Carrier detection of the X-linked primary immunodeficiency diseases using X-chromosome inactivation analysis

Carrier detection of the X-linked primary immunodeficiency diseases using X-chromosome inactivation analysis Jerry

A. Winkelstein,

MD, and Eric Fearon

Baltimore,

Md.

Carrier detection of three of the X-linked primary immunodeficiency diseases (X-linked agammaglobulinemia, X-linked severe combined immunodejciency disease, and the Wiskott-Aldrich syndrome) is possible by analyzing patterns of X-chromosome inactivation in those cells affected by the disorder. Normal women have balanced patterns of X-chromosome inactivation; that is, in a given population of cells, approximately half of their active X chromosomes are of paternal origin and half of their active X chromosomes are of maternal origin. In contrast, female carriers of these X-linked immunodejiciency disorders have an unbalanced pattern of X-chromosome inactivation in those cell lineages that are affected by the disorder; that is, all the active X chromosomes in affected cell lineages are the X chromosomes that carry the normal allele. Two techniques are available for X-chromosome inactivation analysis. One technique depends on methylation difSerences between the active and inactive X chromosome, and the other technique uses somatic cell hybrids that selectively retain the active X chromosome. In either case, carrier detection can be performed in individuals from families in which only one member of the family has been affected, since neither of these methods depends on linkage analysis. (J ALLERGY CLIN IMMUNOL 1990;85:1090-7.)

Since the initial description of XLA in 1952,’ over 50 primary immunodeficiency diseases have been identified.’ This diverse group of diseases affects virtually every component of the normal immune system. Although patients with these disorders most commonly present with an increased susceptibility to infection, they can also present with a variety of inflammatory and rheumatic disorders. Most of the primary immunodeficiency diseases are genetically determined. Eight of these diseases are inherited in an X-linked recessive fashion. In many cases, they have been mapped to specific regions of the X chromosome (Fig. 1).3 Although detection of female carriers has been possible in one of the disorders, X-linked CGD, until recently there have been From the Eudowood Division of Immunology, the Department of Pediatrics, aud from the Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Md. Supported iu part by a grant from the March of Dimes, a grant from the Immune Deficiency Foundation, and National Institutes of Health GrantsGM-07309, GM-07184, and CA-35494. Presentedat the Aspen Allergy Conference, Aspen, Colo., July 1l15, 1989. Reprint requests: Jerry A. Winkelstein, MD, Eudowood Division of Immunology, Department of Pediatrics CMSC 1103, The Johns Hopkins Hospital, Baltimore, MD 21205. 111/2@774

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Abbreviations

used

EBV: Epstein-Barr virus G6PD: Glucose-6-phosphatedehydrogenase RFLP: Restriction fragment length Polymorphism PGK: Phosphoglycerate kinase HPRT: Hypoxanthine guanine phosphoribosyl transferase SCID: Severe combined immunodeficiency disease CGD: Chronic granulomatous disease XLA: X-linked agammaglobulinemia WAS: Wiskott-Aldrich syndrome

no methods available for carrier detection of the other X-linked primary immunodeficiency diseases. Recent advances in the fields of molecular biology and genetics have been successfully applied to the Xlinked immunodeficiency diseases and made detection of female carriers possible in many of these disorders. In the present article we will review the X-linked immunodeficiency diseases, outline the conceptual framework on which carrier detection of these disorders is based, and then present the molecular genetic techniques that make carrier detection of these disorders possible.

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X-linked

primary

immunodeficiency

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THE X-UNlCED t’lWlARY IMMUNODE~MY DISEASES granulomatous disease

The X-linked primary immunodeficiency diseases are a heterogenous group of inborn errors of metabolism. Six of these disorders affect the function of T cells and/or B cells, one affects phagocytic cell function, and one disorder affects the complement system.

X-linked

Properdin deficiency Wiskott-Aldrich syndrome

!t-

Severe combined immunodeficiency

agammaglobulinemia

Agammaglobulinemia

XLA affects the B cell system.4 It is characterized by an absence of plasma cells from tissues and the bone marrow and an absence of B cells from the peripheral blood. Serum immunoglobulins of all classes are markedly reduced as are the serum levels of functional antibodies. Patients with XLA most commonly have an increased susceptibility to infection with a variety of both gram-positive and gram-negative encapsulated bacteria. In addition, patients can develop chronic systemic infections with a number of different enteroviruses.

Lymphoproliferative syndrome

X FIG. 1. The the X-linked

X-linked severe combined immunodeficiency disease One form of SCID is inherited in an X-linked recessive fashion.’ This disease affects both the T cell and B cell systems. Patients with this disorder usually have a marked reduction in both B cell and T cell number and in B cell and T cell function. They often have lymphopenia and usually have marked hypogammaglobulinemia. Patients with SCID have a marked susceptibility to a wide variety of bacteria, viruses, fungi, and protozoa.

Wiskott-Akhich

syndrome

This disorder appears to affect T cells, B cells, monocytes, and platelets, and each to a variable degree.6Patients commonly have decreased T cell function, decreased levels of serum IgM, and reduced levels of antibody to polysaccharide antigens. Patients with WAS typically are first observed with the clinical triad of eczema, tbrombocytopenia, and an increased susceptibility to infection. Although the original description of this disorder highlighted their difficulty with otitis media, these patients have subsequently been demonstrated to have an increased susceptibility to a wide variety of bacteria, fungi, and viruses.

The X-linked

lymphopfolifefative

syndrome

Patients with this disease are usually clinically well until infection with EBV.’ After EBV infection, patients with the X-linked lymphoproliferative syndrome may have a variety of clinical outcomes, even within the same kindred. Although some patients die of their initial EBV infection, other patients develop hypogammaglobulinemia or aplastic pancytopenia, and still other patients progress to a B cell lymphoma.

X-linked

lmmunodeficiency with hyper IgM

immunodeficiency

with normal

IgM

This disorder affects B cell function.* Affected individuals have markedly reduced serum levels of IgG and IgA,

human primary

X chromosome immunodeficiency

and

the locations

ot

diseases.

but their IgM levels are normal or elevated. Patients with this disease most commonly have an increased susceptibility to infection with pyogenic bacteria, but some patients have also developed Pneumocystis curinii pneumonia.Autoimmunediseases, suchasautoimmune hemolyticanemia,have alsobeenobserved. x-iiied

agwn

gfowth

hormone

Patientswith this disorderhave both p~h~~globulinemia andisolatedgrowthhormonedeficiency.*Only two kindredswith this disorderhave beendescribed.It appears to bedistinctfromclassicalXLA, bothat theclinical level, that is, the presenceof isolatedgrowthhormonedeficiency, andat the moleculargeneticlevel.

X-linked

chronic

granubma@us

d

One form of CGD is inheritedin an X-linked recessive fashion.” The disorderaffectsthe oxidative met&olismof phagocyticcells.Thesecellsareunableto reducerno&tiar oxygen to producethe reactive oxygen products,suchas hydrogenperoxide,whicharenecessary for the intmc&luhir killing of bacteriaandfungi. As a result,patientswith this disorderhave an increasedsusceptibilityto thosebacteria and fungi that are catalasepositive and have na net productionof H,O,.

Pfopefdin

deficiency

This disorderaffects one of the impomt regulatory proteins of the alternative complementpathway, properdin.” Patients usually have little proper&n in their serumand, as a consequence, abnormalfunction of the alternativepathway.Although relatively few pa&n@ with thisdeficiencyhavebeenidentified,theyall appearto have a uniquesusceptibilityto systemicmeningococcaliafections.

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NORMAL

CARRIER

FEMALE

I

-B-CELLS T-CELLS GRANULOCYTES

I

ZYGOTE

FIG. 2. The pattern of X-chromosome cells of a normal female subject. The somes are represented by the larger Two populations of a given cell lineage the maternal X chromosome is active in which the paternal X chromosome

X-CHROMOSOME

cm

’

inactivation in the active X chromoX chromosomes. exist, one in which and one population is active.

INACTIVATION

WL

1

B-CELLS T-CELLS GRANULOCYTES

Normal Function

SPERM

WA, PAT HH Q

ZYGOTE

I

OF CGD

EGG

SPERM

EGG

CLIN. IMMUNOL. JUNE 1990

IN MAN

Early in the embryogenesis of each female subject, inactivation of one of the two X chromosomes occurs in each somatic cell. ‘*. I3The process of X-chromosome inactivation is random with respect to which of the two X chromosomes is inactivated. Thus, in some somatic cells the maternal X chromosome is active and the paternal X chromosome is inactive, whereas in other somatic cells, the paternal X chromosome is active and the maternal X chromosome is inactive (Fig. 2). The pattern of X-chromosome inactivation in each progenitor cell is transmitted to its progeny in a highly stable manner. Thus, each woman is a mosaic with some of her somatic cells expressing alleles on the maternal X chromosome and other somatic cells expressing alleles on the paternal X chromosome. The ratio of cells in which the maternal X chromosome is active to cells in which the paternal X chromosome is active varies from woman to woman but is normally distributed around a mean ratio of 50% : 50%. The ratio within each woman is usually the same in all cell lineages. Direct evidence that normal women have two populations of cells, with respect to which of their two X chromosome is active (i.e., are mosaic), was first offered by Davidson

GRANULOCYTES (Function Impaired)

GRANULOCYTES (Function Normal)

FIG. 3. The pattern of X-chromosome inactivation in granulocytes of a female carrier of X-linked CGD. The active X chromosomes are represented by the larger X chromosomes. Two populations of granulocytes exist, one in which the maternal X chromosome is active and function is impaired and one population in which the paternal X chromosome is active and function is normal.

et aLI in 1963. These authors examined women who were heterozygous for allelic protein variants of the X-linked gene, G6PD, and were ableto demonstrate that these women had two populations of fibroblasts, one population in which one allelic variant was produced and thus in which only one X chromosome was active, and another population in which the other allelic variant was produced and thus in which only the other X chromosome was active. Since then, additional evidence that women are mosaics, with respect to which of their X chromosomes is active in a given cell, has been obtained from a number of studies, including studies of fibroblasts of female carriers of X-linked HPRT deficiency” and adrenoleukodystrophy.‘6

THE PAlTERN OF X-CHROMOSOME INACTlVATlON IN CARRIERS OF X-LINKED IMMUNODEACIENCY DISEASES If the mutant gene that causes an X-linked immunodeficiency disease does not interfere with the normal development of the cell lineage (or lineages) affected by the disease, then a female carrier should have two populations of that cell lineage. In one pop-

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ulation of cells, the X chromosome carrying the normal allele is active (i.e., her paternal X chromosome) and the function of those cells is normal. In the other population of cells, the X chromosome carrying the mutant allele is active (i.e., her maternal X chromosome) and the function of those cells is in some fashion abnormal. Such a situation exists in X-linked CGD (Fig. 3). This disorder is charcterized by a failure of phagocytic cells to reduce molecular oxygen to a variety of toxic oxygen metabolites necessary for the killing of intracellular bacteria.” Female carriers have two populations of granulocytes and monocytes, one population in which the production of Hz02 and other reduced forms of molecular oxygen is normal and another population in which they are not produced. In fact, one method of detecting female carriers of this X-linked disorder relies on histochemical techniques to demonstrate that female carriers have two populations of phagocytic cells with respect to the metabolic abnormality. 17.‘* In some of the X-linked immunodeficiency diseases, the mutant gene interferes with the normal development of those cell lineages that are affected by the disorder (Fig. 4). Presumably, the cells in which the X chromosome carrying the mutant allele is active are at a selective disadvantage during their development, whereas the cells in which the X chromosome carrying the normal allele is active are at a selective advantage during their development. As a consequence, a woman who is a carrier will not have two balanced populations of affected cells, with respect to which X chromosome is active, but rather will have unbalanced populations of affected cells. In this situation, the affected cell population will be predominantly derived from progenitor cells in which the active X chromosome is the X chromosome that carries the normal allele. The first direct evidence that female carriers of some of the X-linked immunodeficiency diseases have unbalanced populations of affected cells came from a number of different studies that examined female carriers who were heterozygous for isoenzymes of the X-linked gene, G6PD. Studies of women who were obligate carriers of WAS and who were also heterozygous for G6PD isoenzymes have demonstrated markedly unbalanced patterns of X-chromosome inactivation in their peripheral blood cells. In one study, the carrier’s granulocytes, platelets, monocytes, Blymphocytes, and T-lymphocytes demonstrated only one isoenzyme variant, whereas her cultured skin fibroblasts, used as a control, demonstrated the two isoenzymes in equal proportions.” In another study, the platelets and T-lymphocytes of the carrier of WAS were exclusively of one isoenzyme, whereas her

X-linked

primary

CARRIER EGG

immunodeficiency

diseases

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OF XLA SPERM

ZYQOTE

&-CELLS (Normal Function)

FIG. 4. The pattern of X-chromosome inactivation in the B cells of a female carrier of X-linked agam~~v~i~nemia. The active X chromosomes are represented by the larger X chromosomes. Only one popdation of B cells exists, the population of B cells derived from progenitor cells in which the paternal X chromosome carrying the normal allele is active.

erythrocytes, granulocytes, monocytes, and “non-T”lymphocytes contained approximately 85% of that same isoenzyme and 15% of the other isoenzyme.” A recent study of women who were carriers of XLA and also heterozygous for G6PD isoenzymes demonstrated that the B cells of these women contained isoenzymes of only one type, whereas their T cells and neutrophils contai5ed both isoenzymes.”

Unfortunately, heteFozygosity for G6PD isoenzymes, or for other alIutypic protein markers of Xlinked genes, is found in only a smdl minority of female subjects (~3% in the United States) and thus cannot be widely applied to carrier detection of the X-linked primary immunodeficiency diseases. Therefore, two new strategies that capitalize on molecular genetic differences between active and inactive X chromosomes have been developed for carrier detection of these disorders.

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FIG. 5. Patterns of X-chromosomes inactivation in a normal female subject and a female carrier of XLA. Lane 1, T cells of a normal female subject; lane 2, B cells of a normal female subject; lane 3, granulocytes of a carrier of XLA; lane 4, T cells of a carrier of XLA; lane 5, B cells of a carrier of XLA. Each sample of DNA was first digested with the restriction enzyme, Bgl 1, to discriminate between different PGK alleles on the maternal and paternal X chromosome. One aliquot of the cleaved DNA was not digested further (lane A of each sample), and the other aliquot was digested with Hpa II (lane B ofeach sample) to discriminate between the active and inactive X chromosome (see text for explanation).

X-chromosome inactivation analysis difference in methylation patterns.

using

This strategy depends on the fact that certain genes on the X chromosome have different methylation patterns, depending on whether they reside on the active or inactive X chromosome.“~” The technique requires the use of two different restriction endonucleases. Restriction endonucleases are enzymes that cleave DNA at specific sites determined by specific nucleotide sequences. Different restriction endonucleases are specific for different nucleotide sequences. The specific nucleotide sequences of a given gene may vary by a single nucleotide from individual to individual without any known biologic or clinical consequence. When this DNA sequence variation occurs commonly among individuals in a population, it is termed a polymorphism. If a polymorphism affects a restriction enzyme recognition site, the length of the DNA fragments produced by that enzyme will be changed. This genetic variation from individual to individual is called an RFLP and, at some loci, occurs commonly enough to be of use in distinguishing one allele of a gene from the other allele in a given individual. Thus, RFLP analysis of X-linked genes can be used to distinguish the maternal X chromosome from the paternal X chromosome. One restrictive endonuclease, Hpa II, cleaves DNA at the specific nucleotide sequence, cytosine-cytosineguanine-guanine. However, if the second cytosine is methylated, the DNA can not be cleaved by the Hpa II. At some loci on the X chromosome, differences in the methylation of cytosines exist between a given locus on an active X chromosome and that same locus on the inactive X chromosome. For some loci, the inactive X chromosome is methylated and the active X chromosome is unmetbylated, whereas for other loci, the reverse is true. Thus, whether the Hpa II restriction endonuclease is able to cleave the DNA

depends on whether the locus is on the active or inactive X chromosome. In this fashion the Hpa II is able to discriminate between the active and inactive X chromosome. Analysis of X-chromosome inactivation using differences in methylation patterns depends on two restriction enzymes. The jirst endonudease distinguishes copies of the gene that are on the maternal X chromosome from copies of the gene that are on the paternal X chromosome, based on RFLPs. The secondendonuclease,Hpa II, distinguishes copies of the gene that are on the active X chromosomes from copies of the gene that are on the inactive X chromosome through methylation differences in the DNA. Thus, this strategy requires that a woman be heterozygous for a DNA polymorphism at a particular Xlinked locus and that particular locus differ in its methylation pattern between the active and inactive X chromosome. Two X-linked loci, HPRT and PGK, fulfill these requirements. Slightly over 50% of American women are heterozygous for the polymorphisms at one or the other of these loci and thus suitable for carrier detection with this tcchnique.25 An example of the results obtained in a woman who is a carrier of XLA is illustrated in Fig. 5. When DNA is extracted from the T cells or B cells of a normal woman and treated with a restriction enzyme (Bgl I) that detects an RFLP in the PGK gene and then electrophoresed, two fragments are detected after use of a radiolabeled probe specific for the PGK gene; one fragment represents the allele from the maternal X chromosome and the other fragment represents the allele from the paternal X chromosome. These fragments of the PGK gene are methylated only when they are on the inactive X chromosome. The methylationsensitive enzyme, Hpa II, is able to cleave the fragments only when they are from the active X chromosome, that is, the unmetbylated locus. Thus, when

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the two fragments obtained after Bgl I digestion are further digested with Hpa II, electrophoresed, and then tested with the radiolabeled PGK probe, the intensity of both fragments is reduced by approximately 50%. reflecting the fact that half the maternal alleles are active and half the paternal alleles are active, that is, that there is balanced X-chromosome inactivation. The findings in an obligate carrier of XLA are similar to a normal female subject when DNA from the carrier’s T cells and granulocytes is digested (Fig. 5). Approximately half of each Bgl I fragment is cleaved by the methylation-sensitive Hpa II, indicating that half the maternal alleles and half the paternal alleles are on the active X chromosome, that is, balanced Xchromosome inactivation. However, when DNA from the B cells of a female carrier is digested with Hpa II, only one of the two fragments is completely digested, whereas the other fragment is left intact. Thus, only one of the two alleles is on the active X chromosome, demonstrating an unbalanced Xchromosome inactivation pattern. X-chromosome inactivation analysis using differences in methylation patterns was first used to detect carriers of XLA.26 In this study, the B cells from female carriers were revealed to have unbalanced patterns of X-chromosome inactivation, whereas the T cells and granulocytes had balanced patterns. In a subsequent study, the technique was used to detect carriers of X-linked SCID by demonstrating unbalanced X-chromosome inactivation in their T cells.” Finally, the technique has also been used to detect carriers of WAS by demonstrating unbalanced patterns of X-chromosome inactivation in the carriers’ granulocytes ,‘8 T cells ,‘*. 29and B cells ,28,29but not in their fibroblasts.28 This technique is rapid and relatively simple. However, since only slightly >50% of American women are heterozygous for the RFLP at either the PGK or HPRT loci, it can not be applied to all women at risk for carrying these disorders. However, as new X-linked polymorphic loci are discovered that demonstrate consistent differences in methylation patterns between the active and inactive X chromosomes, the technique should be applicable to a larger proportion of women at risk.

X-chromosome inactivation somatic cell hybrids.

analysis

using

This strategy depends on the use of somatic cell hybrids between human and rodent cells to differentiate between the active and inactive human X chromosome and RPLPs on the X chromosome to distinguish the paternal X chromosome from the maternal X chromosome. The human cell population (e.g., T cells and B cells) to be analyzed for balanced or unbalanced X-

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chromosome inactivation is fused with hbroblasts of Chinese hamsters deficient in the X-linked gene for HPRT.M When the hybrids are grown m a selective medium, only those hybrids that retain the active human X chromosome, and thus can produce HPRT, are able to survive. Individual clones of the surviving hybrids that contain the active human X chromosome are then expanded, and the DNA is isolated. Aliquots of the DNA from a number of different hybrid clones are then digested with restriction enzymes and tested with a number of different probes for polymorphic regions of the X chromosome to discrimmate between the maternal and paternal copies of the X chromosome through RFLP analysis. In this fashion. it can be determined whether all the individual clones have retained the same X chromosome (maternal or paternal) as the active X chromosome or whether some clones have retained the maternal X chromosome as the active X chromosome, while other clones have retained the paternal X chromosome as the active X chromosome, that is, whether X-chromosome inactivation in the affected cell lineage is unbalanced or balanced. This technique has been used to detect carriers of XLA by demonstrating markedly unbalanced Xchromosome inactivation in the I3 cells of carrier women.3i It has also been used to detect carriers of X-linked SCID by demonstrating unbalanced Xchromosome inactivation in their T ceils’” and B cells.“2 Because there are so many polymorphic Xlinked loci (> 1OO),33this form of X-chromosome inactivation analysis can be applied to virmally every woman. However, the technique depends on the production of many somatic cell hybrids aud their clonal expansion before DNA analysis can be performed and thus requires substantial time and effotr

APPLICATIONS

AND IMPLJCATlONS

Carrier detection in three of the X-linked primary immunodeficiency diseases is now possible by analyzing the pattern of X-chromosome inactivation in those cell lineages affected by the disorder. Two techniques are available for X-chromosome inactivation analysis. One technique depends on methylation differences between the active and inactive X chromosome, and the other technique makes use of somatic cell hybrids that selectively retain the active X chromosome. Both techniques are reliable and both possess certain applications and implications that deserve additional comment. Linkage analysis has also been used to detect carriers of some of the X-linked immunodeficiency diseases .34.35 Linkage analysis, however, requires DNA samples from individuals in several generations as well as the affected male subject. Many families may have no affected male subjects available for testing

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or only a single affected male subject. In such situations, X-chromosome inactivation analysis may be especially helpful because women at risk may be tested directly for the carrier state. Analysis of X-chromosome inactivation in carrier women has also been valuable in defining the cellular defect in some of the X-linked primary immunodeficiency diseases. Analysis of carriers of XLA has demonstrated the defect to be restricted to B cells.21~ 26, 3’ Similar studies in female carriers of WAS have demonstrated that the primary defect affects all lymphohematopoietic cell lineages . I99‘O*“2 29Analysis of carriers of X-linked SCID has demonstrated the primary defect to reside in both T cells*‘~ 3oand B cells3’ in this disorder. Finally, X-chromosome inactivation analysis has the potential in some instances to aid in the specific diagnosis of a child with an immunodeficiency disease. For example, in the absence of a positive family history, it can be difficult to determine whether a male infant with eczema and thrombocytopenia has WAS or just the chance association of eczema and thrombocytopenia, since the immunologic findings in WAS vary from patient to patient. Similarly, in the absence of a positive family history, it may be difficult to determine if a male infant with agammaglobulinemia or a male infant with SCID have the X-linked forms of these disorders. By examining the appropriate female relatives of male infants, such as these, it can be determined if they are carriers of these disorders and use that information to make a specific genetic diagnosis in the child. REFERENCES 1. Bruton OC. Agammaglobulinemia. Pediatrics 1952;9:722-8. 2. The primary immunodeficiency diseases. Report of a WHOsponsored meeting. Immunol Rev 1989;1:173-205. 3. McKusick VA. Mendelian inheritance in man. 8th ed. Baltimore.: The Johns Hopkins University Press, 1989. 4. Lederman HM, Winkelstein JA. X-linked agammaglobulinemia: an analysis of % patients. Medicine 1985;64:145-56. 5. Gelfand EW, Dosch HM. Diagnosis and classification of severe combined immunodeficiency disease [Original articles series]. Birth Defects 1983;19:65-72. 6. Blaese RM, Strober W, Brown RS, Waldmann TA. The Wiskott-Aldrich syndrome: a disorder with a possible defect in antigen processing or recognition. Lancet 1968;1:1056-61. 7. Purtillo DT, DeFlorio D, Hutt LM, et al. Variable phenotypic expression of an X-linked recessive lymphoproliferative syndrome. N Engl J Med 1977;297:1077-81. 8. Stiehm ER, Fudenberg HI-I. Clinical and immunologic featu+s of dysgammaglobulinemia type 1. Am J Med 1966;40:805- 15. 9. Fleisher TA, White Rh4, Broder S, et al. X-linked hypogammaglobulinemia and isolated growth hormone deficiency. N Engl J Med 1980;301: 1429-34. 10. Galrm JI, Beuscher ES, Seligmann BE, Nath J, Gaither TE, Katz P. Recent advances in chronic granulomatous disease. AM Intern Med 1983;99:657-74.

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11. Ross SC, Densen P. Complement deficiency states and infection: epidemiology, pathogenesis, and consequences of neisserial and other infections in an immune deficiency. Medicine 1984;63:243-73. 12. Lyon MF. Gene action in the X-chromosome of the mouse. Nature 1961;190:372-3. 13. Lyon MF. X-chromosome inactivation and developmental patterns in mammals. Biol Rev 1972;47:1-35. 14. Davidson RG, Nitowsky HM, Childs B. Demonstration of two populations of cells in the human female heterozygous for glucose-6-phosphate dehydrogenase variants. Proc Nat1 Acad Sci USA 1963;50:481-5. 15. Migeon BR, DerKaloustian VM, Nyhan WL, Young WJ, Childs B. X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: heterozygote has two clonal populations. Science 1968;160:425-7. 16. Migeon BR, Moser HW, Moser AB, Axelman J, Sillence D, Norum RA. Adrenoleukodystrophy: evidence for X-linkage, inactivation, and selection favoring the mutant allele in heterozygous cells. Proc Nat1 Acad Sci USA 1981;78:5066-70. 17. Windhorst DB, Holmes B, Good RA. A newly defined Xlinked trait in man with demonstration of the Lyon effect in carrier females. Lancet 1967;1:737-9. 18. Ochs HD, Igo RP. The NBT slide test: a simple screening method for chronic granulomatous disease and female carriers. J Pediatr 1973;83:77-82. 19. Prchal JT, Carroll AJ, Prchal JF, et al. Wiskott-Aldrich syndrome: cellular impairments and their implication for carrier detection. Blood 1980;56:1048-54. 20. Gealy WJ, Dwyer JM, Harley JB. Allelic exclusion of glucose6-phosphate dehydrogenase in platelets and T-lymphocytes from a Wiskott-Aldrich syndrome carrier. Lancet 198O;l: 63-5. 21. Conley ME, Brown P, Pickard AR, et al. Expression of the gene defect in X-linked agammaglobulinemia. N Engl J Med 1986;315:564-7. 22. Yen PH, Pate1 P, Chinault AC, Mohandas T, Shapiro LJ. Differential methylation of hypoxanthine phosphoribosyl transferase genes on active and inactive human X chromosomes. Proc Nat1 Acad Sci USA 1984;81:1759-63. 23. Wolf SF, Jolly DJ, Lunnen KD, Friedmann T, Migeon BR. Methylation of the hypoxanthine phosphoribosyl transferase locus on the human X chromosome: implications for Xchromosome inactivation. Proc Nat1 Acad Sci USA 1984, 81:2806-10. 24. Keith DH, Singer-Sam J, Riggs AD. Active X chromosome DNA is unmethylated at eight CCGG sites clustered in a guanine-plus-cytosine-rich island at the 5’ end of the gene for phosphoglycerate kinase. Mol Cell Biol 1986;6:4122-5. 25. Vogelstein B, Fearon ER, Hamilton SR, Feinberg AP. Use of restriction fragment length pOlyIIIOrphiSm8 to determine the clonal origin of human tumors. Science 1985;227:642-5. 26. Fearon ER, Winkelstein JA, Civin CI, Pardoll DM, Vogelstein B Carrier detection in X-linked agammaglobulinemia by analysis of X-chromosome inactivation. N Engl J Med 1987; 316:427-31. 27 Goodship J, Malcolm S, Lau YL, Pembrey ME, Levinsky RJ. Use of X-chromosome inactivation analysis to establish carrier statusfor X-linked severecombined immunodeficiency. Lancet 1988;1:729-32. 28. Fearon ER, Kohn DB, Winkelstein JA, Vogelstein B, Blaese RM. Carrier detection in the Wiskott-Aldrich syndrome. Blood 1988;72:1735-9. 29. Greer WL, Kwong PC, Peacocke M, Ip P, Rubin LA, Siminovitch KA. X-chromosome inactivation in the Wiskott-

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Aldrich syndrome: a marker for the detection of the carrier state and identification of cell lineages expressing the gene defect. Genomics 1989;4:60-7. 30. Puck JM, Nussbaum RL, Conley ME. Carrier detection in Xlinked severe combined immunodeficiency based on patterns of X-chromosome inactivation, J Clin Invest 1987;79: 1395 1400 3 I Conley ME. Puck JM. Carrier detection in typical and atypical X-linked agammaglobulinemia. J Pediatr 1988;112:688-94. 32. Conley ME, Lavoie A, Briggs C, Brown P, Guerra C, Puck JM. Nonrandom X-chromosome inactivation in B cells from carriers of X chromosome-linked severe combined immunodeficiency. Proc Nat1 Acad Sci USA 1988;85:3090-4.

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33. Goodfellow PN, Davies KE, Ropers HH. Replrt on the committee on the genetic constitution of the X and Y chromosomes. Cytogenet Cell Genet 1985;40:296-352. 34. Kivan S-P, Km&e1 L, Bruns G, Wedgewood K.I. Latt S. Kosen FS. Mapping of the X-linked agammaglohuliaemia locus by use of restriction fragment length polymorphism. J Clin invest 1986;77:649-52. 35. Peacocke M, Siminovitch KA. Linkage of the Wiskott-Aldrich syndrome with polymorphic DNA sequences from the human X chromosome. Proc Nat1 Acad Sci IiS. 19X1:84:3430-3.

AVAILABLE NOW! The PROCEEDINGSOF THE INTERNATIONAL CONGRESSOF ALLERGOLOGY AND CLINICAL IMMUNOLOGY canbe purchased from the PubJisher. This collectionof “state-of-the-art” presentations Born the XII CongressheldOctober2025, 1985,in Washington,D.C., bringstogetherthe currentadvancesin basicand aspectsof allergy andallergicdiseases. It includes528 pagescoveringsuchtopicsasIgE, &es of the different cell types andtheir products,chnicalproblems,asthma,rhinitis, and reactionsto foods and drugsand occup&ionalagents,collectedand reviewedby Ed&u CharlesE. ReedMD (USA) andAssociateEditorsJosephBeBand,MD (USA), RobertJ. Davies, MD (UK), Sidney Fridaender, MD (USA), Albert Oehling, MD (Spain), and RaymondG. Slavin, MD (USA). To purchase,call or write: Mosby-Year Book, Inc., 11830 Westline IndustriaJDr., St. Louis, MO 63146-3318,or telephoneFREE 1-800-325-4177, JournalFult#lment, ext. 7351(in Missouricall collect at 314-872-8370,JournalFulfithnent, ext. 7351). Prepayment required.Make checkspayableto Mosby-YearBook, Inc. (All paymentsmustbe in US funds drawn on a US bank.) Price: $36.50 in the US, $40.50 in Canada,and $41.50 international(surfaceshippingchargesincluded).