Heritable urea cycle enzyme deficiency-liver disease in 16 patients

April 1979 580 The Journal o f P E D I A T R I C S

Heritable urea fiver disease in

cycle enzyme 16 patients

deficiency-

The liver mitochondrial enzymes ornithine transcarbamylase and carbamyl phosphate synthetase I

catalyze the first two steps in the Kiebs-Henseleit pathWay from ammonia to urea. Inherited deficiency

of either of these enzymes has been associated with severe and sometimes fatal hyperammonemia in

infancy and childhood, but liver histology has usually been described as normal. The Reye syndrome also

causes severe hyperammonemia and produces characteristic light microscopic changes in the river. In

addition, OTC and CPS activities have been shown to be depressed and it has been suggested that some

cases of the Reye syndrome may reflect an inborn defect in OTC which predisposes the patient to the

Reye syndrome. Review of the histopathology of 15 river specimeas from 13 patients with O TC deficiency

and three river specimens from three patients with CPS deficiency has disclosed the following: (1)

hemizygous male infants with X-linked OTC deficiency had essentially normal liver histology, although

fatally affected with extreme hyperammonemia; (2) in contrast, almost all heterozygous female children

and adults had some histologic abnormalities, including nine with minor degrees of steatosis and eight

with focal cell necrosis and inflammation, two of whom had piecemeal necrosis and stellate portal

scarring," (3) rivers from heterozygotes had discrete clusters of larger, pale-staining hepatocytes," (4) female

heterozygotes had abnormal liver function, even after the hyperammonemia was controlled; (5) CPS

deficient patients had essentially normal liver histology," and (6) neither OTC nor CPS deficiency was

associated with a histologic picture which resembled that seen in the Reye syndrome.

Douglas R. LaBrecque, M.D.,* Patricia S. Latham, M.D.,

Caroline A. Riely, M.D., Y. Edward Hsia, B.M., M.R.C.P., and

Gerald Klatskin, M.D. , N e w Haven, Conn.

ORNITHINE TRANSCARBAMYLASE is the second enzyme in the Krebs-Henseleit pathway from ammonia to urea. Absence of OTC is X-linked ~ and in the hemiz)'gous male, fatal hyperammonemia develops shortly after birth. In the heterozygous female, symptoms vary from none to

From the Departments of Medicine and Human Genetics, Yale University School of Medicine.

Supported in part by United States Public Health Service AM 05180-17 and by Grant RR125, GCRC Branch, Division of Research Resources, National Institutes of Health. Dr. Riely is a Teaching and Research Scholar of the American College of Physicians. *Reprint address: Division of Gastroenterology and Hepatology, Department of Medicine, University of Iowa, Apt. 3, Bldg. 3. VA Hospital. lowa City, 1.4 52240.

severe, presumably because of selective lyonization of the X-chromosome. ~

Abbreviations used OTC: ornithine transcarbamylase CPS: carbamyl phosphate synthetase I SGOT: serum glutamic oxloacetic transaminase

The abrupt presentation of OTC deficiency in an older child is similar clinically and biochemicaUy to that of the Reye syndrome, an illness characterized by encephalopathy, liver dysfunction, and a high mortality? It has been Suggested that at least some cases of Reye syndrome may be caused by an inherited abnormality of OTC?

A deficiency of other urea cycle enzymes, including

Vol. 94, No. 4, pp. 580-587 0022-3476/79/400580+08500.80/0 �9 1979 The C. V. Mosby Co.

Volume 94 Heritable urea enzyme deficiency 58 1 Number 4

Table I. Clinical findings in OTC and CPS deficiency

Patient* Growth and

A get developments Duration

of symptomsw

Severity of symptomsl[

At biopsy Liver (cm) �82

OTC L.C. 21 mo F R---~N 7 mo 3 2 5 R.C. 42 yr F N 42 yr 1 1 NP P.D. 48 yr F N 48 yr 1 1 NP

L.B. 21 mo F R---~N 7 mo 3 2 4 BB1 3 days M Died 3 days 4 4 - BB2 15 days, 5 mo M Died 5 mo 2 2,4 NP

J.L. 19m F R---~N 1 yr 3 2 2

T.Md. 20m, 26m F R 12 mo 3 2,2 4

S.P. 16m F R 11 mo 3 2 2

BMI 3 days M Died 3 days 4 4 NP BM3 5 days M Died 5 days 4 4 NP BM4 9 days M Died 9 days 4 4 NP BM5 3 days M Died 3 days 4 4 NP

CPS B.Z. 3 days M Died 3 days 4 4 NP

L.A. 16 yr F R 15 yr 3 2 NP K.A. 10 yr F R - 1 1 NP

*Unrelated patients are separated by horizontal lines. rage at which liver tissue was obtained. SR = Retarded development; N = normal development. w of symptoms prior to diagnosis. IIClinical severity is defined as: 0= Asymptomatic; 1 = history of protein intolerance with self-imposed protein restriction; 2 = Irritable and lethargic, with vomiting and developmental regression when untreated; asymptomatic on protein restriction • lactulose as controlled by physician; 3 = Obtunded; seizures, coma; 4 = Death. �82 Liver size is recorded in centimeters below the right costal margin; NP = nonpalpable; the spleen was palpable only in Patient S.P. in whom a tip was detected.

carbamyl phosphate synthetase, the first enzyme in the

Krebs-Henseleit pathway, ~.6 argininosuccinic synthetase,

argininosuccinase, and arginase, ~ can also produce hyper-

ammonemia and coma.

Liver histology in OTC deficiency, though reported

infrequently, is said to be normal. This is surprising in

view of the severe course and often fatal outcome. We had

noted that liver biopsies obtained at Yale New Haven

Hospital f rom several clinically affected heterozygotes

were not normal. We report the histopathology in 16

patients with OTC or CPS deficiency and compare our

findings with previous descriptions.

P A T I E N T S

Fifteen liver specimens from 13 patients with complete

(6) or partial (7) OTC deficiency and three specimens

from three patients with CPS deficiency were available for

examinat ion (Tables I and II).

O T C families, Detai led clinical and genetic studies of

Families C, M, P, L, and B with OTC deficiency have been reported elsewhere. 1. 8-1o

Family Md. Patient T.Md. was the second daughter of a

24-year-old Caucasian mother. The product of a term,

uncomplicated pregnancy, she displayed normal growth

and intellectual development until weaned at 14 months.

Episodic vomiting and irritability developed and motor

and intellectual skills gradually regressed. At 21 months of

age an SGOT level of 600 U was found at another

hospital. Our retrospective review of a liver biopsy

obtained at that t ime revealed normal histology except for

several groups o f large, pale hepatocytes. An extensive

radiographic and biochemical work-up was unrevealing,

but an electroencephalogram showed generalized slow-

ing. Maternal deprivat ion syndrome or childhood schiz-

ophrenia were considered likely diagnoses. Two months

later the ftrst venous blood ammonia level was obtained

(535/~g/dl), and she was transferred. On arrival she was

comatose and decerebrate. A firm liver edge was felt 3 to 4

cm below the right costal margin. The spleen was not

palpable. Blood ammonia value was 692 /~g/dl initially

and 90 ~tg/dl eight days later; the SGOT level fell from

192 to 62 U in the same interval; bilirubin and alkaline

5 8 2 LaBrecque et al. The Journal of Pediatrics April 1979

Table II. Laboratory and histologic findings in OTC and CPS deficiency

Peak

SGOT NH~ Patient* (U) (#g/dl)

Biopsy

SGOT j NH~ CPS~f OTCf

OTC L.C. -980 1,120 28 118 - 10.2

R.C. 22 251w 19 124 1.52 13.1

P.D, 19 358w 19 129 0.45 22.7

L.B. 55 1,900 45 112 1.33 4.48

BBI . . . . . .

BB2 60 526 55 160 2.53 0.03 60 526 57 526

J.L. 105 300 51 144 1.33 11.5

T.Md. 600 692 . . . . 600 692 71 198 1.44 2.56

S.P. 162 346 58 - 1.74 8.3

BM1 . . . . . . BM3 62 1,608 - - 2.42 0.00 BM4 142 2,570 - - 1.23 0.04 BM5 80 1,200 - - N 0.10

B.Z. - 1,480 - - 0 N

L.A. - 318 19 155 0.18 28.6 K.A. - 270w - 140 0.16 36.9

Hbtoiogy~

Moderate piecemeal necrosis and stellate portal scarring with I fibrous bridge, 2 + microvesicular fat

Diffuse glycogen deposition 1 + microvesicular fat

Normal

Mild 4 portal fibrosis, minor nonspecflic changes

Minor, nonspecific changes, 2 + microvesicular fat

1. Minor, nonspecilic changes (15 days) 2. Same (5 mo)

1 + microvesicular fat

1. Normal (21 too) 2. Mild piecemeal necrosis and portal fibro-

sis, 2 + microvesicular fat (27 mo)

Very mild, nonspecific hepatitis, slight 4' portal fibrosis

Mild inflammation, 1 + microvesicular fat 1 + microvesicular fat Normal 2+ microvesicular fat, 1+ portal inflamma-

tion

1 + microvesicular fat

Normal Normal

*Unrelated patients separated by horizontal lines. tReported as /tmoles of citruUine formed/hour/mw of fiver protein. Normal values for CPS - 1.0 to 8.0 /~rn/hour/m 8 and for OTC - 42 _+ 5 /an/hour/mw ~Presence of fat is estimated on a scale of 0 to 4 + with 0 - absence; 1 + - <5% of hepatocytes; 2 + = 6% - 25%; 3 + - 26% - 75%; 4 + - > 75%. w obtained during an ammonium chloride tolerance test. All other NH3 levels are fasting values.

phosphatase determinations were normal; blood urea nitrogen concentration was low at 3 to 6 mg/dl . The urine glutamate to glutamine ratio and orotic acid value were

increased; other urinary amino acid concentrations were normal. The electroencephalogram again showed pro- nounced generalized slowing. Computerized axial tomog-

raphy revealed dilated ventricles and reduced cortical thickness. She was treated with hydration, protein restric-

tion, and oral neomycin and rapidly became alert.

Discharged on a diet restricted to 1.25 g m/ k g / d a y of protein, she showed motor and intellectual improvement; however, at 27 months of age psychometric testing

showed her to be functioning as a 12-month-old child. A second percutaneous liver biopsy at that time revealed reduced OTC but normal CPS and arginase activities

(Table II). Liver histology revealed several enlarged triads with stellate scarring, moderate mononuclear inflamma-

tory reaction, and segmental erosion of their limiting plates. The parenchyma showed moderate pleomorphism and thick plates in some areas and a large group of pale,

foamy cells which contained fine, PAS-positive strands. The proband's 4-year-old sister is completely asympto-

marie with normal urinary orotic acid excretion. Her

mother has no history of protein intolerance, normal urinary orotic acid values and ammonia tolerance. There is no family history of neonatal deaths. The patient is

presumed to represent a new mutation.

CPS families. Patient B.Z. has been described previously? A second family, described in detail elsewhere, 11 includes two mentally retarded sisters 10 and 16 years

Volume 94 Heritable urea enzyme deficiency 583 Number 4

Fig. 1. Liver biopsy from Patient L.C. treated with the Laidlaw silver reticulin stain. The reticulin fibers are stained black. Illustration demonstrates increased reticulin fibers radiating from portal tracts (P) with some fine portal-portal bridging (B) by the fibrous tracts.

of age. The father and mother have mildly depressed CPS activities, suggesting a recessive mode of inheritance.

M E T H O D S

Liver specimens for enzyme assay were obtained by percutaneous needle biopsy, surgery, or immediate autop- sy. Portions were rapidly frozen on dry ice for later enzyme analysis 8 and in four instances a small portion was simultaneously fixed for electron microscopy. The rest of the biopsy was processed for light microscopy and stained with hematoxylin and eosin, Mason trichrome, Laidlaw silver reticulin, and periodic acid-Schiff (undigested and digested with diastase). All slides were analyzed in detail. Blood and urine were studied by methods previously described 8 as was ammonia tolerance. ~

Specimens for electronmicroscopy were placed in 3.0% gluteraldehyde buffered with 0.1M phosphate and fixed for one hour. They were transferred to phosphate buffer alone (0.1M) overnight and processed for Zeiss electronmicroscopy.

R E S U L T S

OTC deficient patients. Clinical and laboratory findings (Tables I and II). Five

of the six hemizygous males died in coma within a few days of birth. Patient R. B. lived almost five months on a special dietary regimen? ~ a~ The heterozygous females, two adults aged 49 and 53 and five girls aged 1 to 10 at the time of study, are still alive. Both adults are asymptomatic. All of the girls initially had seizures or coma or both but these did not recur once blood ammonia became normal. Two of the girls remain mentally retarded; all have shown improvement.

All 13 patients have had elevated blood ammonia values. However, all heterozygotes returned to the normal range with protein restriction. Several were initially treated with oral lactulose, and two have required main-

tenance lactulose. The SGOT level was increased in all of the children. All five surviving children have continued to show modest elevations of SGOT, despite control of the hyperammonemia. Bilirubin values were never increased in the heterozygotes. Several of the male infants had

5 8 4 LaBrecque et al. The Journal of Pediatrics April 1979

Fig. 2. Liver biopsy from Patient S.P. Hematoxylin and eosin stain reveals a large cluster of cells, paler than the surrounding parenchyma. Inset, a higher power view of the junction of these pale cells with the normal hepatocytes. The pale cells are somewhat larger with pale, foamy cytoplasm and a centrally placed nucleus.

increased unconjugated bilirubin levels preterminally. Liver pathology. Hepatomegaly without splenomegaly

occurred with the acute episodes in all of the heterozygotes. It tended to regress with clinical improvement, but three of the five girls continue to have large livers. Three biopsies were completely normal, and an additional four showed only minor microvesicular fatty infiltration of hepatocytes (confirmed by Oil Red O stain in Patient L. C.). Six specimens showed mild, nonspecific hepatitis, with focal cell necrosis, inflammation, and a mild increase in periportal fibrous tissue. Two had more extensive lesions with piecemeal necrosis, inflammation, and portal fibrosis. One also had significant stellate portal and central scarring and two fibrous bridges (Patient L. C.) (Fig. 1).

Nine of 15 specimens had minor fatty infiltration and 5(~o showed increased glycogen deposition and glycogen nuclei. Five of the six heterozygotes also had one or more clusters of slightly larger, paler staining cells, with central

nuclei and foamy cytoplasm (Fig. 2). On PAS staining these cells were generally dear but had fine strands of PAS positive material traversing the cytoplasm. Clusters of pale cells were also seen with the electron microscope (Fig. 3). Their pale appearance is apparently due to a decrease in density of the ground substance. These cells otherwise exhibited normal ultrastructure, including normal mitochondria. None of the six hemizygous males had abnormal histology. The OTC activity was less than 0.5% of normal in all hemizygous males. In the heterozygous females OTC activity varied from 6 to 53% of normal (Table II).

CPS deficient patients. Clinical and laboratory findings (Tables I & II). The one

male (Patient B.Z.) died at three days of age with extreme hyperammonemia (1,480/tg/dl). The two sisters (Patients C.A. and K.A.) had lower blood NH 3 levels which are well controlled by diet.

Liver pathology. None of the three had a palpable liver

Volume 94 Heritable urea enzyme deficiency 5 8 5 Number 4

Fig. 3. E|ectronmicrograph of liver biopsy from Patient L.B. A normal liver parenchymal cell is seen on the left, A. On the right is a somewhat paler cell with apparent decreased contrast of the ground substance, B. Other cell organdies, including the mitochondria, are normal in appearance. Several areas of dilated rough endoplasmic reticulum may be noted in both the pale and the normal cells. This is often seen as a nonspeeific finding on liver biopsy. (10,152 x .)

586 LaBrecque et aL The Journal of Pediatrics April 1979

or spleen and all had essentially normal liver histology. Electron microscopy of specimens from Patients C.A. and K.A. revealed normal liver ultrastructure. The male infant had no detectable CPS activity; the two sisters had levels less than 10% of normal.

D I S C U S S I O N

Despite the severe and often fatal consequences of deficiency of OTC, liver histopathology was noted in only 15 of 35 previously reported cases. (Table & references available from author on request.) Five of our six hemizygous males with OTC deficiency developed extreme hyperammonemia followed by coma and death within a few days of birth. Despite this fulminant course, none of them had significant liver pathology, nor did the infant dying with CPS deficiency. Thus, acute hyperammonemia alone does not produce liver damage discernible by light microscopy. Electron microscopy in one of the patients who died in coma (BB2) was also normal.

In contrast, almost all heterozygous females had histologic abnormalities, including two with piecemeal necrosis and stellate portal fibrosis. The severity of the lesion showed no clear correlation with liver function tests, prior to or at the time of biopsy. The persistent, mild elevations of SGOT values in our five heterozygous children, despite good control of their hyperammonemia, suggests continu- ing damage unrelated to ammonia concentrations. Fur- ther evidence that the histologic lesion evolves slowly is provided by Patient T.Md. whose first biopsy was essentially normal but whose subsequent biopsy revealed piecemeal necrosis and portal fibrosis.

If the presentation is acute, OTC deficiency may be confused with Reye syndrome. Gall et al ~3 and Hug et al 1~ have noted the failure of clinical and biochemical findings alone to deafly identify Reye syndrome. The latter reported that four of 17 patients originally diagnosed as Reye syndrome on clinical grounds, actually had inherited defects of urea cycle enzymes. The hallmark of Reye syndrome is the sudden onset of nausea and vomiting with a rapidly deteriorating mental state. Characteristic biochemical abnormalities include elevated blood ammonia and SGOT values in the presence of a normal bilirubin determination. The liver is usually enlarged, the spleen not palpable and the prothrombin time may be prolonged. When first seen, all of our heterozygotes, except the two asymptomatic adults, exhibited encephalopathy with increases in SGOT and ammonia levels and a normal bilirubin value. All had large livers and four of the five had prolonged prothrombin times.

Both CPS and OTC are moderately depressed in the acute phase of Reye syndrome? . . . . The liver mitochondria also show characteristic ultrastructural changes. 17

Thaler ~ has suggested that Reye syndrome reflects a predisposing inborn biochemical abnormality of OTC. However, the impaired OTC and CPS activity in Reye syndrome do not appear to cause mitochondrial changes, since electron microscopy of the liver in two patients with OTC deficiency and two with CPS deficiency showed the mitochondria to be normal. TM Nor can OTC and CPS deficiency be implicated in the general pathology of Reye syndrome, s ince even complete deficiency did not produce the striking microvesicular fatty infiltration of the liver seen in Reye syndrome. It seems more likely that whatever causes Reye syndrome produces mitochondrial damage which in turn leads to the depression of OTC and CPS activities.

The significance of the large, pale cells is unclear. None of our hemizygous males displayed these cells, suggesting that they developed only after survival for a period of time. These cells may represent the first histologic expres- sion of the disease since they were already present in the first biopsy from Patient T.Md., when no other abnormalities were observed. By light microscopy they resemble cells seen in glycogen storage disease but they show only thin strands of PAS-positive material. Ultrastructurally, the only consistent abnormality was an apparent decrease in density of the ground substance. The liver of one patient (R.C.) was assayed histochemically for OTC and showed distinct clusters of OTC deficient cells. 1~ The pale cells may be the OTC deficient ones. Histochemical studies have not been performed on serial sections, however, so this speculation cannot be substantiated.

The authors gratefully acknowledge the help of Dr. John MeReynolds who performed the assays for OTC, CPS, arginase, and urine orotate.

R E F E R E N C E S

1. Short EM, Coma HO, Snodgrass HJ, et al: Evidence for X-linked dominant inheritance of omithine transcarbamylase deficiency, N Engl J Med 288:7, 1973.

2. Lyon MF: Gene action in the X-chromosome of the mouse (Mus musculus L.), Nature 190:.372, 1961.

3. Partin JC: Reye's syndrome (encephalopathy and fatty fiver), diagnosis and treatment, Gastroenterology 69:51 l, I975.

4. Thaler MM: Reye's syndrome: Cause-and-effect relation- ships, In Berenberg SR, editor: Liver diseases in infancy and childhood, Baltimore, 1976, The Williams & Wilkins Company, pp 72-83.

5. Gelehrter TD, and Snodgrass PJ: Lethal neonatal deficiency of carbamyl phosphate synthetase, N Engl J Med 290:430, 1974.

6. Batshaw M, Brusilow S, and Walser MI Treatment of carbamyl phosphate synthetase deficiency with keto analogues of essential amino acids, N Engl J Med 292:1085, 1975.

Volume 94 Heritable urea enzyme deficiency 5 8 7 Number 4

7. Hsia YE: Inherited hyperammonemic syndromes, Gastro- enterology 67:347, 1974.

8. Campbell AGM, Rosenberg LE, Snodgrass PF, et al: Ornithine transearbamylase deficiency, a cause of lethal neonatal hyperammonemia in males, N Engl J Med 288:1, 1973.

9. Gelehrter TD, and Rosenberg LE: OTC Deficiency. Unsuc- cessful therapy of neonatal hyperammonemia with N- carbamyl-L-glutamate and L-arginine, N Engl J Med 292:351, 1975.

10. McReynolds J, Mantagos S, Brusilow S, et al: Treatment of complete ornithine transcarbamylase deficiency with nitrogen-free analogues of essential amino acids, J PEDI TR 93:421, 1978.

11. McReynolds J, Crowlcy B, Mahoncy M J, et al: Evidence for autosomal recessive inheritance of mitochondrial carbamyl phosphate synthetase deficiency, Pediatr Res 12:453 (A536), 1978.

12. McReynolds J, Mantagos S, Walser M, et al: Early treatment of complete ornithine transcarbamylase deficiency with keto analogues of essential amino acids, Pediatr Res 11:459 (A528), 1977.

13. Gall DG, Cutz E, McCling H J, et al: Acute liver disease and encephalopathy mimicking Reye syndrome, J PEDIATR 87:869, 1975.

14. Hug G, Lansky L, Bove K, et al: Human hepatocytes in acquired versus heritable defects of urea cycle enzymes, J Cell Biol 70:157a, 1976.

15. Brown T, Hug G, Lansky L, et al: Transiently reduced activity of carbamyl phosphate synthetase and ornithine transcarbamylase in liver of children with Reye's syndrome, N Engl J Med 294:861, 1976.

16. Sinatra F, Yoshida T, Applebaum M, et al: Abnormalities of carbamyl phosphate synthetase and ornithine transcarbamylase in liver of patients with Reye's syndrome, Pediatr Res 9:829, 1975.

17. Partin JC, Schubert WK, and Partin JS: Mitochondrial ultrastructure in Reye's syndrome (encephalopathy and fatty degeneration of the viscera), N Engl J Med 285:1339, 1971.

18. Latham PS, LaBrecque DR, Kashgarian L, et al: A compa- rative ultrastructural study of inherited urea cycle enzyme deficiency states and Reye's syndrome (RS), Gastroenterol- ogy 73:1230, 1977.

19. Ricciuti FC, Gelehrter TD, and Rosenberg LE: X-chromosome inactivation in human liver: Confirmation of X- linkage of ornithine transcarbamylase, Am J Hum C, enet 28:332, 1976.

Heritable urea cycle enzyme deficiency-liver disease in 16 patients

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