The patient was born to apparently normal, 21-year-old parents on April 1, 1965, and weighed
(Received january 19; accepted March 30, 1968.)
Supported by grants from the U.S. Public Health Service 5 TOl HE05478-07 and M01-FR-30.
ADDRESS: (F.S.P.) I)epartment of Pediatrics, Duke University Medical Center, Durham, North Caro-liiia 27706.
PEDIATRICS, Vol. 42, No. 3, September 1968
415
HEREDITARY
OROTIC
ACIDURIA
I. A
NEW
CASE
WITH
FAMILY
STUDIES
Lon E. Rogers, M.D., Lloyd R. Warford, M.D., Richard B. Patterson, M.D.,
and F. Stanley Porter, M.D.
Departments of Pediatrics, Duke University Medical Center, Durham, North Carolina and Bowman Gray School of Medicine, Winston-Salem, North Carolina
ABSTRACT. A new case of hereditary orotic
aci-duria is reported. Clinical manifestations included growth retardation, orotic acid crystallunia, and a
megaloblastic anemia. Uridine therapy produced a prompt hematologic response and a rapid
accelera-tion of growth. Orotic acid excretion on treatment
ranged from 0.96 gin to 3.89 gin orotic acid per
gram creatinine, and no decrease in excretion was observed when the uridine dosage was increased
from 0.75 to 2.0 gm per day. Studies of the
pa-tient’s ervthrocytes revealed a virtual absence of the activity of orotidylic pyrophosphorylase and
orotidylic decarboxylase. Previously undescribed
studies of liver revealed the enzymatic deficiency in this tissue as well.
Four generations of the patient’s family were
evaluated with erythrocyte enzyme assays and with a urinary screening test developed during the
course of these studies. A subject apparentl
unre-lated to the propositus was unexpectedly found to
be a carrier of the disorder. Investigation of his family revealed other heterozygous individuals. With the pedigrees of these families, an autosomal,
recessive mode of inheritance was demonstrated.
Pediatrics, 42:415, 1968, hEREDITARY OROTIC ACIDU-RIA, OROTIC ACID, FAMILY STUDIES.
H
EREDITARY orotic aciduria is a raredis-order characterized by megaloblastic anemia, retarded growth and development, and excessive excretion of orotic acid in the
urine.’ Tile enzymatic defect involves two
essential enzymes of normal pyrimidine
synthesis, orotidylic pyrophosphorylase, and orotidylic decarboxylase.’ These enzymes
have been shown deficient in erythrocytes,’
leukocytes,’ fibroblasts,’ and saliva’ of af-fected mdividuals. This unique double
enzyme defect aIld its genetic implications
have been discussed at length.’ To date
only 3 cases homozygous for this condition
have been reported.1( It is the purpose of
this paper to present a fourth case with
studies of the erythrocyte orotidylic
en-zymes in tile patients and four generations of
her family. Previously undescribed studies of liver show the enzymatic defect to be
present in this tissue of the homozygote.
CASE REPORT
6 lb, 7% oz. The mother’s pregnancy and the
in-fant’s early weeks of life were normal. At 2
months of age, however, the patient’s hemoglobin
was found to be 7.7 gm/100 ml. Treatment of the
anemia with oral iron (30 mg elemental iron per
day) and vitaniin C (50 mg per day) was begun
at that time and continued during the subsequent
3 months without response.
Upon admission to the North Carolina Baptist Hospital, Winston-Salem, North Carolina, in Oc-tober 1965, the patient appeared normal, except for pallor and bilateral strabismus. Physical ex-amination revealed a harsh precordial systolic
mur-mur. The liver and spleen were not enlarged. The infant was active and alert. The infant’s weight
was 7.4 kg (50th percentile) and length was 68 cm (90th percentile). Laboratory examination revealed the following: hemoglobin, 7.8 gm/100 ml;
he-matocrit, 28%; ervthrocytes, 2.9 million per cubic millimeter; reticulocvtes, 1.5%; platelets, 570,000 per cubic millimeter; leukocytes, 3,300 per cubic millimeter; mean corpuscular hemoglobin
concen-tration, 28%; niean corpuscular volume, 97 cubic
microns; mean corpuscular hemoglobin, 27 tqsg.
METHODS
Erythrocyte Orotidylic Enzymes
The assay used is a modification of
previ-ously reported methods” and measures the
rate of conversion of orotic acid to uridylic acid, a process requiring the activity of both affected enzymes (Fig. 3). Orotic acid-7-C” was used as the substrate, and carbon dioxide-C’ released by the second enzymatic step was collected and measured. Hemolysates were prepared as follows: whole blood was anticoagulated with EDTA. Plasma and huffy coat were sep-arated by centrifugation, and the
erythro-a1
(I) a,
>% (1
0
C)
a,
Fic. 2. Hemoglobin and neticulocyte response following unidine
administra-tion. Lack of response to treatment with iron, vitamin C, vitamin B12, and folic acid is demonstrated.
Fic. 1. Megaloblasts of bone marrow from
homo-zygous patient with hereditary orotic aciduria.
The patient was treated with folic acid, 15 mg/
day for 5 days followed by 7.5 mg/day for 5 days
intramuscularly; thereafter, 5 mg/day was
admin-istered by mouth for 14 days. Vitamin B1, was
ad-ministered intramuscularly in the dosage of 30 tg
every other day for a total of three doses. Packed
enythrocyte transfusions were required on two
oc-casions. Early in 1966 orotic acid crystalluria was first noted during an episode of minimal
dehydra-tion accompanying gastroentenitis. By this time the
patient’s weight was at the 10th percentile and
the length was at the 50th percentile. In March
1966 oral unidine (1.5 gm/day) was begun. The
treatment was followed by a reticulocyte response
within 48 hours which peaked at 14% on the fifth
day of therapy and was followed by a rise of
hemoglobin to 12.3 gm/100 ml (Fig. 2). The
leukocyte count, which ranged from 1,900 to 6,000
pen cubic millimeter before unidine therapy,
in-creased to a range of 3,900 to 12,000 per cubic
millimeter following therapy. A bone marrow
aspirate obtained 6 weeks following the initiation
of uridine therapy was normal. After treatment the
overall activity of the child increased, the appetite
improved, and the rate of weight gain was
ac-celerated. By age 18 months the infant’s weight
was again in the fiftieth percentile and the length
was in the ninetieth percentile. The patient’s
men-tal development is apparently normal at age 23i
years. All appropriate developmental milestones
have been achieved. The patient has subsequently
been hospitalized for a correction of stnabismus
and for a percutaneous liver biopsy. The liver was
normal histologically. Further evaluation of the
Orotic Acid
ARTICLES 417
Orotidine-5--P04Uridine-5’-P04
ii 02 II
0 0 c’4 0
________________
HN
CH
H1N C1 ,,H1N I II
0N )-C’00H N ,,,C.CI4OOH ON”
H OROTIDYLIC
I
OROTIDYLIC IPYROPHOSPHORYLASE HC DECARBOXYLASE HC
HCOH HC OH
I
I
0
HCOH
]
HG o
II
HG-OP-OH HI
OH HG-OP-OH
HI OH
FIG. 3. Affected enzymes in hereditary orotic aciduria resulting in deficient unidine-5’-P04 production.
cytes were washed three times with 0.155
M potassium chloride-4% dextrose solution.
Erythrocytes were resuspended in a volume
of 0.155 M potassium chloride-4% dextrose solution to approximate the original blood volume. Erythrocyte counts were made with a Coulter Counter,#{176} and the cells were hemolyzed by freezing and thawing. Sam-ples not to be determined immediately were stored at -10#{176}C. All determinations were made within 48 hours after the blood was collected. A volume of 0.1 ml hemolysate was added to 0.9 ml of 0.062 M sodium phosphate buffer, pH 7.5 containing 0.25 p.
moles (0.5 p.Ci) orotic acid-7-C’4, 3 p. moles magnesium chloride, and 0.25 p. moles 5-phosphorylribose-1-pyrophosphate (mag-nesium salt). The sample was incubated in the outer chamber of a Warburg-type ap-paratus for 1 hour at 37#{176}Cin a shaking water bath. Hydroxide of Hyamine, 0.5 ml, was placed in the center well. The re-action was halted by the addition of 1 ml
of 0.3N sulfuric acid. An additional
45-minute incubation time was allowed for the absorption of the carbon dioxide-C14 into the Hyamine. The Hyamine was then re-moved into 10 ml Bray’s solution8 and the radioactivity was determined in a Packard
* Coulter Counter Electronics, Inc., Hialeah,
Florida.
Tri-Carb Liquid Scintillation Spectrometer.t A blank was prepared by substituting an equal volume of the buffer for the hemoly-sate. The results are expressed as mp. moles carbon dioxide per 10’ erythrocytes per hour. Control blood samples were from random donors at the hospital blood bank. All samples were determined in duplicate.
Urinary Orotic Acid Excretion
An isotope dilution method was used.”9 Orotic acid-6-C’4 of known specific activity was added to the urine sample. Orotic acid was then separated from the urine by col-umn chromatography on Dowex-chloride 2 X 8-400. By determining the specific activ-ity of the separated orotic acid, the concen-tration of the acid in the urine could be cal-culated. A colorimetric method was also used for the assay of urinary orotic acid and is described in the companion article.’0
Urinary creatinine concentration was measured by a standard picric acid colon-metric method.h1 The results are expressed as grams orotic acid per gram of creatinine.
Liver Orotidylic Enzymes
Liver was obtained from the patient by percutaneous biopsy. weighed and
#{149}...
>10
9
(-)
5 II 0
A #{149} I’
-a
.SE .‘
0
#{149}I.
...
Controls
.
I
offlG)
oo0
“I”Fomily
? ? /
IG
77 .
FIG. 4. Solid circles represent presumed normals and
open circles presumed heterozygotes; triangle in “H” Family represents propositus. Subject JIG
(Fig. 5) is identified in “I” Family.
enized with a dounce homogenizer in 0.07
M sodium phosphate buffer, pH 7.5 to form a liver suspension diluted 1:20 vt/vol. For the measurement of liver orotidylic pyro-phosphorylase, 0.1 ml of the liver homog-enate was added to 0.9 ml 0.062 M sodium phosphate, pH 7.5 containing 0.25 p. moles
(0.5 p.Ci) orotic acid-7-C”, 3 p. moles mag-nesium chloride, 0.25 p. moles
5-phosphoryl-ribose-1-pyrophosphate (magnesium salt), and an excess of yeast orotidvlic decar-boxylase. The decarboxylase enzvne ‘Vas
extracted from yeast by the method of Ilep-pel and Hilmoe.12 For the blank 0.1 ml buffer was substituted for the liver homog-enate.
Liver orotidylic decarboxylase activity
was measured by incubating 0.1 ml of tile
liver suspension in 0.9 ml 0.062 M sodium
phosphate buffer, pH 7.5 containing 0.05
p. moles (1 p.Ci)
(tniammonium salt). For the blank 0.1 ml buffer was substituted for the liver homog-enate. The assay was carried out thereafter as described for the erythrocyte enzymes.
Measurement of conversion of orotic acid to unidylic acid (both enzymes required) was performed as for the erythrocyte enzyme assay. A 0.1 ml volume of the liver
suspension was substituted for the
hemoly-sate.
All samples were measured in duplicate. Control specimens for these assays were ob-tained by open liver biopsy from subjects who were undergoing abdominal surgery.
RESULTS
Erythrocyte Enzymes
Erythrocyte enzyme assays were per-formed on 23 blood relatives of tile
proposi-I.
n.
In.
FIG. 5. Stippled circle = propositus; cross-hatched circles = heterozygous females; cross-hatched squares
= heterozygous males; open circles and squares = unaffected females and males; ? = living but not
TABLE I
ItESUI.TS OF ERYTIIROCYTE ENZYME ASSAYS AND URINARY SCREENING TESTS FOR PRFSUMF.D
UFTEROZYGOTES WITH HEREDITARY Ooic ACIDI’RIA
Suhject*
Normal (19 controls) Normal (100 controls)
IE F II B D G J Erythro-cyte Enzyme Assay 2.4-14.9 0.8 0.8 0.8 0.8 1.5 0.5 0.8 0.7 0.9 0.9 0.6 0.7 0.6 02 Urinary Screening Test 48O:J12 Ratio”
0.33 (S.l).= .09)
1.00 0.94 0.51 0.68 0.56 0.85 0.61 0.74 0.64 0.68 1. 1.03 o.90 ARTICLES 419
tus. All additional five in-laws were studied.
Enzyme levels are expressed as mp. moles
carbon (liOXi(le collected per 10’
erythro-c’tes per hour. Of the 2.3 blood relatives
stti(liecl, 10
(
including the patient and bothparents
)
had enzyme activities less than 1.0whereas 13 ranged from 2.0 to 10.2
(
Fig. 4,“H” family
)
. The range for 19 controlsam-pies was from 2.4 to 14.9. Those family members with values under 1.0 were con-sidered probable heterozygotes, and those with values of 2.0 or greater were consid-ered probable normal individuals. The ho-mozygote’s enzyme activity was 0.2. The p value for the difference of the means of the presumed normals and presumed heterozy-gotes is <.01. Of the five in-laws studied, the ellZyllle activities of one
(
Fig. 5, Sub-ject hG) were at an intermediate level of 1.5 which prompted an examination of the erythrocyte enzymes of his father and of four siblings. Two of these siblings were also found to be probable heterozygotes (Fig. 4, “I” Family). A scattergram (Fig. 4) illustrates the enzymatic activities of both families and the controls. Subjects IIIB and IIID are included in both fami-lies. The enzyme activities for the in-laws are not represented in the scattergram but are listed in Tables I and II.A screening test for the presence of ex-cessive orotic acid in urine was used in the study of these families. This test is de-scribed in another article.b0 The values ob-tained for the screening test and for the enzyme assays are tabulated in Tables I and II. On the basis of the combined
re-suits of urinary screening test and the
enzyme assays, a pedigree indicating the propositus, her family, and the unrelated family studied was constructed and is shown in Figure 5.
Urinary Orotic Acid Excretion
Orotic acid and creatinine concentrations were measured in random urine samples and the results are expressed as grams of orotic acid per gram of creatinine. Initially orotic acid was measured by both the iso-tope dilution method and the colorimetric assay. Subsequently, the colorimetric assay
III A
B F
1
IV E (homozygote)
* Numbers identifying subjects correspond to those
of Figure 5. IV E refers to the homozygous propositus.
was used alone because the results obtained by both methods were in close agree-ment.’0
Initial studies of urinary orotic acid ex-cretion were undertaken while the patient was receiving 0.75 gm oral unidine daily. The dosage was increased to 2 gm daily over a period of several weeks. With the smaller dosage the orotic acid excretion on three separate occasions was 1.11, 3.57, and 1.20 gm per gram creatinine. Increasing the unidine dosage to 2.0 gm daily failed to change the level of orotic acid excretion, which ranged from 0.96 to 3.89 gm per gram of creatinine.
Liver Enzymes
TABLE II
RESULTS OF ERYTHROCYTE ENZYME ASSAYS ANI)
URINARY SCREENING TESTS FOR UNAFFECTED MEMBERS OF AFFECTED FAMILIES WITH
HEREDITARY OROTIC ACIDURIA
Normal (19 controls) Normal (100 controls)
A G H J K L M II C B F H K M N 0 III C D B G H L M N .4-14.9 3.8 7.9 5.7 4.9,4.6 4.9 1.7 5.7 3.0 3.7 4.1 4.1 5.3 10.2 4.4 2.6
0.33 (S.D.= .09)
0.30 0.43 0. 5 0.68, 0.4 0.38 0 .3 0.33 0.48 0. 0.36 0. 0.40 0.33 0 .9 0 .3 0.45 0.33 0.40 0.38 0.47 0.40 0.39 Su&.iect* Eryt hr cyte Enzyme Assay Urinary Screening Test 4SO:12 Ratio’#{176} IV A C
* Numbers identifying subjects correspond to those
of Figure 5.
control values. These values are reported as m moles carbon dioxide collected per 5 mg
wet
weight
of
liver per hour and are asfol-lows:
onotidylic
pyrophosphonylase,
patient0.02, controls 0.79 and 1.9; orotidylic decan-boxylase, patient 0.96, controls 5.4, 4.1 and
3.6. When
the
conversion of orotic acid tounidine-5’-P04 was measured (both enzymes
required), the result for the patient was 0.02
as compared to the values of the controls of 0.83, 1.5, and 2.4.
DISCUSSION
The enzymatic defect of hereditary orotic aciduria involves two consecutive enzymes of the pynimidine synthetic pathway, orot-idylic pyrophosphorylase, and orotidylic decarboxylase
(
Fig. 3 ).2 The first enzyme catalyzes the formation of orotidine-5’-PO4 from the reaction of orotic acid with 5-phosphorylribose-1-phosphate. The second enzyme catalyzes the decarboxylation of orotidine-5’-P04 to form unidine-5’-P04. The deficient activities of these enzymes re-sult in an inadequate de novo synthesis ofuridine-5’-P04 and other pynimidine
nucleo-tides. This metabolic block may be circum-vented in the patient by the supplementa-tion of the diet with superabundant amounts of the pyrimidine nucleoside, unidine.
Hereditary orotic acidunia is manifested
clinically by megaloblastic anemia, in-creased urinary orotic acid excretion, and
retardation of growth and development.
The anemia is refractory to the usual
hema-tinics but responds when unidine or other pynimidine derivatives are administered.’ In the patient herein reported, the administra-tion of uridine corrected the anemia and the megaloblastic changes of the bone mar-row. At the same time the infant’s appetite
ARTICLES 421
two of the three previously reported cases.6’7
This child has excreted elevated levels of orotic acid, although crystalluria was not recognized until the patient became slightly
dehydrated during a bout of gastroenteritis.
Orotic acid excretion in other cases studied has approached 1,000 times that of the nor-ma! adult, which is 1.4 mg per 24 hours.7’9 The maximum measured excretion for the patient was 3.89 gm orotic acid per gram creatinine; the minimum was 0.96 gm per gram creatinine. Based upon a daily creati-nine excretion of 20 mg per kg body weight,’3 the output of orotic acid would range from approximately 0.25 to 1 gm daily. The patient’s initial orotic acid deter-minations were obtained during treatment with 0.75 gm uridine daily. Increasing this dosage to 2.0 gm daily over a period of sev-eral weeks failed to lessen urinary orotic acid levels. In contrast, an increase of un-dine dosage from 0.75 gm to 1.5 gm daily in the case of Becroft and Phillips reduced orotic acid excretion by approximately 60%. Greater dosages of uridine, however, did not further lessen the orotic acid output. It would seem preferable to administer an amount of uridine resulting in the lowest possible levels of orotic acid excretion. This finding is of especial importance because of the danger of urinary tract obstruct-tion by orotic acid crystals in these
chil-dren.1,6
The enzymatic defect in hereditary orot-ic aciduria has been shown present in all tissues studied thus far.24 This report dem-onstrates the deficient activities of oroti-dylic pyrophosphorylase and orotidylic de-carboxylase in the hepatic tissue of the ho-mozygous patient. The activity of orotidylic pyrophosphorylase was 1.5% that of the mean of the controls. The activity of oroti-dylic decarboxylase measured 22% that of the mean control values. When the conver-sion of orotic acid to uridine-5’-P04 was measured, a process requiring the activities of both enzymes, the activity of the pa-tient’s hepatic tissue was 1.3% of that of the mean of the controls.
The erythrocyte enzyme studies and the urinary screening test allowed the study of the inheritance of this disorder. As in the family studied by Fallon, et al.,’ the inheri-tance is autosomal recessive in type. Only the homozygous patient was overtly af-fected. The transmission of the trait from fathers to sons on three occasions excludes x-linked inheritance as do four instances of nontransmission from fathers to daughters. One subject
(
Fig.
5, Subject IlL) was found to be heterozygous, but neither of his parents was an apparent carrier of the trait. This finding suggests reduced penetrance of the gene, questionable paternal identity, or the emergence of a new mutation.A family (“I” Family) not apparently re-lated to the propositus was unexpectedly found to be affected. Subject IIIB was found to be heterozygous whereas his mother, a blood relative of the propositus (“H” Family), was normal by both enzy-matic and urinary studies. The enzymatic
activities of the father’s erythrocytes,
how-ever, were borderline, as were the results of his urinary screening test. Additional sam-ples were obtained with similar results. Studies of this man’s siblings and a niece revealed other affected members in the family. Consanguinity between these fami-lies was not demonstrable, although the families have resided in the same western
North Carolina community for genera-tions.
The rarity of this disorder is indicated by the discovery of only three previous cases”7 since its description by Huguley,
et al. in 1959.1 A case similar to hereditary
orotic aciduria has been reported.15 This patient’s illness, however, was responsive to folic acid and probably represents another
genetic
disorder. The infrequency of thehomozygous condition may imply a gene
frequency
of
greater rarity than is, in fact,the case. In both this study and a previous
investigation,2 a subject unrelated to the
propositus was quite unexpectedly found to
sug-gested l)y Smith, et a!.’ is that the homozy-gous state often results in intra-uterine or neonatal death. If this is the case, the de-fective gene could, in fact, not be rare at all, but only unrecognized.
SUMMARY
A patient with hereditary orotic aciduria
is presented. Clinical manifestations in-cluded a megaloblastic anemia, orotic acid crystalluria, and retardation of growth. Un-dine administration produced a prompt he-matologic response and acceleration of growth. Previously undescnibed studies of liver demonstrate the enzymatic defect to be present in this tissue of the homozygous patient. Erythrocyte enzyme assays and a urinary screening test developed during this investigation were used to evaluate four generations of the patient’s family. Au-tosomal, recessive inheritance was demon-strated. A subject apparently unrelated to
the propositus was found to be a carrier of
this disorder, leading to the discovery of an
additional affected family.
REFERENCES
1. Huguley, C. M., Jr., Bain, J. A., Rivers, S. L.,
and Scoggins, R. B.: Refractory
megaloblas-tic anemia associated with excretion of orot-ic acid. Blood, 14:615, 1959.
2. Smith, L. H., Jr., Sullivan, M., and Huguley,
C. M., Jr.: Pyrimidine metabolism in man.
IV. The enzymatic defect of orotic aciduria.
J. Clin. Invest., 40:656, 1961.
3. Fallon, H. J., Lotz, M., and Smith, L. H., Jr.:
Congenital orotic aciduria: demonstration of
an enzyme defect in leukocytes and
compar-ison with drug-induced orotic aciduria.
Blood, 20:700, 1962.
4. Krooth, R. S.: Properties of diploid cell strains
developed from patients with an inherited
abnormality of uridine biosynthesis. Sympos.
Quant. Biol., 29:189, 1964.
5. Smith, L. H., Jr., Huguley, C. M., Jr., and
Bain, J. A. : Hereditary orotic aciduria. in
Stanbury, J. B., Wyngaarden, J. B., and
Fredrickson, D. S., ed. : The Metabolic
Basis of Inherited Disease, ed. 2. New York:
McGraw-Hill, pp. 739-758, 1966.
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he-reditary disorder of pyrimidine metabolism
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7. Becroft, D. M. 0., and Phillips, L. I.:
Heredi-tary orotic aciduria and megaloblastic
anae-mia: a second case, with response to uridine.
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8. Bray, G. A.: A simple efficient liquid
scintilla-tor for counting aqueous solutions in a
li-quid scintillation counter. Anal. Biochem.,
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Excretion of orotic acid and orotidine in
heterozygotes of congenital orotic aciduria.
Nature (London), 197:194, 1963.
10. Rogers, L. E., and Porter, F. S.: Hereditary
orot-ic aciduria: II. A urinary screening test.
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11. Hawk, P. B., Oser, B. L., and Summerson, W.
H.: Practical Physiological Chemistry, ed. 2.
New York: Blakiston, p. 899, 1954.
12. Heppel, L. A., and Hilmoe, R. j.: Purification
of yeast inorganic pvrophosphatase. J. Biol.
Chein., 192:87, 1951.
13. Nelson, W. E., ed.: Textbook of Pediatrics, ed.
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1964.
14. Fallon, H. J., Smith, L. II., Graham, J. B., and
Burnett, C. H.: A genetic study of
heredi-tary orotic aciduria. New Eng. J. Med.,
270:878, 1964.
15. Neimann, N., Najean, Y., Scialom, C., Boulard,
M., Pierson, M., and Bernard, J.: Etude
d’un cas d’an#{233}mie m#{233}galoblastique de
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