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Congenital nonspherocytic hemolytic anemia due to glucose-6-phosphate dehydrogenase East Harlem: A new deficient variant
Volume 90 Number 1
4. Granoff DM, and Roskes S: Urinary tract infection due to Haemophilus influenzae type b, J PEDIATR84:414, 1974. 5. Doolittle KH, et al: Epididymitis in the prepubertal boy, J Urol 96:364, 1966. 6. Haahr J, et al: Epididymitis in children, Acta Paediatr Scand 60:16, 248, 1971. 7. McDonald JH, et al: Acute pneumococcal epididymitis, Ill Med J 95:304, 1949.
Congenital nonspherocytic hemolytic anemia due to glucose-6-phosphate dehydrogenase East Harlem: A new deficient variant Robert Feldman, M.D.,* Donald S. Gromisch, M.D., A. Leonard Luhby, M.D., New York,
N.Y., and Ernest Beutler, M.D., Duarte, Calif.
G L U C O S E - 6 - P H O S P H A T E DEHYDROGENASE was the first enzyme to be identified as abnormal in congenital nonspherocytic hemolytic anemia'; it is probably also the most frequently encountered enzymatic defect in this disorder. Although occasionally found in patients who have the c o m m o n Mediterranean type of G-6-PD deftciency,'-' C N S H A is usually associated with rare structural mutants of G-6-PD which can be characterized on the basis of their altered biochemical and kinetic properties. With very few exceptions, the reported cases of rare variants affect Caucasians of non-Mediterranean origin. A m o n g blacks, so far only four of these "rare variants" have been described. Another variant, differing distinctly from those previously reported, is described in this report. From the Department of Pediatrics, New York Medical College-Metropolitan Medical Center, and the Department of Medicine, City of Hope Medical Center. *Reprint address: Department of Pediatrics, Metropolitan Hospital, Room 505, 1901 First Ave.. New York, NY 10029.
Brief clinical and laboratory observations
89
8. Davis WH, and Scardino PL: Meningitis presenting as epididymitis, Southern Med J 65:9360, 1972. 9. DeLeon W: Report of a case of meningitis, arthritis, and epididymitis due to mixed Pfeiffer bacillus and pneumococcus infection, J Phillipine Islands Med Assn 1:195, 1921. 10. Samellas W: Acute epididymo-orchitis associated with bacteremic shock, J Urol 89:370, 1963.
CASE REPORT Patient D.O., a black boy, was born prematurely at home and was admitted to the Intensive Care Nursery. Weight was 1,473 gm; length was 34 cm. On admission he was in good general condition and had the following laboratory findings: hemoglobin 17.3 gm/dl, hematocrit 52%, red cells 6,000,000, reticulocytes 21%. His and his mother's blood type were A Rh-positive; his direct Coombs test was negative. On the third day of life he was markedly jaundiced with a total serum bilirubin of 15.6 mg/dl with 1 mg/dl direct reacting. Hemoglobin was 17.4 gm/dl, reticulocytes were 20%, and nucleated red cells were 28 per 100 leukocytes. By the seventh day, the bilirubin concentration had receded to 3.4 mg/ dl. However, at the age of one month he was markedly pale and had a firm, enlarged spleen (3 cm below left costal margin). There was no jaundice. His hemoglobin had fallen to 7.2 gm/dl and he had 25% reticulocytes. The stained blood smear showed moderate aniso-poikilocytosis, marked polychromasia, and absence of spherocytes. Abbreviations used G-6-PH: glucose-6-phosphate dehydrogenase CNSHA: congenital nonspherocytic hemolytic anemia Hgb: hemoglobulin G-6-P: glucose-6-phosphate NADP: nicotinamide adenine dinucleotide phosphate A sickling test was negative. However, a screening test for G-6PD deficiency (dye decolorization test) was strongly positive on two occasions. The bone marrow showed marked erythroid hyperplasia, with 52.8% of all nucleated cells belonging to the erythroid series. During the remaining hospital stay, he continued to develop normally but his hemoglobin and reticulocytes fluctuated within a range of 8.0 to 10.0 gm/dl and 5.0 to 15.0%, respectively. He was discharged at the age of 4% months, to be followed in the outpatient clinic. At 20 months his weight and height corresponded to that of a premature infant with satisfactory development. Motor and mental development was normal. Roentgen survey showed normal bone age and none of the bone changes frequently associated with severe chronic hemolysis. Hemoglobin was 9.0 gm/dl, red cells 2,880,000, reticulocytes 7%, hematocrit 26.0, mean corpuscular volume 90 tt~, mean corpuscular hemoglobin
Brief clinical and laboratory observations
90
The Journal of Pediatrics January 1977
Table I
Variant
RBC enzyme activity (% of normal)
Electrophoretic mobility (% of normal)
B
100
East Harlem
l0
Km G-6-P
Deamino NADP utilization (% of NADP rate)
(#M)
K'n 2dG-6-P NADP utilization (% (l~M) of G-6-P rate)
100
50-70
2.9-4.4
4
55-60
Normal
104 (EBT) 102 (Tris-HC1) 102 (PO4)
107
8.2
2.1
34.5
Moderately thermolabile
30 #/~g, mean corpuscular hemoglobin concentration 34%, white cells 6,100 with 72% lymphocytes, 24% neutrophiles, and 4% monocytes; the platelet count was 360,000. On the smear there was aniso-poikilocytosis, polychromasia, and absence of spherocytes. The bone marrow again showed erythroid hyperplasia with a myeloid:erythroid ratio of 1.2:1. Serum and whole blood folate and serum vitamin B,~ were normal, serum iron was 102 mg/dl, and total iron-binding capacity was 237 mg/dl. Osmotic red cell fragility without and with incubation was normal; the 5*Cr red cell half-life was 19.5 days. A test for formation of Heinz bodies after incubation with acetyl phenylhydrazine was strongly positive. Serum bilirubin was 2.4 mg/dl (all indirect reacting), hemoglobin electrophoresis showed Hgb A. Hgb F, by the alkali denaturation method was 4% and a heat stability test for unstable hemoglobin was normal. At the age of two years, he looked somewhat pale, his spleen was enlarged (palpable), his Hgb was 9.0 gm/dl, red blood cells 3,100,000 reticulocytes 8%. The maternal grandmother, the only family member available for study, had a hemoglobin of 13.0 gm/dl, a reticulocyte count of 0.5%, and a normal blood smear. There was no history of jaundice or chronic anemia in the family. Biochemical and kinetic studies. Partial purification and characterization of the enzyme was carried out by the WHO standard methods ~as described in detail elsewhere? RESULTS Enzyme activity in the red cells was 0.56 U / g m Hgb, approximately 10% of mean normal. The enzyme was electrophoretically slightly rapid, giving a mobility of 104% of normal on the EDTA-borate-tris system, 102% of normal in the tris HC1 system, and 102% of normal in the phosphate-buffered system. The K,n NADP, measured spectrophotometrically, was slightly elevated to 8.2 gM, while the Km G-6-P was considerably elevated to 107/~M. The deamino N A D P utilization was moderately decreased to 34.5% of N A D P utilization. 2-Deoxy G-6-P utilization was normal, at 2.1% of G-6-P utilization. The pH activity curve was slightly more peaked than normal with an optimum at the 8.5 to 9 range. The enzyme was moderately thermolabile, with 45% of activity being lost on heating at 46 ~ for 20 minutes, compared with 12.6% loss of control enzyme activity under identical conditions.
Thermal stability
pH optima Normal (truncate) 8.5-9.0
The characteristics of the enzyme are summarized in Table I. DISCUSSION Glucose-6-phosphate dehydrogenase deficiency is perhaps the most c o m m o n cause of congenital nonspherocytic hemolytic anemia, ranking close to pyruvate kinase deficiency as a cause of this disorder. Elsewhere '~ we have summarized data regarding 24 variants of G-6-PD which have been found to be associated with this clinical disorder. Only a few occurred in Negro families: G-6-PD Charleston, ~ G-6-PD Ohio, 7 and G-6-PD San Diego? In addition, a previously u n n a m e d variant (now designated G-6-PD Lawndale) resulting in chronic hemolysis was reported by Grossman and associates? The variant described in this paper, which we have designated G-6-PD East Harlem, is probably unique."' Of the variants which have been associated with nonspherocytic congenital hemolytic anemia only Ohio, Torrance, Charleston, San Diego, and Lawndale are electrophoretically fast. With the possible exception of G-6-PD Lawndale, the characteristics of these abnormal enzymes are very different from those of G-6-PD East Harlem. However, G-6-PD Lawndale had no detectable activity in the erythrocytes, whereas RBC G-6-PD East Harlem was almost 10% of normal activity. Furthermore, the Km for this enzyme for N A D P and for glucose-6-P was almost twice that which we have found for G-6-PD East Harlem. Finally, G-6-PD East Harlem is only mildly thermolabile, whereas Lawndale is extremely thermolabile. Indeed, G-6-PD Lawndale closely resembles G-6-PD Oklahoma, except for its morerapid-than-normal electrophoretic mobility.
REFERENCES
1. Newton WA, and Frajole WJ: Drug sensitive chronic hemolytic anemia. Family studies, Clin Res 6:392, 1955. 2. Ben-Ischay D, and Izak G: Chronic Hemolysis associated with glucose-6-phosphate dehydrogenase deficiency in Israel, J Lab Clin Med 63:1002, 1964. 3. WHO Scientific Group: Standardization of procedures for
Volume 90 Number 1
Brief clinical and laboratory observations
the study of glucose-6-phosphate dehydrogenase. WHO Tech Rep Ser 366, Geneva, 1967. 4. Beutler E, Mathai CK, and Smith JE: Biochemical variants of glucose-6-phosphate dehydrogenase giving rise to congenital nonspherocytic hemolytic disease, Blood 31:131, 1968. 5. Beutler E: Disorders due to enzyme defects in the red blood cell, in Adv Metab Disord 6:131, 1972. 6. Beutler E, Grooms AM, Morgan SK, and Trinidad F: Chronic severe hemolytic anemia due to G-6-PD Charleston: A new deficient variant, J PEDIATR 80:1005, 1972. 7. Pinto PVC, Newton WA Jr, and Richardson KE: Evidence for four types of erythrocyte glucose-6-phosphate dehydro-
91
genase from G-6-PD deficient human subjects, J Clin Invest 45:823, 1966. 8. Howell EB, Nelson AJ, and Jones OW: A new G-6-PD variant associated with chronic non-spherocytic haemolytic anaemia in a Negro family, J Med Genet 9:160, 1972. 9. Grossman A, Ramanathan K, Justice P, Gordon J, Shahidi NT, and Hsia D: Congenital nonspherocytic hemolytic anemia associated with erythrocyte G-6-PD deficiency in a Negro family, Pediatrics 37:624, 1966. 10. Feldman R et al: G-6-PD East Harlem, unpublished data, Cited in Beutler E, and Yoshida A: Human G-6-PD variants: a supplementary tabulation, Ann Hum Genet (London) 37:151, 1973.
Popliteal pterygium syndrome: Report of a
m a n a g e m e n t o f a b a b y with cleft lip or p a l a t e ? W e wish to describe a black family in which two affected siblings were b o r n to a father with cleft lip a n d palate. These defects are part o f a s y n d r o m e which includes popliteal webbing. This syndrome, recognized as far back as 18695 is an autosomal d o m i n a n t trait a n d carries a 50% risk o f recurrence.
family Gary S. Frohlieh, B.A., M.S., Kathryn L. Starzer, A.B., M.S., and
CASE REPORTS
John M. Tortora, M.D., Brooklyn, N.Y.
Case 1. Baby D.3, a two-day-old girl weighing 2,466 gm, was referred after birth for consultation concerning various congenital anomalies. These anomalies included a posterior cleft palate and popliteal pterygium (Fig. 1). In addition, the nails of the great toes of the left foot and the great and second toe on the right foot were hypoplastic. At four months Baby D.3 was admitted for corrective surgery of the popliteal web. Examination of the genitals at this time showed bilateral hypoplastic labia majora with normal size labia minora and clitoris. The infant had a posterior cleft palate 2 cm long, involving the soft and posterior aspect of the hard palate. The popliteal web of the right leg was described as a dense
CLEFT LIP AND PALATE are a m o n g the m o s t c o m m o n congenital m a l f o r m a t i o n s detectable u p o n surface examination at birth. Usually cleft lip a n d palate occur in otherwise n o r m a l children, with a recurrence risk o f less than 10%. However, cleft lip or palate m a y occur as part o f a s y n d r o m e in which the recurrence risk is m u c h higher. Genetic counseling for the parents is a part o f the
From the Department of Obstetrics and Gynecology The Brooklyn-Cumberland Medical Center.
Fig. 1.
4. Granoff DM, and Roskes S: Urinary tract infection due to Haemophilus influenzae type b, J PEDIATR84:414, 1974. 5. Doolittle KH, et al: Epididymitis in the prepubertal boy, J Urol 96:364, 1966. 6. Haahr J, et al: Epididymitis in children, Acta Paediatr Scand 60:16, 248, 1971. 7. McDonald JH, et al: Acute pneumococcal epididymitis, Ill Med J 95:304, 1949.
Congenital nonspherocytic hemolytic anemia due to glucose-6-phosphate dehydrogenase East Harlem: A new deficient variant Robert Feldman, M.D.,* Donald S. Gromisch, M.D., A. Leonard Luhby, M.D., New York,
N.Y., and Ernest Beutler, M.D., Duarte, Calif.
G L U C O S E - 6 - P H O S P H A T E DEHYDROGENASE was the first enzyme to be identified as abnormal in congenital nonspherocytic hemolytic anemia'; it is probably also the most frequently encountered enzymatic defect in this disorder. Although occasionally found in patients who have the c o m m o n Mediterranean type of G-6-PD deftciency,'-' C N S H A is usually associated with rare structural mutants of G-6-PD which can be characterized on the basis of their altered biochemical and kinetic properties. With very few exceptions, the reported cases of rare variants affect Caucasians of non-Mediterranean origin. A m o n g blacks, so far only four of these "rare variants" have been described. Another variant, differing distinctly from those previously reported, is described in this report. From the Department of Pediatrics, New York Medical College-Metropolitan Medical Center, and the Department of Medicine, City of Hope Medical Center. *Reprint address: Department of Pediatrics, Metropolitan Hospital, Room 505, 1901 First Ave.. New York, NY 10029.
Brief clinical and laboratory observations
89
8. Davis WH, and Scardino PL: Meningitis presenting as epididymitis, Southern Med J 65:9360, 1972. 9. DeLeon W: Report of a case of meningitis, arthritis, and epididymitis due to mixed Pfeiffer bacillus and pneumococcus infection, J Phillipine Islands Med Assn 1:195, 1921. 10. Samellas W: Acute epididymo-orchitis associated with bacteremic shock, J Urol 89:370, 1963.
CASE REPORT Patient D.O., a black boy, was born prematurely at home and was admitted to the Intensive Care Nursery. Weight was 1,473 gm; length was 34 cm. On admission he was in good general condition and had the following laboratory findings: hemoglobin 17.3 gm/dl, hematocrit 52%, red cells 6,000,000, reticulocytes 21%. His and his mother's blood type were A Rh-positive; his direct Coombs test was negative. On the third day of life he was markedly jaundiced with a total serum bilirubin of 15.6 mg/dl with 1 mg/dl direct reacting. Hemoglobin was 17.4 gm/dl, reticulocytes were 20%, and nucleated red cells were 28 per 100 leukocytes. By the seventh day, the bilirubin concentration had receded to 3.4 mg/ dl. However, at the age of one month he was markedly pale and had a firm, enlarged spleen (3 cm below left costal margin). There was no jaundice. His hemoglobin had fallen to 7.2 gm/dl and he had 25% reticulocytes. The stained blood smear showed moderate aniso-poikilocytosis, marked polychromasia, and absence of spherocytes. Abbreviations used G-6-PH: glucose-6-phosphate dehydrogenase CNSHA: congenital nonspherocytic hemolytic anemia Hgb: hemoglobulin G-6-P: glucose-6-phosphate NADP: nicotinamide adenine dinucleotide phosphate A sickling test was negative. However, a screening test for G-6PD deficiency (dye decolorization test) was strongly positive on two occasions. The bone marrow showed marked erythroid hyperplasia, with 52.8% of all nucleated cells belonging to the erythroid series. During the remaining hospital stay, he continued to develop normally but his hemoglobin and reticulocytes fluctuated within a range of 8.0 to 10.0 gm/dl and 5.0 to 15.0%, respectively. He was discharged at the age of 4% months, to be followed in the outpatient clinic. At 20 months his weight and height corresponded to that of a premature infant with satisfactory development. Motor and mental development was normal. Roentgen survey showed normal bone age and none of the bone changes frequently associated with severe chronic hemolysis. Hemoglobin was 9.0 gm/dl, red cells 2,880,000, reticulocytes 7%, hematocrit 26.0, mean corpuscular volume 90 tt~, mean corpuscular hemoglobin
Brief clinical and laboratory observations
90
The Journal of Pediatrics January 1977
Table I
Variant
RBC enzyme activity (% of normal)
Electrophoretic mobility (% of normal)
B
100
East Harlem
l0
Km G-6-P
Deamino NADP utilization (% of NADP rate)
(#M)
K'n 2dG-6-P NADP utilization (% (l~M) of G-6-P rate)
100
50-70
2.9-4.4
4
55-60
Normal
104 (EBT) 102 (Tris-HC1) 102 (PO4)
107
8.2
2.1
34.5
Moderately thermolabile
30 #/~g, mean corpuscular hemoglobin concentration 34%, white cells 6,100 with 72% lymphocytes, 24% neutrophiles, and 4% monocytes; the platelet count was 360,000. On the smear there was aniso-poikilocytosis, polychromasia, and absence of spherocytes. The bone marrow again showed erythroid hyperplasia with a myeloid:erythroid ratio of 1.2:1. Serum and whole blood folate and serum vitamin B,~ were normal, serum iron was 102 mg/dl, and total iron-binding capacity was 237 mg/dl. Osmotic red cell fragility without and with incubation was normal; the 5*Cr red cell half-life was 19.5 days. A test for formation of Heinz bodies after incubation with acetyl phenylhydrazine was strongly positive. Serum bilirubin was 2.4 mg/dl (all indirect reacting), hemoglobin electrophoresis showed Hgb A. Hgb F, by the alkali denaturation method was 4% and a heat stability test for unstable hemoglobin was normal. At the age of two years, he looked somewhat pale, his spleen was enlarged (palpable), his Hgb was 9.0 gm/dl, red blood cells 3,100,000 reticulocytes 8%. The maternal grandmother, the only family member available for study, had a hemoglobin of 13.0 gm/dl, a reticulocyte count of 0.5%, and a normal blood smear. There was no history of jaundice or chronic anemia in the family. Biochemical and kinetic studies. Partial purification and characterization of the enzyme was carried out by the WHO standard methods ~as described in detail elsewhere? RESULTS Enzyme activity in the red cells was 0.56 U / g m Hgb, approximately 10% of mean normal. The enzyme was electrophoretically slightly rapid, giving a mobility of 104% of normal on the EDTA-borate-tris system, 102% of normal in the tris HC1 system, and 102% of normal in the phosphate-buffered system. The K,n NADP, measured spectrophotometrically, was slightly elevated to 8.2 gM, while the Km G-6-P was considerably elevated to 107/~M. The deamino N A D P utilization was moderately decreased to 34.5% of N A D P utilization. 2-Deoxy G-6-P utilization was normal, at 2.1% of G-6-P utilization. The pH activity curve was slightly more peaked than normal with an optimum at the 8.5 to 9 range. The enzyme was moderately thermolabile, with 45% of activity being lost on heating at 46 ~ for 20 minutes, compared with 12.6% loss of control enzyme activity under identical conditions.
Thermal stability
pH optima Normal (truncate) 8.5-9.0
The characteristics of the enzyme are summarized in Table I. DISCUSSION Glucose-6-phosphate dehydrogenase deficiency is perhaps the most c o m m o n cause of congenital nonspherocytic hemolytic anemia, ranking close to pyruvate kinase deficiency as a cause of this disorder. Elsewhere '~ we have summarized data regarding 24 variants of G-6-PD which have been found to be associated with this clinical disorder. Only a few occurred in Negro families: G-6-PD Charleston, ~ G-6-PD Ohio, 7 and G-6-PD San Diego? In addition, a previously u n n a m e d variant (now designated G-6-PD Lawndale) resulting in chronic hemolysis was reported by Grossman and associates? The variant described in this paper, which we have designated G-6-PD East Harlem, is probably unique."' Of the variants which have been associated with nonspherocytic congenital hemolytic anemia only Ohio, Torrance, Charleston, San Diego, and Lawndale are electrophoretically fast. With the possible exception of G-6-PD Lawndale, the characteristics of these abnormal enzymes are very different from those of G-6-PD East Harlem. However, G-6-PD Lawndale had no detectable activity in the erythrocytes, whereas RBC G-6-PD East Harlem was almost 10% of normal activity. Furthermore, the Km for this enzyme for N A D P and for glucose-6-P was almost twice that which we have found for G-6-PD East Harlem. Finally, G-6-PD East Harlem is only mildly thermolabile, whereas Lawndale is extremely thermolabile. Indeed, G-6-PD Lawndale closely resembles G-6-PD Oklahoma, except for its morerapid-than-normal electrophoretic mobility.
REFERENCES
1. Newton WA, and Frajole WJ: Drug sensitive chronic hemolytic anemia. Family studies, Clin Res 6:392, 1955. 2. Ben-Ischay D, and Izak G: Chronic Hemolysis associated with glucose-6-phosphate dehydrogenase deficiency in Israel, J Lab Clin Med 63:1002, 1964. 3. WHO Scientific Group: Standardization of procedures for
Volume 90 Number 1
Brief clinical and laboratory observations
the study of glucose-6-phosphate dehydrogenase. WHO Tech Rep Ser 366, Geneva, 1967. 4. Beutler E, Mathai CK, and Smith JE: Biochemical variants of glucose-6-phosphate dehydrogenase giving rise to congenital nonspherocytic hemolytic disease, Blood 31:131, 1968. 5. Beutler E: Disorders due to enzyme defects in the red blood cell, in Adv Metab Disord 6:131, 1972. 6. Beutler E, Grooms AM, Morgan SK, and Trinidad F: Chronic severe hemolytic anemia due to G-6-PD Charleston: A new deficient variant, J PEDIATR 80:1005, 1972. 7. Pinto PVC, Newton WA Jr, and Richardson KE: Evidence for four types of erythrocyte glucose-6-phosphate dehydro-
91
genase from G-6-PD deficient human subjects, J Clin Invest 45:823, 1966. 8. Howell EB, Nelson AJ, and Jones OW: A new G-6-PD variant associated with chronic non-spherocytic haemolytic anaemia in a Negro family, J Med Genet 9:160, 1972. 9. Grossman A, Ramanathan K, Justice P, Gordon J, Shahidi NT, and Hsia D: Congenital nonspherocytic hemolytic anemia associated with erythrocyte G-6-PD deficiency in a Negro family, Pediatrics 37:624, 1966. 10. Feldman R et al: G-6-PD East Harlem, unpublished data, Cited in Beutler E, and Yoshida A: Human G-6-PD variants: a supplementary tabulation, Ann Hum Genet (London) 37:151, 1973.
Popliteal pterygium syndrome: Report of a
m a n a g e m e n t o f a b a b y with cleft lip or p a l a t e ? W e wish to describe a black family in which two affected siblings were b o r n to a father with cleft lip a n d palate. These defects are part o f a s y n d r o m e which includes popliteal webbing. This syndrome, recognized as far back as 18695 is an autosomal d o m i n a n t trait a n d carries a 50% risk o f recurrence.
family Gary S. Frohlieh, B.A., M.S., Kathryn L. Starzer, A.B., M.S., and
CASE REPORTS
John M. Tortora, M.D., Brooklyn, N.Y.
Case 1. Baby D.3, a two-day-old girl weighing 2,466 gm, was referred after birth for consultation concerning various congenital anomalies. These anomalies included a posterior cleft palate and popliteal pterygium (Fig. 1). In addition, the nails of the great toes of the left foot and the great and second toe on the right foot were hypoplastic. At four months Baby D.3 was admitted for corrective surgery of the popliteal web. Examination of the genitals at this time showed bilateral hypoplastic labia majora with normal size labia minora and clitoris. The infant had a posterior cleft palate 2 cm long, involving the soft and posterior aspect of the hard palate. The popliteal web of the right leg was described as a dense
CLEFT LIP AND PALATE are a m o n g the m o s t c o m m o n congenital m a l f o r m a t i o n s detectable u p o n surface examination at birth. Usually cleft lip a n d palate occur in otherwise n o r m a l children, with a recurrence risk o f less than 10%. However, cleft lip or palate m a y occur as part o f a s y n d r o m e in which the recurrence risk is m u c h higher. Genetic counseling for the parents is a part o f the
From the Department of Obstetrics and Gynecology The Brooklyn-Cumberland Medical Center.
Fig. 1.