4 163 I. MATSUDA

(%) 100 Conclusion In OTCD, gene mutations that severely affect the structure and function of OTC enzyme pro- 50 tein are associated with the complete absence

Survival rate of enzyme activity and neonatal onset, whereas gene mutations affecting the protein surface are associated with a 10–20% enzyme activity 051015 20 25 (Years) level and onset in late childhood or adulthood. Age In addition, a close association has been found Fig. 2 Survival rate in patients with late-onset OTCD between protein intake and disease onset. These Death of female patients (thin line) begins at about 5 years and findings represent a useful set of information follows a course similar to that in male patients (thick line). (From Uchino, T. et al.: Neurodevelopmental outcome of for genetic counseling in relation to treatment long-term therapy of urea cycle disorders in Japan. J Inherit of the patient and prenatal diagnosis of the Metab Dis 1998; 21(Suppl1): 151Ð159) disease. addition to the usual essential amino acids, REFERENCES arginine (400 mg/kg/day) should be given to 1) Matsuda, I., Arashima, S., Nanbu, H. et al.: the patient in conjunction with other essential Hyperammonemia due to a mutant enzyme of amino acids. ornithine transcarbamylase. Pediatrics 1971; With regard to prognosis, most neonatal- 48: 595–600. onset cases end in death within several months 2) Hata, A., Tsuzuki, T., Shimada, K. et al.: Struc- after birth, as mentioned previously. In contrast, ture of the human ornithine transcarbamylase gene. J Biochem 1988; 103: 302–308. male patients with late-onset disease and female 3) Haraguchi, Y., Uchino, T., Takiguchi, M. et al.: patients show various courses of illness ranging Cloning and sequence of a cDNA encoding from a complete lack of symptoms to death human carbamyl phosphate synthetase I: Mo- after the initial onset, under the strong influ- lecular analysis of hyperammonemia. Gene ences of both the gene mutation (remaining 1991: 107: 335–340. enzyme activity) and treatment.13) In general, 4) Hoshide, R., Matsuura, T., Haraguchi, Y. et al.: patients with an initial blood ammonia level Carbamyl phosphate synthetase I deficiency. exceeding 1,000␮g/dl have a poor prognosis. One base substitution in an exon of the CPS 1 Figure 2 shows the survival rate of Japanese gene causes a 9-basepair deletion to aberrant splicing. J Clin Invest 1993; 91: 1884–1887. patients. 5) Uchino, T., Snyderman, S.E., Lambert, M. et al.: In recent years, liver transplantation has been Molecular basis of phenotypic variation in performed with the aim of radical treatment, patients with argininemia. Hum Genet 1995: achieving successful results in many cases. 96: 255–260. Although gene therapy is still in the experi- 6) Haraguchi, Y., Aparicio, J.M., Takiguchi, M. mental stage, AdexCAGhOTC, developed in et al.: Molecular basis of argininemia. Identifi- Japan, has been demonstrated to be a consid- cation of two discrete frame-shift deletions in erably more efficient vector than AdexSR- the liver-type arginase gene. J Clin Invest 1990: 86: 347–350. ␣hOTC, developed in the US.14) However, ap- 7) Matsuda, I., Nagata, N., Matsuura, T. et al.: plication to human subjects is not practical as Retrospective survey of urea cycle disorders: long as basic immunological issues associated Part 1. Clinical and laboratory observations of with adenovirus vectors remain unsolved. thirty-two Japanese male patients with ornithine transcarbamylase (OTC) deficiency. Am

164 JMAJ, April 2004—Vol. 47, No. 4 ORNITHINE TRANSCARBAMYLASE DEFICIENCY

J Med Genet 1991; 38: 85–89. 11) Tuchman, M., Matsuda, I., Munnich, A. et al.: 8) Matsuda, I. and Tanase, S.: The ornithine Proportions of spontaneous mutations in males transcarbamylase (OTC) gene: Mutations in and females with ornithine transcarbamylase 50 Japanese families with OTC deficiency. Am deficiency. Am J Med Genet 1995; 55: 67–70. J Med Genet 1997; 71: 378–383. 12) Matsuda, I.: Ethical issues around prenatal 9) Matsuura, T., Hoshide, R., Setoyama, C. et al.: diagnosis: From the standpoint of molecular Expression of four mutant human ornithine biology. Shusanki Igaku 1998; 28: 999–1003. transcarbamylase genes in cultured Cos-I cells (in Japanese) relates to clinical phenotypes. Hum Genet 13) Uchino, T., Endo, F. and Matsuda, I.: Neuro- 1994; 93: 129–134. developmental outcome of long-term therapy 10) Matsuda, I., Matsuura, T., Nishiyori, A. et al.: of urea cycle disorders in Japan. J Inherit Phenotype variability in male patients car- Metab Dis 1998; 21(Suppl1): 151–159. rying the mutant ornithine transcarbamylase 14) Kiwaki, K., Kanegae, Y., Saito, I. et al.: Correc- (OTC) allele, Arg40His, ranging from a child tion of ornithine transcarbamylase deficiency with an unfavorable prognosis to a sympto- in adult Spfash mice and in OTC-deficient matic older adult. J Med Genet 1996; 33: 645– human hepatocytes with recombinant adeno- 648. viruses bearing the CAG promoter. Hum Gene Ther 1996; 7: 821–830.

JMAJ, April 2004—Vol. 47, No. 4 165