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G-6-PD worcester
G-6-PD Worcester A New Variant, Associated with X-Linked Optic Atrophy
L. MICHAEL SNYDER, M.D. THOMAS F. NECHELES, M.D. WILLIAM J. REDDY, S.C.D. with the technical assistance of KENNETH CALCAGNI, M.T. FRANK REED, B.A. Worcester, Massachusetts
A new variant of glucose-6-phosphate dehydrogenase (G-6-PD), G-6-PD Worcester, is described in a Caucasian family with associated congenital, nonspherocytic hemolytic anemia, absent erythrocyte G-6-PD activity and optic atrophy. The inheritance of this form of optic atrophy is closely linked to the G-6-PD locus and totalcolor blindness. G-6-PD Worcester is distinguished from previously characterized variants by absent erythrocyte G-6-PD activity, slow electrophoretic mobility, thermofability, a sharp peak at pH 8 and a Michaelis constant for TPN of 11.2 micromolar. Deficiency of erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity is an x-linked [1] genetic disorder with close linkage to the genes for color blindness [2], XgA locus [3] and hemophilia A [4]. Extreme variability of the clinical expression of G-6-PD deficiency is paralleled by the different biochemical characteristics in the G-6-PD enzyme types. A number of attempts have been made to relate erythrocyte G-6-PD deficiency to nonhematologic conditions, such as regional enteritis [5], diabetes mellitus [6], catatonic schizophrenia [7], pernicious anemia [8] and neonatal jaundice [9]. Only in neonatal jaundice is there a true correlation. In certain patients with erythrocyte G-6-PD deficiency, the enzyme is decreased in lens tissue [10,11], leukocytes [12], platelets [13], skin cultures [14] and the liver [15]. Leber's optic atrophy 1"16,17] is a rare, familial, x.linked disorder. Acute or subacute bilateral failure of central vision and restriction of the peripheral fields develop classically in young males; however, there is a wide range of age of onset through the seventh decade. The initial loss is usually followed by a period of stabilization and even improvement in vis. ual acuity. Demonstration of the presumed x.linked mode of inheritance has not been conclusively shown [18]. We now report a new variant of G-6-PD deficiency--"G.6-PD Worcest e r " - - w i t h absent erythrocyte G-6-PD activity, nonspherocytic congenital hemolytic anemia and optic atrophy, probably of the Leber's type.
MATERIALS AND METHODS
From the Departments of Hematology and Chemistry, Saint Vincent Hospital, Worcester. This study was supported by the Saint Vincent Hospital Research Foundation. Requests for reprints should be addressed to Dr. L. Michael Snyder, Department of Hematology, Saint Vincent Hospital, 25 Winthrop Street, Worcester, Massachusetts 01604. Manuscript received October 20, 1969.
Volume 49, July 1970
The G-6-PD enzyme was characterized and partially purified by modification of the methods described by Beutler et al. [191 and in the WHO scientific report on G-6-PD [20], dialysis being performed for only four hours due to enzyme instability. Partial purification of 200 cc of variant packed erythrocytes with intact buffy coat, gave a 45 per cent yield of the enzyme. Storage at 4~ for twelve hours resulted in 80 per cent loss of specific activity. Standard vertical starch gel electrophoresis with an ethylenediaminetetracetic acid (EDTA) tris-borate buffer, pH 8.6, was supplemented by electrophoresis on cellulose acetate strips using an EBT system buffered to pH 9.1 as described by Sparkes et al. [21], with direct application of 10 lambda of partially purified enzyme (specific activity, delta 0.02 OD units per minute) to the cellulose acetate strips. Erythrocyte chromium sl survival, hematologic studies, erythrocyte enzyme assays, glutathione and Heinz body studies (reported as number of cells con-
125
G-6-PD WORCESTER ~
SNYDER ET AL.
taining 4 or more Heinz bodies after incubation with phenylhydrazine) were conducted according to previously published methods [22-25]. G-6-PD assay on crude hemolysate was carried out utilizing the method of Kornberg and Horecker [26], with the addition of 10 micromolar of triphosphopyridine nucleotide (TPN), 7 millimolar of 2-mercaptoethanol and 2.7 millimolar of neutralized EDTA to the hemolyzing solution and expressed as micromoles of T P N / I O 1~ red blood cells or white blood cells/minute. Graded estimations of G6-PD activity in the individual erythrocytes were made using Fairbanks' and Lampes' method [27]. Normal spinal fluid from nine male subjects, aged nineteen to seventy-two, with normal erythrocyte G-6-PD, and spinal fluid from the propositus, R.Z., were assayed for G-6-PD using a modification of the method of Kornberg and Horecker [26]. The results are expressed as international enzyme units per 100 cc of spinal fluid. All enzyme assays and kinetics were performed on a Beckman 'Kintrac '| with the temperature stabilized at 25~
one day a loss of vision in his right eye. At that time, superior temporal fields in the right eye and both upper quadrant fields in the left eye remained intact. Complete neurologic examination, including a roentgenogram of the skull, an electroencephalogram, brain scan, lumbar puncture, serologic test for syphilis (VDRL) and two hour postprandial blood sugar levels, were within normal limits. Gradual improvement in vision was noted in the right eye following a combination of ACTH and prednisone therapy. During a recent viral illness, the patient was admitted to the hospital with increasing jaundice and anemia. Physical examination revealed a thin Caucasian man with moderate pallor and conjunctival icterus. Eye examination showed normal extraocular movements, no evidence of nystagmus and normal pupillary reaction bilaterally. Funduscopic examination revealed bilateral, white pale atrophic discs with normal vessels and intact retinas. Visual fields showed marked peripheral field constriction with central scotomas. Results of the Ishihara test revealed total color blindness, but with recognition of red and blue on gross color vision. Visual acuity was 2 0 / 8 0 corrected; optical densiLy: finger counting only at 2 feet, right eye. Laboratory data (Table I) disclosed a hemoglobin of 12.1 gm per cent, hematocrit of 36 to 38 per cent, platelet count of 400,O00/cu mm, reticulocyte count of 8.8 per cent, white blood cell count of 11,000/cu mm with a normal differential. The peripheral smear showed marked aniso- and poikilocytosis, target cells, Howell-Jolly bodies and Heinz bodies in 90 per cent of the red cells counted. His serum creatinine, calcium, phosphorus, alkaline phosphatase were all within norreal limits. A three hour oral glucose tolerance test showed the fasting blood sugar to be 100 mg per cent, 202 mg per cent at 30 minutes, 220 mg per cent at 60 minutes, 118 mg
CASE REPORTS R.Z. The propositus, a twenty-six year old Caucasian man of French-Polish-German extraction, required transfusions soon after birth for an uncompensated hemolytic anemia with jaundice. During the first fifteen years of his life he received fifteen to twenty transfusions for numerous episodes of jaundice, dark colored urine and hemolytic anemia, precipitated by upper respiratory tract infections and aspirin ingestion. In 1957 (age fourteen) he underwent splenectomy with a subsequent decrease in his transfusion requirement. At the age of thirteen permanent blindness developed in his left eye over a period of three to four weeks, and at twenty-four years of age, after cholecystectomy, he experienced in
TABLE
I
Hematologic Studies on Selected Family Members
Subject Propositus 111-3 Son IV-4 Mother 11-6 Sister
Age(yr) and Sex
Packed Hemoglobin Cell Volume (gin/100 cc) (%)
Bilirubin (mg %) Total
Direct
Reticu10cytes (%)
Heinz Body (%)
Or57 T 1/2 (days)
1.4
8.8
90
...
3
G-6-PD* RBC
WBC
4.5
0
110
10
...
4.7
--
24, M
12.1
38
3.4
NB, M
16.0
55
12.1
56, F
10.4
33
1.0
0.6
5.6
30
...
0.88
215
28, F
15.2
46
0.8
0.6
0.8
15
...
2.6
350
70, F
10.0
30
1.8
0.6
6.2
31
. ..
1.18
--
48, F
12.2
36
2.6
1.3
1.8
33
...
1.7
235
15, M
10.8
33
5.0
1.2
10.8
45
...
0
120
19, M
11.6
35
5.4
2.7
9.2
92
...
0
130
111-2
Maternal aunt 11-13 Maternal first cousin 111-15 Maternal second cousin IV-8 Maternal second cousin IV-9
G-6-PD _-- #moles of TPN r e d u c e d / m i n u ; e / l O 1~ red blood ceils (RBC) at 25 ~ Values for twenty normal volunteer subjects = 2.38 to 3.91, mean 2.9. # moles of TPN r e d u c e d / m i n u t e / l O 10 white blood cells (WBC) at 25 ~ Values for twenty normal volunteer subjects ~- 245 to 495.
126
The American Journal of Medicine
G-6-PD WORCESTER ~
per cent at 120 minutes and 67 mg per cent at 180 minutes, without glycosuria. The serologic test for syphilis (VDRL), and direct and indirect Coombs' test were all nonreactive. Serum total bilirubin was 3.4 mg per cent with 114 mg per cent direct reacting. Serum glutamic oxalacetic acid transaminase (SGOT) was 65 units (normal 8 to 40 units, Reitman-Frankel method, modified). Serum glutamic pyruvic acid transaminase (SGPT) was 55 units (normal 5 to 30 units, ReitmanFrankel method, modified). Chest and skull roentgenograms, an electroencephalogram and lumbar puncture were all within normal limits. Serum iron was 192 mg per cent with 73 per cent saturation. A liver biopsy demonstrated mild to moderate hemosiderosis. A bone marrow aspirate showed intense erythroid hyperplasia and increased iron but it was otherwise within normal limits. Special hematologic data included an autohemolysis test result of 1.55 per cent (normal is 0 to 1.14 per cent), with added ~glucose 1.21 per cent (normal is 0 to 0.3 per cent), with added adenosine triphosphate (ATP) 1.18 per cent (normal is 0 to 0.75 per cent). Osmotic fragility (fresh and incubated) and the sugar water test were normal. T~e activity of red cell enzymes of the Embden-Myerhoff pathway from hexokinase to lactic dehydrogenase, ATP levels and the remaining hexose-monophosphate shunt enzymes, were either normal or increased. Assay of erythrocyte G-6-PD revealed no activity; the white cells contained 110 micromoles of TPN/1010 cells/minute. R.M.Z. The newborn son of the propositus was born after thirty-eight weeks gestation, complicated by premature rupture of the membranes forty-eight hours prior to delivery associated with endometritis. At birth, there was no evidence of hepatosplenomegaly. Apgar score was 9. Laboratory data included a hematocrit of 55 to 60 per cent, hemoglobin 16.5 gm per cent, white blood cells 12,000/cu mm with a normal differential for a newborn. Reticulocyte count was 3 per cent. Bilirubin in cord blood was 1.5 mg per cent initially. The Coombs test, direct and indirect, was negative. There was no evidence of ABO incompatability. Because of sepsis in the mother, the patient was treated with kanomycin and penicillin. On the third hospital day his serum bilirubin level rose to 6 mg per cent and then climbed to a high of 12 mg per cent by the sixth day of life. Red cell G-6-PD activity was 4.7 (normal for newborns, 4.1 to 8.28 with a mean of 5.4). By the eighth day the bilirubin had dropped to 6 mE per cent and the patient continued to do well clinically, his hemogram remaining stable. B.Z. The mother of the propositus, age fifty-six, has worn glasses for twenty-five years, but she has no ophthalmologic complaints. Physical examination was within normal limits. Laboratory data included a hemato@rit of 33 per cent, hemoglobin 10.4 gm per cent, reticulocyte count 5.6 per cent. Examination of red cell morphology showed normocytic, normochromic cells with a mild polychromasia. Heinz body preparation revealed 30 per cent positive red cells. The serum total bilirubin was 1.0 mg per cent with 0.6 mg per cent indirect reacting. Red cell G-6-PD activity was 0.88; white cell G-6-PD activity was 240. H.B. The maternal aunt, aged seventy, experienced sudden blindness of unknown etiology at the age of thirty-five years. She had a long history of anemia accompanied by one episode of jaundice associated with pneumococcal pneumonia. Physical examination was within normal limits except for her eyes, which demonstrated bilateral optic atrophy with com-
Volume 49, July 1970
SNYDER ET AL.
plete loss of vision in the left eye and visual acuity corrected to 2 0 / 7 0 in the right. Laboratory data included a hematocrit of 30 per cent, hemoglobin 10 gm per cent, reticulocyte count 6.2 per cent. Heinz bodies were found in 31 per cent of the red cells. Erythrocyte G-6-PD activity was 1.18. Serum total bilirubin was 1.8 mg per cent with 0.6 mg per cent direct reacting. O.P. A maternal first cousin, aged forty-eight, jaundiced shortly after birth, required transfusions at that time but none since. Examination revealed conjunctival icterus with a pale atrophic disc in the right eye, compatable with mild optic atrophy. Visual acuity in both eyes was 20/30 corrected. Laboratory data included a hematocrit of 36 per cent, hemoglobin 12.2 gm per cent, reticulocyte count 0.8 per cent; Heinz bodies were present in 33 per cent of the red cells. Erythrocyte G-6-PD activity was 1.7; white blood cell G-6-PD activity was 235. Serum total bilirubin was 2.6 mg per cent with 1.3 mg per cent direct reacting. D.P. A maternal second cousin, aged fifteen, son of O.P., had jaundice shortly after birth, for which he required exchange transfusions. He experienced one episode of jaundice as a child and recently was told that he was anemic. Physical examination revealed scleral icterus, bilaterally pale optic discs, with visual acuity reduced to 20/30 corrected bilaterally. There was mild constriction of the peripheral fields and a small central scotoma in the left eye. The spleen was palpated 3 cm below the left costal margin. Total color blindness was noted. Laboratory data included a hematocrit of 33 per cent, hemoglobin 10.8 gm per cent, reticulocyte count 10.8 per cent. A one hour postprandial blood sugar level was 110 mg per cent. Erythrocyte G-6-PD activity was absent and white cell G-6-PD activity was reduced to 120. Serum total bilirubin was 5.0 mg per cent with 1.2 mg per cent direct reacting. Heinz bodies were 45 per cent. L.P. A maternal second cousin, aged eighteen, son of O.P., was jaundiced after birth and required exchange transfusions; subsequently he had episodes of jaundice at three years of age. He had three hospitalizations between the ages of four and seven years for anemia and jaundice, and received four transfusions. No further transfusions were required after a splenectomy at the age of seven. At the age of ten sudden blindness developed in both eyes over a ten day to two week period, with gradual improvement over the next few months. Cholecystectomy was performed when he was eighteen years old. Examination revealed conjunctival icterus, bilaterally pale optic discs with reduced visual acuity to 2 0 / 2 0 0 in the right eye and 4 / 2 0 0 in the left (corrected), with bilateral central scotomas, severely restricted peripheral fields and total color blindness by the Ishihara test. Laboratory data included a hematocrit of 35 per cent, hemoglobin 11.6 gm per cent and a reticulocyte count of 9.2 per cent. Examination of red cell morphology showed anisocytosis and poikilocytosis, target cells and Howell-Jolly bodies. Heinz body preparation revealed 92 per cent positive cells. There was absent erythrocyte G-6-PD activity and white blood cell G-6-PD activity was reduced to 130. Serum total bilirubin was 5.4 mg per cent with 2.7 mg per cent direct reacting. One hour and two hour blood sugar levels after a 100 gm glucose load were 100 and 90 mg per cent, respectively. R ESU LTS Hematologic studies (Table I) show that the three affected male subjects have mild, chronic, nonspherocytic
127
G-6-PD WORCESTER - - SNYDER ET AL.
Ophthalmologic Findings in Selected Family Members
TABLE II
Color Blindness IshiharaTest
Visual Acuity Pedigree
Subject
III _-- 3
R.Z.
20/80 corrected
III _-- 1 II _-- 6
J.S. B.Z.
20/20 20/40 corrected 20/70 corrected
II ~ 13
H.B.
III _-- 15
O.P.
IV _-- 4 IV _-- 7
R.M.Z . L.P.
IV ---- 8
D.P.
O.D.
O.S.
20/30 corrected . . . . . 20/200 corrected
Finger Total - counting bilateral at 2 feet 20/20 ... 20/40 ... corrected Finger ... counting at 2 feet 20/30 ... corrected . . . . 4/100 Total (-- 1.00 = --1.25 X
120) 20/30
20/30
Total
hemolytic anemia associated with clinical jaundice; the propositus has a chromium 51 survival of four and a half days. In addition, the three heterozygotes have evidence of hemolysis as evidenced by reticulocytosis and bilirubinemia. The ophthalmologic findings (Table II) are characterized by acute or subacute onset of visual impairment occurring between ten and thirty-eight years of age. After the initial visual deterioration, a period of stabilization followed with subjective improvement in visual acuity. The fundi of those affected revealed pale, atrophic discs with varying degrees of central scotomas and restricted TABLE III
RBC(% Activity of MeanNormal)
peripheral vision correlating with the degree of clinical abnormality. In addition, total color blindness, as measured by the Ishihara test, was present in the three affected males. Cytochemical staining of the erythrocytes of the heterozygotes B.Z. and O.P. showed two distinct populations with 266 erythrocytes (55 per cent) possessing zero granules, 29 (5.8 per cent) with 1+ granules, 195 (39 per cent) with 2-t- granules and 10 (2 per cent) with 3 + granules (500 cells counted). The mode of inheritance for G-6-PD Worcester is typically sex-linked (Fig. 1), the heterozygotes displaying intermediate erythrocyte G-6-PD levels, two distinct cytochemical erythrocyte populations and two separate electrophoretic bands (each band reflecting the gene product of one x chromosome). The detailed family study allows discussion of the nature of the optic atrophy, its mode of inheritance and whether or not it is related to the same gene as G-6-PD Worcester. Biochemical results are shown in Table II1. The thermal stability of the variant enzyme was very low (Fig. 2). After incubation for forty minutes at 46~ all specific activity was lost. The electrophoretic mobility on starch gel was 86 per cent of the the normal B + enzyme (Fig. 3) whereas the partially purified heterozygote showed two distinct bands, a normal band migrating at 9 cm and a slower band migrating at 7.8 cm from the origin. On cellulose acetate, the difference in mobility was even more distinct, the variant displaying 70 per cent mobility of the normal B' enzyme. The pH optimum for G-6PD Worcester showed a sharp peak at 8 as compared with the normal truncate pattern with a broad peak at pH 9 (Fig. 4). The mean Michaelis constant (Kin) for glucose-6-r)hosphate (G6P) was 61 micromolar with a range of 58 to 62 micromolar. The mean K,,, for TPN was 11 micromolar with a range of 9 to 12.4 micromolar (Fig. 5). The utilization of 2-deoxy-glucose-6.phosphate
Biochemical Aspects of "G-6-PD Worcester" Electrophoretic Mobility (% of Normal)
Heat Lability
pH OPtimum
K., TPN (#M)
K,, G6P (#M)
2dG6P Utilization (% of G6P)
42-58
2
DTPN Utilization (% of TPN)
DPN Uti!ization (% of TPN)
N o r m a l (Five Subjects)
100
Normal
100
0
Very labile
Slow 70
9.0 Truncate
3.5-7.5
50-55
0
21
0
G-6-PD Worcester (Three Subjects)
86
EBTpH 9.1 cellulose acetate EBTpH 8.6 starch gel
8,0 Sharp
11.2
61
<2
peak
NOTE: Km TPN _-- Michaelis constant for triphosphopyridine nucleotide. Kn, G6P ---- Michaelis constant for glucose-6-phosphate. 2d G6P ----2 deoxyglucose-6-phosphate. DTPN ----diamotriphosphopyridine nucleotide. DPN = diphosphopyridine nucleotide.
128
The American Journal of Medicine
G-6-PD WORCESTER - - SNYDER ET AL.
as a substrate was less than 1 per cent for both the variant and normal; however the utilization of deaminoTPN was 21 per cent for the variant with a range of 50 to 55 per cent for the normal enzyme. The mean normal level of G-6-PD in spinal fluid was 33 I.U. per 100 cc of spinal fluid with a range of 15 to 60 I.U., standard deviation (S.D.I.) ___14, The propositus I
(R.Z.) had 20 I.U. of G-6-PD activity per 100 cc of spinal fluid. COMMENTS More than fifty distinct variants of erythrocyte G-6-PD have been recognized but few result in significant nonspherocytic hemolytic anemia [22, 2 8 - 3 0 ] , Types asso-
2
I
3
5
6
7
4
8
II
12
13
0
0
LEGEND (~ OBLIGATE CARRIER [] BILATERAL OPTIC ATROPHY AND RBC G-6-PD DEFICIENCY (~) PROVEN CARRIER STATE FOR OPTIC ATROPHY AND G-6-PD DEFICIENCY (~ []
DIED AT CHILDBIRTH DEAD, AGE 3, OF PNEUMONIA AND JAUNDICE
~}
ALIVE, NOI STUDIED, BUT PRESUMED NORMAL BY HISTORY
2}
DECEASED,BUT PRESUMED NORMAL BY HISTORY
SUBSCRIPT REPRESENTS ACTIVITY Ol~' G-6-PD ~MOLES OF TPN REDUCED/MINUTE/IOi~ RBC AT ~S'C
Family pedigree. G.6-PD deficiency, chronic nonspherocytic hemolytic anemia, optic atrophy. Fig. 1.
.05 I0 .04'
9
\
E
O
\ .o3
\ ~"NORMAL 8~ VARIANT
o
E8
\
<~
i'~'~ X
NORMAL
7
.01~
6
ORIGIN
20
40
T I ME (MINUTES) Fig. 2. Thermal stability exp r e s s e d as OD/minute (B) 46~ using dialyzed enzyme.
Volume 49, July 1970
I
1
I
2
I
3 Fig. 3. Electrophoresis. Vertical starch gel electrophoresis of partially purified G-6-PD Worcester, using EDTA, tris.borate buffer system, p H 8.6. Slot ] contains normal B" enzyme. S l o t 2 contains G-6-PD Worcester. Slot 3 contains heterozygote f o r G-6-PD Worcester.
129
G-6-PD WORCESTER - -
SNYOER F_.T At.
120
IO0
\
I
~80 G-6-PD WORCESTEF
-
-
60
NORMAL B G-6-PD
W > -
-
40
W
20
/ I
5.0
5.5
I
60
I
I
I
6.5
7D
Z5
I
80
I
I
[
8.5
90
9.5
i
I0.0
pH Fig. 4.
Effects of pH on rates of normal and variant G-6-PD.
Activity expressed relative to activity at pH 7.5 which is taken as 100 per cent. Each point represents mean of three assays on variant and mean of five normal.
ciated with hemolytic anemia include G-6-PD Chicago, Oklahoma, Duarte, Bangkok, Mediterranean, Albuquerque, Torrance and the less well defined "Eyssen," "Tubingen" and "Berlin." Completely absent or markedly diminished erythrocyte G-6-PD activity, as described in G-6-PD "Eyssen" [30], Albuquerque, Mediterranean and Torrance, is rare. Although G-6-PD "Union and Markham" show extremely low levels of G-6-PD, they are not associated with chronic hemolysis. The new variant, G6-PD Worcester, is similar to G-6-PD "Eyssen" with regard to its slow electrophoretic mobility and absent erythrocyte G-6-PD activity, but it can be distinguished by the Michaelis constant for TPN, thermal stability and pH optimum. G-6-PD Milwaukee, Panay, Seattle, West Bengal, Madrona, Kerala, Capetown, ~badan-Austin, Athens, Baltimore-Austin and TelHashomer all have slow electrophoretic mobility but have biochemically distinct Michaelis constants for TPN, pH optimum, thermal stability and only moderate to mild enzyme deftciency, differentiating them from G-6-PD Worcester. Complete absence of G-6-PD activity in a fifty-nine year old Caucasian man with nonspherocytic hemolytic anemia, optic atrophy and neurologic manifestations has beerx reported previously [31], but detailed family studies were not included, and the enzyme was not biochemically characterized. Cataracts have been described in the lenses of affected patients with G-6-PD deficiency but appear to be the result of a local disturbance of carbohydrate metabolism [11].
130
In the present family, the low G-6-PD enzyme levels i~, the white blood cells may reflect a more generalized deficiency, but the acute onset of the optic atrophy with a normal level of G-6-PD in the spinal fluid are against a cause and effect relationship. The lesions may be the resuit of a single gene abnormality, but the presence of two closely aligned genes of the same chromosome is more likely. Evidence of a diffuse neurologic disorder is required by some investigators [32] for the diagnosis of Leber's optic atrophy whereas others do not consider this necessary [16]. The diagnosis of Leber's optic atrophy in t h e present family is likely despite the absence of other neurologic manifestations. Behr's optic atrophy [16,17] is excluded in view of the age of onset, type of inheritance and the absence of ataxia with dementia. Autosomal recessive optic atrophy usually presents in early infancy and autosomal dominant type in early childhood, and both have a more benign clinical course [33]. Optic atrophy with Friedreich's ataxia and spastic paraplegia is overshadowed by the other neurologic abnormalities [17]. The hematologic and ophthalmologic defects both appear to be due to mutations on the x chromosome. It is too early to tell if the male patients have affected grandsons through their daughters, but of the eight sons of the three proved carriers three are affected (we may consider this 50 per cent for purposes of discussion). This indicates either x-chromosome linkage or autosomal dominant inheritance with reduced penetrance in the male. The close linkage with G-6-PD deficiency implies probable x-chromosome inheritance for the optic atrophy. Both defects appear to be carried on the same chromosome, since in the twelve offspring, all are either 220L-
180 =
160
\ a o
140
~
t
f
'~
o
/:
<3
\
~
8o
60 40 2(
20
I0
0
I0
20 30 40 50 60 70 80 90 I00 SUBSTRATE (micromolar) Fig. 5. Michaelis constant for G-6-PD Worcester with TPN. Hatched area represents range of five normal B" partially purified G-6-PD.
The American
Journal of Medicine
G-6-PO WORCESTER- - SNYDER ET AL
completely normal or exhibit both defects. Only with continued study of subsequent generations will these questions be truly resolved.
ACKNOWLEDGMENT We wish to express our appreciation to Dr. E. Beutler, City of Hope Medical Center, Dr. R. E. Sage, Tufts Uni-
versity and Dr. A. Yoshida, University of Washington, for reviewing this manuscript and for their helpful criticisms. We are also indebted to Dr. S. Murphy and Dr. E. Epstein for performing the neurologic and ophthalmologic examinations in this family and to Dr. R. Abodeely for allowing us to study the propositus, R.Z. Our thanks also to Pamela Bohn for secretarial assistance.
REFERENCES 1. Davidson RG, Nitowsky HM, Childs B: Demonstration ~f two populations of cells in the human female heterozygote for glucose-6-phosphate dehydrogenase of variants. Proc Nat Acad Sci USA 50: 481-485, 1963. 2. Adam A: Linkage between deficiency of glucose-6-phosphate dehydrogenase and colour blindness. Nature (London) 189: 686, 1961. 3. Adam A, Sheba C, Race RR, Sanger R, Tippet P, Wampner J, Gavin J: Linkage relationships of the X-borne genes responsible for glucose-6-phosphate dehydrogenase and for the XgA blood group. (Letter) Lancet 1: 1188, 1962. 4. Boyer SH, Graham JB: Linkage between the X-chromosome loci for glucose-6-phosphate dehydrogenase electrophoretic variation and Hemophilia A. Amer J Hum Genet 17: 320, 1965. 5. Sheehan RG, Lindeman RJ, Meyer J, Patterson JF, Necheles TF: The possible association of erythrocyte glucose-6-phosphate dehydrogenase deficiency and regional enteritis. J Clin Invest 44: 1098, 1965. 6. Chanmugan D, Frumin AM: Abnormal oral glucose tolerance response in erythrocytes--glucose-6-phosphate dehydrogenase deficiency. New Eng J Med 27: 12021204, 1964. 7. Dern RJ, Glynn MF, Brewer GJ: Studies on the correlation of the genetically determined trait, glucose-6-phosphate dehydrogenase deficiency, with behavioral manifestations in schizophrenia. J Lab Clin Med 62: 319, 1963. B. McCurdy PR: An apparent association between red cell glucose-6-phosphate dehydrogenase deficiency and pernicious anemia in Negro males. Clin Res 1: 91, 1966. 9. Naiman JL, Kosoy MH: Red cell glucose-6-phosphate dehydrogenase deficiency--a newly recognized cause of neonatal jaundice and kernicterus in Canada. Canad Med Ass J 91: 1243, 1964. 10. Zinkham WH: Enzyme studies on lenses from persons with primaquine-sensitive erythrocytes. Amer J Dis Child 100: 525, 1960. 11. Zinkham WH: A deficiency of glucose-6-phosphate dehydrogenase activity in lens from individuals with primaquine sensitive erythrocytes. Bull Johns Hopkins Hosp 109: 206, 1961. 12. Ramot B, Fisher S, Szeinberg A, Adam A, Sheba C, Gafni D: A study of subjects with erythrocyte glucose-6phosphate dehydrogenase deficiency. II. Investigation of leukocyte enzymes. J Clin Invest 38: 2234, 1959. 13. Ramot B, Seinberg A, Adam A, Sheba C, Gafni D: A study of subjects with erythrocyte glucose-6-phosphate dehydrogenase deficiency. I. Investigation of platelet enzymes. J Clin Invest 38: 1659, 1959.
Volume 49, July 1970
14. Gartler SM, Gandini E, Ceppellini R: Glucose-6-phosphate dehydrogenase deficient mutant in human cell culture. Nature (London) 193: 602, 1962. 15. Chan TK, Todd D, Wong CC: Tissue enzyme levels in erythrocyte glucose-6-phosphate dehydrogenase deficiency. J Lab Clin Med 66: 937-942, 1965. 16. Waardenburg PJ, Franceschetti P, Klein D: Genetics and Ophthalmology, vol. 2, Assen, Holland, Royal Gorcum & Co, 1963, p 1617. 17. Bereday M, Cobb S: Relation of hereditary optic atrophy (Leber) to other familial degenerative diseases of the central nervous system. Arch Ophthal (Chicago) 48: 669-680. 18. McKusick VA: On the X-chromosome of man. Quart Rev Biol 37: 69-175, 1962. 19. Beutler E, Mathai CK, Smith JE: Biochemical variants of glucose-6-phosphate dehydrogenase giving rise to congenital non-spherocytic hemolytic disease. Blood 31: 131-150, 1968. 20. World Health Organization Technical Report Series: Standardization of procedures for the study of glucose-6-phospha'e dehydrogenase. WHO Report, 1967, No. 366: 5-53. 21. Sparkes RS, Baluda MC, Townsend DE: Cellulose acetate electrophoresis of human glucose-6-phosphate dehydrogenase. J Lab Clin Med 73: 531-534, 1969. 22. Tanaka KR, Valentine WN, Miwa S: Pyruvate kinase (PK) deficiency hereditary nonspherocytic hemolytic anemia. Blood 19: 267, 1962. 23. Koutras GA, Hattori M, Schneider AS, Ebaugh FJ Jr, Valentine WN: Studies on chromated erythrocytes. Effect of sodium chromate on erythrocyte glutathione reductase. J Clin Invest 43: 323, 1964. 24. Beutler E, Duron O, Kelly BM: Improved method for the determination of blood glutathione. J Lab Clin Med 63: 715, 1963. 25. Beutler E, Dern RJ, AIving AS: The hemolytic effect of primaquine. Vl. An in vitro test for sensitivity of erythrocytes to primaquine. J Lab Clin Med 45: 40, 1955. 26. Kornberg A, Horecker BL: Glucose-6-phosphate dehydrogenase. Methods of Enzymology, Vol I (Colowick SP and Kaplan NO, ed) New York, Academic Press, 1955, p 323. 27. Fairbanks VF, Lampe LT: A tetrazolium-tinked cytochemical method for estimation of glucose-6-phosphate dehydrogenase activity in individual erythrocytes: applications in the study of heterozygotes for glucose6-phosphate dehydrogenase deficiency. Blood 31: 589--602, 1968. 28. Motulsky AG, Yoshida A: Methods for the study of red cell glucose-6-phosphate dehydrogenase. Biochemi-
131
G-6-PD WORCESTER - - SNYDER ET AL.
cal Methods in Red Cell Genetics (Yunis J J, ed) New York, Academic Press, 1969, p 52--65. 29. Giblett ER: Glucose-6-phosphate dehydrogenase. Genetic Markers in Human Blood, Philadelphia, F.A. Davis Co., 1969, p 453. 30. Boyer SH, Porter IH, Weilbacher RG: Electrophoretic heterogeneity of glucose-6-phosphate dehydrogenase and its relationship to enzyme deficiency in man. Proc Nat Acad Sci USA 48: 1868, 1962.
132
31. Escobar MA, Heller P, Trobaugh FE: "Complete" erythrocyte glucose-6-phosphate dehydrogenase deficiency. Arch Intern Med 113: 428, 1964. 32. Wilson J: Leber's hereditary optic atrophy--some clinical and aetiological considerations. Brain 86: 3 4 7 362, 1963. 33. Kjer P: Infantile optic atrophy with dominant mode of inheritance. Acta Ophthal (Kobenhavn) supp 54, p 1, 1959.
The American Journal of Medicine
L. MICHAEL SNYDER, M.D. THOMAS F. NECHELES, M.D. WILLIAM J. REDDY, S.C.D. with the technical assistance of KENNETH CALCAGNI, M.T. FRANK REED, B.A. Worcester, Massachusetts
A new variant of glucose-6-phosphate dehydrogenase (G-6-PD), G-6-PD Worcester, is described in a Caucasian family with associated congenital, nonspherocytic hemolytic anemia, absent erythrocyte G-6-PD activity and optic atrophy. The inheritance of this form of optic atrophy is closely linked to the G-6-PD locus and totalcolor blindness. G-6-PD Worcester is distinguished from previously characterized variants by absent erythrocyte G-6-PD activity, slow electrophoretic mobility, thermofability, a sharp peak at pH 8 and a Michaelis constant for TPN of 11.2 micromolar. Deficiency of erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) activity is an x-linked [1] genetic disorder with close linkage to the genes for color blindness [2], XgA locus [3] and hemophilia A [4]. Extreme variability of the clinical expression of G-6-PD deficiency is paralleled by the different biochemical characteristics in the G-6-PD enzyme types. A number of attempts have been made to relate erythrocyte G-6-PD deficiency to nonhematologic conditions, such as regional enteritis [5], diabetes mellitus [6], catatonic schizophrenia [7], pernicious anemia [8] and neonatal jaundice [9]. Only in neonatal jaundice is there a true correlation. In certain patients with erythrocyte G-6-PD deficiency, the enzyme is decreased in lens tissue [10,11], leukocytes [12], platelets [13], skin cultures [14] and the liver [15]. Leber's optic atrophy 1"16,17] is a rare, familial, x.linked disorder. Acute or subacute bilateral failure of central vision and restriction of the peripheral fields develop classically in young males; however, there is a wide range of age of onset through the seventh decade. The initial loss is usually followed by a period of stabilization and even improvement in vis. ual acuity. Demonstration of the presumed x.linked mode of inheritance has not been conclusively shown [18]. We now report a new variant of G-6-PD deficiency--"G.6-PD Worcest e r " - - w i t h absent erythrocyte G-6-PD activity, nonspherocytic congenital hemolytic anemia and optic atrophy, probably of the Leber's type.
MATERIALS AND METHODS
From the Departments of Hematology and Chemistry, Saint Vincent Hospital, Worcester. This study was supported by the Saint Vincent Hospital Research Foundation. Requests for reprints should be addressed to Dr. L. Michael Snyder, Department of Hematology, Saint Vincent Hospital, 25 Winthrop Street, Worcester, Massachusetts 01604. Manuscript received October 20, 1969.
Volume 49, July 1970
The G-6-PD enzyme was characterized and partially purified by modification of the methods described by Beutler et al. [191 and in the WHO scientific report on G-6-PD [20], dialysis being performed for only four hours due to enzyme instability. Partial purification of 200 cc of variant packed erythrocytes with intact buffy coat, gave a 45 per cent yield of the enzyme. Storage at 4~ for twelve hours resulted in 80 per cent loss of specific activity. Standard vertical starch gel electrophoresis with an ethylenediaminetetracetic acid (EDTA) tris-borate buffer, pH 8.6, was supplemented by electrophoresis on cellulose acetate strips using an EBT system buffered to pH 9.1 as described by Sparkes et al. [21], with direct application of 10 lambda of partially purified enzyme (specific activity, delta 0.02 OD units per minute) to the cellulose acetate strips. Erythrocyte chromium sl survival, hematologic studies, erythrocyte enzyme assays, glutathione and Heinz body studies (reported as number of cells con-
125
G-6-PD WORCESTER ~
SNYDER ET AL.
taining 4 or more Heinz bodies after incubation with phenylhydrazine) were conducted according to previously published methods [22-25]. G-6-PD assay on crude hemolysate was carried out utilizing the method of Kornberg and Horecker [26], with the addition of 10 micromolar of triphosphopyridine nucleotide (TPN), 7 millimolar of 2-mercaptoethanol and 2.7 millimolar of neutralized EDTA to the hemolyzing solution and expressed as micromoles of T P N / I O 1~ red blood cells or white blood cells/minute. Graded estimations of G6-PD activity in the individual erythrocytes were made using Fairbanks' and Lampes' method [27]. Normal spinal fluid from nine male subjects, aged nineteen to seventy-two, with normal erythrocyte G-6-PD, and spinal fluid from the propositus, R.Z., were assayed for G-6-PD using a modification of the method of Kornberg and Horecker [26]. The results are expressed as international enzyme units per 100 cc of spinal fluid. All enzyme assays and kinetics were performed on a Beckman 'Kintrac '| with the temperature stabilized at 25~
one day a loss of vision in his right eye. At that time, superior temporal fields in the right eye and both upper quadrant fields in the left eye remained intact. Complete neurologic examination, including a roentgenogram of the skull, an electroencephalogram, brain scan, lumbar puncture, serologic test for syphilis (VDRL) and two hour postprandial blood sugar levels, were within normal limits. Gradual improvement in vision was noted in the right eye following a combination of ACTH and prednisone therapy. During a recent viral illness, the patient was admitted to the hospital with increasing jaundice and anemia. Physical examination revealed a thin Caucasian man with moderate pallor and conjunctival icterus. Eye examination showed normal extraocular movements, no evidence of nystagmus and normal pupillary reaction bilaterally. Funduscopic examination revealed bilateral, white pale atrophic discs with normal vessels and intact retinas. Visual fields showed marked peripheral field constriction with central scotomas. Results of the Ishihara test revealed total color blindness, but with recognition of red and blue on gross color vision. Visual acuity was 2 0 / 8 0 corrected; optical densiLy: finger counting only at 2 feet, right eye. Laboratory data (Table I) disclosed a hemoglobin of 12.1 gm per cent, hematocrit of 36 to 38 per cent, platelet count of 400,O00/cu mm, reticulocyte count of 8.8 per cent, white blood cell count of 11,000/cu mm with a normal differential. The peripheral smear showed marked aniso- and poikilocytosis, target cells, Howell-Jolly bodies and Heinz bodies in 90 per cent of the red cells counted. His serum creatinine, calcium, phosphorus, alkaline phosphatase were all within norreal limits. A three hour oral glucose tolerance test showed the fasting blood sugar to be 100 mg per cent, 202 mg per cent at 30 minutes, 220 mg per cent at 60 minutes, 118 mg
CASE REPORTS R.Z. The propositus, a twenty-six year old Caucasian man of French-Polish-German extraction, required transfusions soon after birth for an uncompensated hemolytic anemia with jaundice. During the first fifteen years of his life he received fifteen to twenty transfusions for numerous episodes of jaundice, dark colored urine and hemolytic anemia, precipitated by upper respiratory tract infections and aspirin ingestion. In 1957 (age fourteen) he underwent splenectomy with a subsequent decrease in his transfusion requirement. At the age of thirteen permanent blindness developed in his left eye over a period of three to four weeks, and at twenty-four years of age, after cholecystectomy, he experienced in
TABLE
I
Hematologic Studies on Selected Family Members
Subject Propositus 111-3 Son IV-4 Mother 11-6 Sister
Age(yr) and Sex
Packed Hemoglobin Cell Volume (gin/100 cc) (%)
Bilirubin (mg %) Total
Direct
Reticu10cytes (%)
Heinz Body (%)
Or57 T 1/2 (days)
1.4
8.8
90
...
3
G-6-PD* RBC
WBC
4.5
0
110
10
...
4.7
--
24, M
12.1
38
3.4
NB, M
16.0
55
12.1
56, F
10.4
33
1.0
0.6
5.6
30
...
0.88
215
28, F
15.2
46
0.8
0.6
0.8
15
...
2.6
350
70, F
10.0
30
1.8
0.6
6.2
31
. ..
1.18
--
48, F
12.2
36
2.6
1.3
1.8
33
...
1.7
235
15, M
10.8
33
5.0
1.2
10.8
45
...
0
120
19, M
11.6
35
5.4
2.7
9.2
92
...
0
130
111-2
Maternal aunt 11-13 Maternal first cousin 111-15 Maternal second cousin IV-8 Maternal second cousin IV-9
G-6-PD _-- #moles of TPN r e d u c e d / m i n u ; e / l O 1~ red blood ceils (RBC) at 25 ~ Values for twenty normal volunteer subjects = 2.38 to 3.91, mean 2.9. # moles of TPN r e d u c e d / m i n u t e / l O 10 white blood cells (WBC) at 25 ~ Values for twenty normal volunteer subjects ~- 245 to 495.
126
The American Journal of Medicine
G-6-PD WORCESTER ~
per cent at 120 minutes and 67 mg per cent at 180 minutes, without glycosuria. The serologic test for syphilis (VDRL), and direct and indirect Coombs' test were all nonreactive. Serum total bilirubin was 3.4 mg per cent with 114 mg per cent direct reacting. Serum glutamic oxalacetic acid transaminase (SGOT) was 65 units (normal 8 to 40 units, Reitman-Frankel method, modified). Serum glutamic pyruvic acid transaminase (SGPT) was 55 units (normal 5 to 30 units, ReitmanFrankel method, modified). Chest and skull roentgenograms, an electroencephalogram and lumbar puncture were all within normal limits. Serum iron was 192 mg per cent with 73 per cent saturation. A liver biopsy demonstrated mild to moderate hemosiderosis. A bone marrow aspirate showed intense erythroid hyperplasia and increased iron but it was otherwise within normal limits. Special hematologic data included an autohemolysis test result of 1.55 per cent (normal is 0 to 1.14 per cent), with added ~glucose 1.21 per cent (normal is 0 to 0.3 per cent), with added adenosine triphosphate (ATP) 1.18 per cent (normal is 0 to 0.75 per cent). Osmotic fragility (fresh and incubated) and the sugar water test were normal. T~e activity of red cell enzymes of the Embden-Myerhoff pathway from hexokinase to lactic dehydrogenase, ATP levels and the remaining hexose-monophosphate shunt enzymes, were either normal or increased. Assay of erythrocyte G-6-PD revealed no activity; the white cells contained 110 micromoles of TPN/1010 cells/minute. R.M.Z. The newborn son of the propositus was born after thirty-eight weeks gestation, complicated by premature rupture of the membranes forty-eight hours prior to delivery associated with endometritis. At birth, there was no evidence of hepatosplenomegaly. Apgar score was 9. Laboratory data included a hematocrit of 55 to 60 per cent, hemoglobin 16.5 gm per cent, white blood cells 12,000/cu mm with a normal differential for a newborn. Reticulocyte count was 3 per cent. Bilirubin in cord blood was 1.5 mg per cent initially. The Coombs test, direct and indirect, was negative. There was no evidence of ABO incompatability. Because of sepsis in the mother, the patient was treated with kanomycin and penicillin. On the third hospital day his serum bilirubin level rose to 6 mg per cent and then climbed to a high of 12 mg per cent by the sixth day of life. Red cell G-6-PD activity was 4.7 (normal for newborns, 4.1 to 8.28 with a mean of 5.4). By the eighth day the bilirubin had dropped to 6 mE per cent and the patient continued to do well clinically, his hemogram remaining stable. B.Z. The mother of the propositus, age fifty-six, has worn glasses for twenty-five years, but she has no ophthalmologic complaints. Physical examination was within normal limits. Laboratory data included a hemato@rit of 33 per cent, hemoglobin 10.4 gm per cent, reticulocyte count 5.6 per cent. Examination of red cell morphology showed normocytic, normochromic cells with a mild polychromasia. Heinz body preparation revealed 30 per cent positive red cells. The serum total bilirubin was 1.0 mg per cent with 0.6 mg per cent indirect reacting. Red cell G-6-PD activity was 0.88; white cell G-6-PD activity was 240. H.B. The maternal aunt, aged seventy, experienced sudden blindness of unknown etiology at the age of thirty-five years. She had a long history of anemia accompanied by one episode of jaundice associated with pneumococcal pneumonia. Physical examination was within normal limits except for her eyes, which demonstrated bilateral optic atrophy with com-
Volume 49, July 1970
SNYDER ET AL.
plete loss of vision in the left eye and visual acuity corrected to 2 0 / 7 0 in the right. Laboratory data included a hematocrit of 30 per cent, hemoglobin 10 gm per cent, reticulocyte count 6.2 per cent. Heinz bodies were found in 31 per cent of the red cells. Erythrocyte G-6-PD activity was 1.18. Serum total bilirubin was 1.8 mg per cent with 0.6 mg per cent direct reacting. O.P. A maternal first cousin, aged forty-eight, jaundiced shortly after birth, required transfusions at that time but none since. Examination revealed conjunctival icterus with a pale atrophic disc in the right eye, compatable with mild optic atrophy. Visual acuity in both eyes was 20/30 corrected. Laboratory data included a hematocrit of 36 per cent, hemoglobin 12.2 gm per cent, reticulocyte count 0.8 per cent; Heinz bodies were present in 33 per cent of the red cells. Erythrocyte G-6-PD activity was 1.7; white blood cell G-6-PD activity was 235. Serum total bilirubin was 2.6 mg per cent with 1.3 mg per cent direct reacting. D.P. A maternal second cousin, aged fifteen, son of O.P., had jaundice shortly after birth, for which he required exchange transfusions. He experienced one episode of jaundice as a child and recently was told that he was anemic. Physical examination revealed scleral icterus, bilaterally pale optic discs, with visual acuity reduced to 20/30 corrected bilaterally. There was mild constriction of the peripheral fields and a small central scotoma in the left eye. The spleen was palpated 3 cm below the left costal margin. Total color blindness was noted. Laboratory data included a hematocrit of 33 per cent, hemoglobin 10.8 gm per cent, reticulocyte count 10.8 per cent. A one hour postprandial blood sugar level was 110 mg per cent. Erythrocyte G-6-PD activity was absent and white cell G-6-PD activity was reduced to 120. Serum total bilirubin was 5.0 mg per cent with 1.2 mg per cent direct reacting. Heinz bodies were 45 per cent. L.P. A maternal second cousin, aged eighteen, son of O.P., was jaundiced after birth and required exchange transfusions; subsequently he had episodes of jaundice at three years of age. He had three hospitalizations between the ages of four and seven years for anemia and jaundice, and received four transfusions. No further transfusions were required after a splenectomy at the age of seven. At the age of ten sudden blindness developed in both eyes over a ten day to two week period, with gradual improvement over the next few months. Cholecystectomy was performed when he was eighteen years old. Examination revealed conjunctival icterus, bilaterally pale optic discs with reduced visual acuity to 2 0 / 2 0 0 in the right eye and 4 / 2 0 0 in the left (corrected), with bilateral central scotomas, severely restricted peripheral fields and total color blindness by the Ishihara test. Laboratory data included a hematocrit of 35 per cent, hemoglobin 11.6 gm per cent and a reticulocyte count of 9.2 per cent. Examination of red cell morphology showed anisocytosis and poikilocytosis, target cells and Howell-Jolly bodies. Heinz body preparation revealed 92 per cent positive cells. There was absent erythrocyte G-6-PD activity and white blood cell G-6-PD activity was reduced to 130. Serum total bilirubin was 5.4 mg per cent with 2.7 mg per cent direct reacting. One hour and two hour blood sugar levels after a 100 gm glucose load were 100 and 90 mg per cent, respectively. R ESU LTS Hematologic studies (Table I) show that the three affected male subjects have mild, chronic, nonspherocytic
127
G-6-PD WORCESTER - - SNYDER ET AL.
Ophthalmologic Findings in Selected Family Members
TABLE II
Color Blindness IshiharaTest
Visual Acuity Pedigree
Subject
III _-- 3
R.Z.
20/80 corrected
III _-- 1 II _-- 6
J.S. B.Z.
20/20 20/40 corrected 20/70 corrected
II ~ 13
H.B.
III _-- 15
O.P.
IV _-- 4 IV _-- 7
R.M.Z . L.P.
IV ---- 8
D.P.
O.D.
O.S.
20/30 corrected . . . . . 20/200 corrected
Finger Total - counting bilateral at 2 feet 20/20 ... 20/40 ... corrected Finger ... counting at 2 feet 20/30 ... corrected . . . . 4/100 Total (-- 1.00 = --1.25 X
120) 20/30
20/30
Total
hemolytic anemia associated with clinical jaundice; the propositus has a chromium 51 survival of four and a half days. In addition, the three heterozygotes have evidence of hemolysis as evidenced by reticulocytosis and bilirubinemia. The ophthalmologic findings (Table II) are characterized by acute or subacute onset of visual impairment occurring between ten and thirty-eight years of age. After the initial visual deterioration, a period of stabilization followed with subjective improvement in visual acuity. The fundi of those affected revealed pale, atrophic discs with varying degrees of central scotomas and restricted TABLE III
RBC(% Activity of MeanNormal)
peripheral vision correlating with the degree of clinical abnormality. In addition, total color blindness, as measured by the Ishihara test, was present in the three affected males. Cytochemical staining of the erythrocytes of the heterozygotes B.Z. and O.P. showed two distinct populations with 266 erythrocytes (55 per cent) possessing zero granules, 29 (5.8 per cent) with 1+ granules, 195 (39 per cent) with 2-t- granules and 10 (2 per cent) with 3 + granules (500 cells counted). The mode of inheritance for G-6-PD Worcester is typically sex-linked (Fig. 1), the heterozygotes displaying intermediate erythrocyte G-6-PD levels, two distinct cytochemical erythrocyte populations and two separate electrophoretic bands (each band reflecting the gene product of one x chromosome). The detailed family study allows discussion of the nature of the optic atrophy, its mode of inheritance and whether or not it is related to the same gene as G-6-PD Worcester. Biochemical results are shown in Table II1. The thermal stability of the variant enzyme was very low (Fig. 2). After incubation for forty minutes at 46~ all specific activity was lost. The electrophoretic mobility on starch gel was 86 per cent of the the normal B + enzyme (Fig. 3) whereas the partially purified heterozygote showed two distinct bands, a normal band migrating at 9 cm and a slower band migrating at 7.8 cm from the origin. On cellulose acetate, the difference in mobility was even more distinct, the variant displaying 70 per cent mobility of the normal B' enzyme. The pH optimum for G-6PD Worcester showed a sharp peak at 8 as compared with the normal truncate pattern with a broad peak at pH 9 (Fig. 4). The mean Michaelis constant (Kin) for glucose-6-r)hosphate (G6P) was 61 micromolar with a range of 58 to 62 micromolar. The mean K,,, for TPN was 11 micromolar with a range of 9 to 12.4 micromolar (Fig. 5). The utilization of 2-deoxy-glucose-6.phosphate
Biochemical Aspects of "G-6-PD Worcester" Electrophoretic Mobility (% of Normal)
Heat Lability
pH OPtimum
K., TPN (#M)
K,, G6P (#M)
2dG6P Utilization (% of G6P)
42-58
2
DTPN Utilization (% of TPN)
DPN Uti!ization (% of TPN)
N o r m a l (Five Subjects)
100
Normal
100
0
Very labile
Slow 70
9.0 Truncate
3.5-7.5
50-55
0
21
0
G-6-PD Worcester (Three Subjects)
86
EBTpH 9.1 cellulose acetate EBTpH 8.6 starch gel
8,0 Sharp
11.2
61
<2
peak
NOTE: Km TPN _-- Michaelis constant for triphosphopyridine nucleotide. Kn, G6P ---- Michaelis constant for glucose-6-phosphate. 2d G6P ----2 deoxyglucose-6-phosphate. DTPN ----diamotriphosphopyridine nucleotide. DPN = diphosphopyridine nucleotide.
128
The American Journal of Medicine
G-6-PD WORCESTER - - SNYDER ET AL.
as a substrate was less than 1 per cent for both the variant and normal; however the utilization of deaminoTPN was 21 per cent for the variant with a range of 50 to 55 per cent for the normal enzyme. The mean normal level of G-6-PD in spinal fluid was 33 I.U. per 100 cc of spinal fluid with a range of 15 to 60 I.U., standard deviation (S.D.I.) ___14, The propositus I
(R.Z.) had 20 I.U. of G-6-PD activity per 100 cc of spinal fluid. COMMENTS More than fifty distinct variants of erythrocyte G-6-PD have been recognized but few result in significant nonspherocytic hemolytic anemia [22, 2 8 - 3 0 ] , Types asso-
2
I
3
5
6
7
4
8
II
12
13
0
0
LEGEND (~ OBLIGATE CARRIER [] BILATERAL OPTIC ATROPHY AND RBC G-6-PD DEFICIENCY (~) PROVEN CARRIER STATE FOR OPTIC ATROPHY AND G-6-PD DEFICIENCY (~ []
DIED AT CHILDBIRTH DEAD, AGE 3, OF PNEUMONIA AND JAUNDICE
~}
ALIVE, NOI STUDIED, BUT PRESUMED NORMAL BY HISTORY
2}
DECEASED,BUT PRESUMED NORMAL BY HISTORY
SUBSCRIPT REPRESENTS ACTIVITY Ol~' G-6-PD ~MOLES OF TPN REDUCED/MINUTE/IOi~ RBC AT ~S'C
Family pedigree. G.6-PD deficiency, chronic nonspherocytic hemolytic anemia, optic atrophy. Fig. 1.
.05 I0 .04'
9
\
E
O
\ .o3
\ ~"NORMAL 8~ VARIANT
o
E8
\
<~
i'~'~ X
NORMAL
7
.01~
6
ORIGIN
20
40
T I ME (MINUTES) Fig. 2. Thermal stability exp r e s s e d as OD/minute (B) 46~ using dialyzed enzyme.
Volume 49, July 1970
I
1
I
2
I
3 Fig. 3. Electrophoresis. Vertical starch gel electrophoresis of partially purified G-6-PD Worcester, using EDTA, tris.borate buffer system, p H 8.6. Slot ] contains normal B" enzyme. S l o t 2 contains G-6-PD Worcester. Slot 3 contains heterozygote f o r G-6-PD Worcester.
129
G-6-PD WORCESTER - -
SNYOER F_.T At.
120
IO0
\
I
~80 G-6-PD WORCESTEF
-
-
60
NORMAL B G-6-PD
W > -
-
40
W
20
/ I
5.0
5.5
I
60
I
I
I
6.5
7D
Z5
I
80
I
I
[
8.5
90
9.5
i
I0.0
pH Fig. 4.
Effects of pH on rates of normal and variant G-6-PD.
Activity expressed relative to activity at pH 7.5 which is taken as 100 per cent. Each point represents mean of three assays on variant and mean of five normal.
ciated with hemolytic anemia include G-6-PD Chicago, Oklahoma, Duarte, Bangkok, Mediterranean, Albuquerque, Torrance and the less well defined "Eyssen," "Tubingen" and "Berlin." Completely absent or markedly diminished erythrocyte G-6-PD activity, as described in G-6-PD "Eyssen" [30], Albuquerque, Mediterranean and Torrance, is rare. Although G-6-PD "Union and Markham" show extremely low levels of G-6-PD, they are not associated with chronic hemolysis. The new variant, G6-PD Worcester, is similar to G-6-PD "Eyssen" with regard to its slow electrophoretic mobility and absent erythrocyte G-6-PD activity, but it can be distinguished by the Michaelis constant for TPN, thermal stability and pH optimum. G-6-PD Milwaukee, Panay, Seattle, West Bengal, Madrona, Kerala, Capetown, ~badan-Austin, Athens, Baltimore-Austin and TelHashomer all have slow electrophoretic mobility but have biochemically distinct Michaelis constants for TPN, pH optimum, thermal stability and only moderate to mild enzyme deftciency, differentiating them from G-6-PD Worcester. Complete absence of G-6-PD activity in a fifty-nine year old Caucasian man with nonspherocytic hemolytic anemia, optic atrophy and neurologic manifestations has beerx reported previously [31], but detailed family studies were not included, and the enzyme was not biochemically characterized. Cataracts have been described in the lenses of affected patients with G-6-PD deficiency but appear to be the result of a local disturbance of carbohydrate metabolism [11].
130
In the present family, the low G-6-PD enzyme levels i~, the white blood cells may reflect a more generalized deficiency, but the acute onset of the optic atrophy with a normal level of G-6-PD in the spinal fluid are against a cause and effect relationship. The lesions may be the resuit of a single gene abnormality, but the presence of two closely aligned genes of the same chromosome is more likely. Evidence of a diffuse neurologic disorder is required by some investigators [32] for the diagnosis of Leber's optic atrophy whereas others do not consider this necessary [16]. The diagnosis of Leber's optic atrophy in t h e present family is likely despite the absence of other neurologic manifestations. Behr's optic atrophy [16,17] is excluded in view of the age of onset, type of inheritance and the absence of ataxia with dementia. Autosomal recessive optic atrophy usually presents in early infancy and autosomal dominant type in early childhood, and both have a more benign clinical course [33]. Optic atrophy with Friedreich's ataxia and spastic paraplegia is overshadowed by the other neurologic abnormalities [17]. The hematologic and ophthalmologic defects both appear to be due to mutations on the x chromosome. It is too early to tell if the male patients have affected grandsons through their daughters, but of the eight sons of the three proved carriers three are affected (we may consider this 50 per cent for purposes of discussion). This indicates either x-chromosome linkage or autosomal dominant inheritance with reduced penetrance in the male. The close linkage with G-6-PD deficiency implies probable x-chromosome inheritance for the optic atrophy. Both defects appear to be carried on the same chromosome, since in the twelve offspring, all are either 220L-
180 =
160
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140
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f
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o
/:
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60 40 2(
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0
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20 30 40 50 60 70 80 90 I00 SUBSTRATE (micromolar) Fig. 5. Michaelis constant for G-6-PD Worcester with TPN. Hatched area represents range of five normal B" partially purified G-6-PD.
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G-6-PO WORCESTER- - SNYDER ET AL
completely normal or exhibit both defects. Only with continued study of subsequent generations will these questions be truly resolved.
ACKNOWLEDGMENT We wish to express our appreciation to Dr. E. Beutler, City of Hope Medical Center, Dr. R. E. Sage, Tufts Uni-
versity and Dr. A. Yoshida, University of Washington, for reviewing this manuscript and for their helpful criticisms. We are also indebted to Dr. S. Murphy and Dr. E. Epstein for performing the neurologic and ophthalmologic examinations in this family and to Dr. R. Abodeely for allowing us to study the propositus, R.Z. Our thanks also to Pamela Bohn for secretarial assistance.
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14. Gartler SM, Gandini E, Ceppellini R: Glucose-6-phosphate dehydrogenase deficient mutant in human cell culture. Nature (London) 193: 602, 1962. 15. Chan TK, Todd D, Wong CC: Tissue enzyme levels in erythrocyte glucose-6-phosphate dehydrogenase deficiency. J Lab Clin Med 66: 937-942, 1965. 16. Waardenburg PJ, Franceschetti P, Klein D: Genetics and Ophthalmology, vol. 2, Assen, Holland, Royal Gorcum & Co, 1963, p 1617. 17. Bereday M, Cobb S: Relation of hereditary optic atrophy (Leber) to other familial degenerative diseases of the central nervous system. Arch Ophthal (Chicago) 48: 669-680. 18. McKusick VA: On the X-chromosome of man. Quart Rev Biol 37: 69-175, 1962. 19. Beutler E, Mathai CK, Smith JE: Biochemical variants of glucose-6-phosphate dehydrogenase giving rise to congenital non-spherocytic hemolytic disease. Blood 31: 131-150, 1968. 20. World Health Organization Technical Report Series: Standardization of procedures for the study of glucose-6-phospha'e dehydrogenase. WHO Report, 1967, No. 366: 5-53. 21. Sparkes RS, Baluda MC, Townsend DE: Cellulose acetate electrophoresis of human glucose-6-phosphate dehydrogenase. J Lab Clin Med 73: 531-534, 1969. 22. Tanaka KR, Valentine WN, Miwa S: Pyruvate kinase (PK) deficiency hereditary nonspherocytic hemolytic anemia. Blood 19: 267, 1962. 23. Koutras GA, Hattori M, Schneider AS, Ebaugh FJ Jr, Valentine WN: Studies on chromated erythrocytes. Effect of sodium chromate on erythrocyte glutathione reductase. J Clin Invest 43: 323, 1964. 24. Beutler E, Duron O, Kelly BM: Improved method for the determination of blood glutathione. J Lab Clin Med 63: 715, 1963. 25. Beutler E, Dern RJ, AIving AS: The hemolytic effect of primaquine. Vl. An in vitro test for sensitivity of erythrocytes to primaquine. J Lab Clin Med 45: 40, 1955. 26. Kornberg A, Horecker BL: Glucose-6-phosphate dehydrogenase. Methods of Enzymology, Vol I (Colowick SP and Kaplan NO, ed) New York, Academic Press, 1955, p 323. 27. Fairbanks VF, Lampe LT: A tetrazolium-tinked cytochemical method for estimation of glucose-6-phosphate dehydrogenase activity in individual erythrocytes: applications in the study of heterozygotes for glucose6-phosphate dehydrogenase deficiency. Blood 31: 589--602, 1968. 28. Motulsky AG, Yoshida A: Methods for the study of red cell glucose-6-phosphate dehydrogenase. Biochemi-
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cal Methods in Red Cell Genetics (Yunis J J, ed) New York, Academic Press, 1969, p 52--65. 29. Giblett ER: Glucose-6-phosphate dehydrogenase. Genetic Markers in Human Blood, Philadelphia, F.A. Davis Co., 1969, p 453. 30. Boyer SH, Porter IH, Weilbacher RG: Electrophoretic heterogeneity of glucose-6-phosphate dehydrogenase and its relationship to enzyme deficiency in man. Proc Nat Acad Sci USA 48: 1868, 1962.
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31. Escobar MA, Heller P, Trobaugh FE: "Complete" erythrocyte glucose-6-phosphate dehydrogenase deficiency. Arch Intern Med 113: 428, 1964. 32. Wilson J: Leber's hereditary optic atrophy--some clinical and aetiological considerations. Brain 86: 3 4 7 362, 1963. 33. Kjer P: Infantile optic atrophy with dominant mode of inheritance. Acta Ophthal (Kobenhavn) supp 54, p 1, 1959.
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