Am J Hum Genet 28:482-488, 1976


Hemophilia has been regarded for many years as the archetype for X-linked recessive inheritance in man. Blood coagulation research in recent years has revealed, however, that the genetic situation is more complex than originally suspected. The first intimation came in 1952 with the discovery of Christmas disease, now hemophilia B, an X-linked type of hemophilia distinguishable from the classical type or hemophilia A by laboratory tests. Since they are distinct phenotypes, the two disorders were presumed from the beginning to be nonallelic, a fact since documented by both genetic and biochemical means. Soon after the discovery of hemophilia B, it became apparent that abnormalities of factor VIII, the factor defective in hemophilia A, could be produced by mutation at several loci. First, it was discovered that deficiency of factor VIII was one feature of a disorder transmitted as an autosomal dominant and now known as von Willebrand syndrome (vWs). Shortly afterwards, an autosomal recessive syndrome, characterized by combined deficiencies of factors VIII and V, was observed. More recently, additional methods of studying hemophilia have revealed that there are many, presumably allelic, vWs phenotypes, although genetic evidence for allelism is nonexistent. A recent review contains details of most of these discoveries [1]. Quite recently, dominant hemophilia A has been reported [2]. This complex genetic picture of the inheritance of factor VIII has been complemented by an increasingly sophisticated understanding of the various factor VIIIrelated activities and their structural correlates [ 1-3 ]. To cope with this complexity a genetic nomenclature was adopted in 1973 [4], and additional phenotypic nomenclature has been tentatively suggested for the several factor VIII-related activities. A task force still receiving suggestions from the scientists concerned has suggested that the factor VIII coagulant activity be referred to as VIII:C, the related antigenic activities as VIIIR:AG, and the activity related to platelet aggregation and adhesion (which is deficient or defective in vWs) as VIIIR:WF [5]. The recent discoveries and designations of the multiple factor VIII-related Received January 8, 1976; revised February 23, 1976. This work was supported in part by research grants HL-06350, RCDA GM-31,732, and GM-16,697 from the U.S. Public Health Service. 1 Program in Hemostasis and Curriculum in Genetics, University of North Carolina, Chapel Hill, North Carolina. 2 The Clinical Pathology Division, Department of Pathology, University of Alabama Medical Center, Birmingham, Alabama. o 1976 by the American Society of Human Genetics. All rights reserved.




activities are of considerable importance to genetic counselors because they have led to effective laboratory tests both for the more accurate diagnosis of vWs and the direct detection of heterozygotes for hemophilia A [6-8]. Current data suggest that hemophilia carriers can be positively identified with high confidence, but that exclusion of the carrier state is hazardous. It appears that as many as 20%o of heterozygotes may be "concealed" (i.e., have normal phenotypes presumably as the result of the vagaries of X-chromosome inactivation [9]). This paper describes three sisters who are obligatory carriers of hemophilia A because their father is an hereditary hemophiliac. When studied by current methods, they exhibited the complete range of phenotypes possible for heterozygotes. One is phenotypically normal but has an affected son. Another has the laboratory findings typical of heterozygotes but is childless. The phenotype of the third, clinically a bleeder, is almost indistinguishable from that of her affected male relatives. MATERIALS AND METHODS

Blood for coagulation studies was obtained from the antecubital vein by syringe and mixed 9:1 with 3.2% sodium citrate solution. After centrifugation at 1,000 g, samples of plasma were divided into 1 ml aliquots, quickly frozen at -70C, stored for transport in solid C02, and then transferred to a Revco freezer at -70'C until assayed. Factor VIII-related activities were assessed by the following four methods. 1. Coagulant factor VIII (VIII:C) was determined on a freshly thawed sample of plasma by a one-stage assay procedure utilizing hemophilic plasma as substrate and "Thrombofax" as the partial thromboplastin (Ortho Diagnostics, Inc., Raritan, N.J.) [10]. The control was the pooled standard plasma described below. 2. Antigenic factor VIII (VIII:AG) was assessed by the quantitative electroimmunoassay described by Laurell [11] as modified for factor VIII of plasma [6-8]. Various dilutions of standard and test plasma are electrophoresed through a gel containing rabbit antibody against human factor VIII; precipitation occurs in the zones of equivalence. The heights of the "rockets" thus produced -are directly, although not linearly, proportional to the factor VIII concentration in each starting well. The quantity of factor VIIIrelated antigen in the unknown is expressed in terms of a standard control prepared by pooling at least 30 plasmas. The pooled plasma is defined as containing 1 U of VIIIR: AG/ml. The standard pooled plasma, aliquoted in 1 ml lots, was stored at -70'C, and an aliquot from the same pool was used as the control for each VIII:C, VIIIR:AG, and VIIIR:WF assay. Aliquots were discarded after a single use. The antibody against factor VIII was prepared by chromatography of 180 mg of Hemophil (Hyland Laboratories, Costa Mesa, Calif.), a highly purified concentrate of plasma factor VIII, through a 2.5 X 45 cm column of Biogel A-15M (200-400 mesh). The eluting fluid was Tris-HCL 0.05 M, NaCl 0.15 M, pH 6.8. The 20 ml immediately following the void volume was precipitated with 30% polyethylene glycol 6,000. Rabbits were immunized with the precipitate reconstituted in 0.5 ml of 0.15 M NaCl mixed with an equal volume of Freund's complete adjuvant, 0.2 ml being placed in each of five separate subcutaneous sites. The immunization, using the complete adjuvant, was repeated three additional times at weekly intervals, and reimmunization was carried out thereafter at monthly intervals without Freund's adjuvant. One week after the fourth series of injections the rabbits were bled, and citrated plasma was prepared from each rabbit and adsorbed with Al(OH)3 suspension, 0.1 ml/ml of plasma [12]. The adsorbed plasma was heated at 560C for 1 hr, and coagulated fibrinogen removed by centrifugation. The treated plasma was absorbed with 3%-8% ethanol precipitate of normal plasma to render the



antibody specific for factor VIII-related antigen [6]. This absorbed "antiserum" was then mixed with 0.9% agarose ("Seravac," Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, N.Y.) at an optimal concentration (in this case 0.5%). Gel plates of 1 mm thickness were prepared for the electroimmunoassay. 3. Factor VIII-related Willebrand factor (VIIIR:WF) of plasma was measured by a modification of the method of Sarji et al. [13] using platelets "fixed" with formalin [14]. Our assay procedure [15] consists of a system containing variable amounts of plasma and constant amounts of fixed platelets and ristocetin. Platelet agglutination is timed in the wells of microflocculation tiles, and the concentration of Willebrand factor in the test is expressed as a proportion of that in the standard control (the same control as for VIII:C and VIIIR:AG) and defined as having 1 U VIIIR:WF/ml. 4. Bleeding times were determined by the method of Ivy [16]. RESULTS

Proband's Clinical History The female proband (IV-1) was born in 1937 to a father with a history of "free-bleeding" and a mother without a bleeding diathesis. A chronically swollen ankle in early childhood (hemarthrosis?) may have been related to defective hemostasis, but she has had no problem with her joints since childhood. She had prolonged bleeding following extraction of two teeth at age 13; bleeding and clotting time tests performed by her family physician before and after this episode produced values in the normal range. She bleeds heavily with menses but did not bleed excessively at age 24 either upon spontaneous abortion of a 16-18 week gestation or from the D. and C. which followed. In February 1974 she developed hemorrhagic shock from an intra-abdominal hemorrhage that was found at laparotomy to be from a small "bleeding point" in a broad ligament. She required treatment with cryoprecipitate after this surgery, and the hematologic diagnosis at that time was von Willebrand syndrome. The proband states that her childlessness is a result of a deliberate decision not to have children.

History of the Kindred The hemophilia in the kindred (fig. 1) can be traced to the village of Glossa on the island of Skopelos in the Aegean Sea. The first person known to have been affected was I-1, the great-grandfather of the proband. He is remembered as having had considerable bleeding with tooth extractions, and two of his daughters (II-8 and II-11) are known to have had hemophilic descendants. The trait was brought to the United States by the proband's father (III-2) who was born in 1902, settled permanently in Alabama in 1924, married a local girl of Anglo-Saxon background, and had three daughters (IV-1, IV-3, and IV-6). There have been no known instances of "free-bleeding" in the mother's family during the last 4 generations. The hemophilic father of the proband has had only slight difficulty and has never required a blood transfusion. He has, however, avoided dentists and has never undergone elective surgery. He has had only minor hemarthroses, always related to obvious trauma; these have tended to involve his elbows rather than his knees, which he relates to closing the doors of his restaurant's refrigerators with his





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FIG. 1.-El = males; 0 = females; solid symbols = clinically abnormal; open symbols = tested normal or untested but without symptoms; 0 = heterozygous carrier; O = potential carrier. Arabic numerals above the symbols indicate pedigree position; arabic numerals below the symbols indicate U/100 ml of the various factor VIII-related activities. C = coagulant factor VIII; R:AG = factor VIII-related antigen; R:WF = factor VIII-related Willebrand factor, in this case measured as "ristocetin co-factor."

elbows. He is presently a semi-invalid because of a pseudotumor in the distal part of his left femur which resulted from a fracture at age 72. Only one daughter has had children, but she has a hemophilic son. On the basis of the family history the hemophilia is regarded as X-linked and moderately severe, and the three daughters of case III-2 are considered obligatory carriers. LABORATORY TESTS

Normal bleeding times were observed on all persons tested. Results of the other laboratory tests are listed in relation to specific individuals on the pedigree chart (fig. 1) which also shows the X-linked mode of inheritance. It should be noted that all persons tested were also normal with respect to factor VIII-related antigen and the Willebrand factor. This is particularly important to note in the case of the proband (IV-1) because it excludes the diagnosis of von Willebrand syndrome,



the diagnosis with which she was referred to us. With respect to coagulation factor VIII, three members of the kindred are clearly abnormal: case III-2, his daughter (IV-1, our proband), and his grandson (V-5). While both males and the female clearly have the hemophilia A phenotype, the woman has a slightly higher VIII: C level than the men (0.09 U/ml vs. 0.06 U and 0.03 U). Chromosomes of the proband, obtained by lymphocyte culture, were 46,XX with a normal G-banding pattern.* The two clinically normal daughters of III-2 are obligatory carriers of hemophilia. One of them (IV-3) is phenotypically normal by our four tests of factor VIII-related activities, but she has demonstrated her heterozygosity by producing a hemophilic son (V-5). The other (IV-6) is childless but shows the typical laboratory findings of heterozygosity (i.e., an VIII:C level (40%0) which is less than half the level of both the factor VIII-related antigen (83%) and the Willebrand factor [92%SO]). We have described elsewhere [17] a linear discriminant for probabilistic classification of potential hemophilia carriers based upon age and two of the tests of factor VIII-related activity, coagulant and antigenic factor VIII. The discriminant is (-0.3746 - 0.0496a) (x) + (4.447 - 0.0055a) (y), where x l_ n VIIIR:AG, y -ln VIII: C, and a- age in years. This discriminant is normally distributed with a mean of 16.16 - 0.2363a and a variance of 3.09 in carriers and is normally distributed with a mean of 20.52 - 0.2696a and a variance of 1.11 in normal women. The discriminant can be applied to the two clinically normal, obligatory carriers, IV-3 and IV-6, if it is assumed that they have equal prior probabilities (.50) of being carriers or noncarriers. (This is not the case, of course, since their father is a hemophiliac.) Under this assumption, the probabilities that individuals IV-3 (aged 36 and with a hemophilic son) and IV-6 (aged 34) are carriers are .32 and .99998, respectively. In the actual case of individual V-3 (aged 13) who does have an equal prior probability of .50, the posterior probability is .31. DISCUSSION

Our study of this kindred illustrates the use of current methods of studying members of families transmitting hemophilia A. The kindred contains three sisters who have inherited X-linked recessive hemophilia A from their father and who demonstrate the entire range of phenotypes possible for heterozygotes. The only caveat concerns the uncertainty about the genotype of the clinically affected daughter of the hemophilic father, our proband (IV-1). She has a coagulant factor VIII level of 0.09 U per ml (9 U/100 ml) and has had less joint disability than her father. The question is whether she is a "manifesting heterozygote" or is homozygous. There is no evidence of hemophilia in 4 generations of her mother's family, and her mother has a normal phenotype. As demonstrated by one of her daughters (IV-3), however, a normal phenotype does not necessarily imply a * Chromosomes of the proband that were prepared by G banding cultured peripheral blood lymphocytes were examined in the laboratory of Dr. W. H. Sternberg, Pathology Department, Tulane University School of Medicine, New Orleans, Louisiana.



normal genotype. Point mutation in the ovum which produced the proband cannot be excluded, but it has been suggested elsewhere that "extreme lyonization" is probably as likely as mutation in these circumstances [9]. A somewhat less severe degree of joint involvement and an VIII:C level which is slightly higher than those of her affected male relatives (0.09 U vs. 0.06 U and 0.03 U) also suggest that she is a manifesting heterozygote rather than a homozygote. Similarly affected women who are also probably heterozygous have been described by others in both hemophilia A and B [1, 18, 19]. Therapeutically, the proband must be treated as though she were a man with moderately severe hemophilia. The birth of a normal son would prove that she is in fact heterozygous, but this is not an experiment which she is inclined to carry out "in the interests of science." Her childlessness suggests that the proband might have an abnormality involving the X chromosome which may cause relative but not absolute infertility. As discussed previously [2], at least two cytologic abnormalities immediately come to mind when one is faced with a woman with hemophilia A or B who is not infertile (i.e., isochromosome-X and a balanced X-autosomal translocation). Either of these in a hemophilia heterozygote might produce a bleeder, but her karyotype is 46,XX with a normal banding pattern. On balance we are inclined to believe that her phenotype is more likely the result of "extreme lyonization" than mutation, but we shall probably never be entirely certain. SUMMARY

We have described the study of a small kindred with X-linked hemophilia A. It was ascertained through a clinically affected female, the daughter of a man with moderately severe hemophilia. The pedigree and the proband's phenotype suggest that she may be a heterozygote in whom most of the normal alleles at the VIII-1 locus are not active. She has two sisters, also obligatory carriers. The three sisters exhibit the three phenotypes possible for heterozygous females: clinically affected, clinically normal but phenotypically abnormal as determined by laboratory tests, and clinically and phenotypically normal. REFERENCES 1. BARROW EM, GRAHAM JB: Blood coagulation factor VIII (antihemophilic factor): with comments on von Willebrand's disease and Christmas disease. Physiol Rev 54:23-74, 1974 2. GRAHAM JB, BARROW ES, ROBERTS HR, WEBSTER WP, BLATT PM, BUCHANAN P, CEDERBAUM AI, ALLAIN JP, BARRETT DA, GRALNICK HR: Dominant inheritance of hemophilia A in three generations of women. Blood 46:175-188, 1975 3. RIZZA CR: Factor VIII-related antigen and von Willebrand's disease. Br J Haematol 31, suppl.: 231-245, 1975 4. GRAHAM JB, BARRETT DA, BLOMBACK B, CANN HM, HARDISTY RM, LARRIEU MJ, RENWICK JH: A genetic nomenclature for human blood coagulation. Thromb Diath Haemorrh 30:2-11, 1973 5. GRAHAM JB, BARROW ES, BLOMBACK B, BLOOM AL, BOUMA BN, FORBES C, HARDISTY RM, HOYER LW, INGRAM GIC, LARRIEU MJ, RENWICK JH: Nomenclature of factor VIII-related activities. Preliminary report of the Task Force, Paris, France, July 21, 1975



6. ZIMMERMAN TS, RATNOFF OD, POWELL AE: Immunologic differentiation of classic hemophilia (factor VIII deficiency) and von Willebrand's disease. J Clin Invest 50:244-254, 1971 7. ZIMMERMAN TS, RATNOFF OD, LITTELL AS: Detection of carriers of classic hemophilia using an immunologic assay for antihemophilic factor (factor VIII). J Clin Invest 50:255-258, 1971 8. BENNETT B, RATNOFF OD: Detection of the carrier state for classic hemophilia. N Engl J Med 288:342-345, 1973 9. GRAHAM JB, BARROW ES, ELSTON RC: Lyonization in hemophilia: a cause of error in direct detection of heterozygous carriers. Ann NY Acad Sci 240:141-146, 1975 10. LANGDELL RD, WAGNER RH, BRINKHOUS KM: Effect of antihemophilic factor on one-stage clotting tests. J Lab Clin Med 41:637-647, 1953 11. LAURELL CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 15:45-52, 1966 12. BARROW EM, AMOS SM, HEINDEL C, GRAHAM JB: Certain biochemical properties of human AHF (factor VIII) separated from fibrinogen with manganous chloride and thrombin. Proc Soc Expl Biol Med 121 :1001-1005, 1966 13. SARJI KE, STRATTON RD, WAGNER RH, BRINKHous KM: Nature of von Willebrand factor: a new assay and a specific inhibitor. Proc Natl Acad Sci USA 71:29372941, 1974 14. ALLAIN JP, COOPER HA, BRINKHOUs KM: Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor. J Lab Clin Med 85:318-328, 1975 15. REISNER HM, KATZ HJ, GRAHAM JB: A rapid quantitative macroscopic assay for Willebrand factor (WF). Fed Proc 35:756, 1976 16. Ivy AC, SHAPIRO PF, MELNICH P: The bleeding tendency in jaundice. Surg Gynecol Obstet 60:781-784, 1935 17. ELSTON RC, GRAHAM JB, MILLER CH, REISNER HM, BOUMA BN: Probabilistic classification of hemophilia A carriers by discriminant analysis. Thromb Res 8:683695, 1976 18. CZAPEK EE, HOYER LW, SCHWARTZ AD: Hemophilia in a female: use of factor VIII antigen levels as a diagnostic aid. J Pediatr 84:485-489, 1974 19. REvEsz TD, SCHULER D, GOLDSCHMIDT B, ELODI S: Christmas disease in one of a pair of monozygotic twins, possibly the effect of lyonization. J Med Genet 9:396400, 1972

The phenotypic range of hemophilia A carriers.

Am J Hum Genet 28:482-488, 1976 The Phenotypic Range of Hemophilia A Carriers JOHN B. GRAHAM,1 CONNIE H. MILLER,1 HOWARD M. REISNER,1 ROBERT C. ELSTO...
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