Clin. exp. Immunol. (1991) 85, 182-192

ADO)NIS 000991049100213G

REVIEW

Genetics of human X-linked immunodeficiency diseases R. W. HENDRIKS & R. K. B. SCHUURMAN Division of Immunobiology and Genetics, Department of Immunohaematology, University Medical Center, Leiden, The Netherlands

(Acceptedfor publication 20 March 1991) INTRODUCTION Since the initial description of X-linked agammaglobulinaemia (XLA) (Bruton, 1952), almost 100 different immunodeficiency diseases have been described in humans (reviewed by Gelfand & Dosch, 1983; Rosen, Cooper & Wedgwood, 1984; Schuurman, Mensink & Schot, 1987a). Most immunodeficiencies involve recessive genetic disorders, both in the antigen-specific immune responses of B and T lymphocytes and in the non-specific immunity mediated by myeloid cells, monocytes or the complement system. Seven of the immunodeficiency diseases show an X-linked mode of inheritance, supported by transmission of the disease along the female lineage for many generations. Within the pedigrees, about half of the males are affected and about half of the females are carriers of the disease. The female carriers do not manifest disease symptoms. Autosomal recessive disorders are usually confined to one generation in one family (except in case of consanguinity), whereas X-linked recessive diseases usually reappear in every next generation. X-linked inheritance is found in about half of the children with severe immunodeficiency diseases. Two of the X-linked immunodeficiencies involve nonspecific immunity. X-linked chronic granulomatous disease (XCGD) affects the phagocytic cells and is a defect in the beta chain of the cytochrome b complex (Teahan et al., 1987; Dinauer et al., 1987). The X-CGD gene was isolated on the basis of its chromosomal location at the Xp2l region to which the XCGD locus had been assigned both by deletion and linkage analysis. Properdin deficiency (PD) is characterized by the absence of a component of the complement system that promotes activation of the alternative pathway by stabilizing C3 convertase (Ross & Densen, 1984). The properdin gene locus has been localized on Xpl 1.23-Xp21.1 (Goundis et al., 1989). Five of the X-linked immunodeficiencies involve differentiation arrests of the lymphoid cell lineages. The exact function or identity of these X-linked lymphoid immunodeficiency (XLID) genes is not known. This review concentrates on the identification of the intrinsically affected haematopoietic cell populations by X-chromosome inactivation analyses in obligate female XLID carriers. The results have significant implications in that they give an insight into the functions of the XLID genes and have clinical relevance in prevention by carrier detection and

prenatal diagnosis as well as in postnatal diagnosis based on genetic analyses.

LOCALIZATION OF XLIDS ON THE X CHROMOSOME The XLIDs include XLA, X-linked immunodeficiency with hyperimmunoglobulinaemia M (XHM), X-linked lymphoproliferative syndrome (XLP), X-linked severe combined immunodeficiency (XSCID) and the Wiskott-Aldrich Syndrome (WAS). Transmission of these diseases along the female lineage provided the first evidences for the X-linked recessive mode of inheritance. In segregation studies within such pedigrees, a series of DNA probes, recognizing restriction fragment length polymorphisms (RFLPs) with known localizations on the X chromosome, established the X chromosomal locations of the XLIDs. The WAS locus was localized on the short arm of the X chromosome, the other XLIDs on the long arm. The frequency of meiotic recombination between the XLID gene and a particular RFLP is used as the measure for the distance between the XLID and RFLP loci (Fig. 1). Distinct X chromosomal deletions provided further data for the localization of the RFLPs and linked XLID genes. The WAS locus was assigned to the Xpl 1.4-Xpl 1.21 region, closely linked to the DXS 14, DXS255 and DXS7 loci (Peacocke & Siminovitch, 1987; De Saint Basile et al., 1989; Greer et al., 1990). The XSCID locus did as yet not manifest recombination with the PGK or DXS72 loci at the Xql2-Xq21.1 region. As males with deletions encompassing the DXS72 and DXS3 loci had no SCID, the XSCID locus is centromeric of DXS72 (De Saint Basile et al., 1987; Goodship, Levinsky & Malcolm, 1989; Puck et al., 1989). XLA is closely linked to DXS178 at Xq22 and is located between DXS3 and DXS94 with a multi-point lod score

exceeding 10 (Mensink et al., 1986b; Kwan et al., 1986; 1990; Malcolm et al., 1987; Guioli et al., 1989). Based on estimations using pulse-field gel electrophoresis, the Xq22 region containing the XLA locus spans about 8-12 mbp of genomic DNA (R.J. Levinsky, personal communication). XLP was found closely linked to the DXS37, DXS42 and DXS12 loci at Xq24-q27 (Skare et al., 1989a, 1989b). An interstitial deletion within Xq25 in an XLP pedigree supported this localization of XLP (Sanger et al., 1990). XHM was localized at the same Xq24-Xq27 region, linked to the DXS42 locus (Mensink et al., 1987a; Hendriks et al., 1991 b).

Correspondence: R. K. B. Schuurman, Division of Immunobiology and Genetics, Department of Immunohaematology, Building I E3-Q, University Hospital, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands.

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Genetics of X-linked immunodeficiency diseases PGK

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H .

Il _

Fig. 2. Restriction map of the 5' region of the human phosphoglycerate kinase (PGK) locus, compiled from Keith et al. (1986) and Vogelstein et al. (1987). The position of the PGK probe is indicated. E, EcoRI; BI, BglI; BI!, BgIII; H, HpaII. The BglI site is polymorphic (*), resulting in restriction fragment sizes of 1-3 and 1-7 kbp, as indicated.

DXS 94 DXS 17

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Fig. 1. Linkage maps of Wiskott-Aldrich syndrome (WAS), X-linked severe combined immunodeficiency (XSCID), X-linked agammaglobulinaemia (XLA), X-linked lymphoproliferative syndrome (XLP) and Xlinked immunodeficiency with hyper-IgM (XHM) and the RFLPs found closely linked to these X-linked immunodeficiencies (XLIDs). Recombination fractions (0) are given between the RFLP loci. For the RFLPs that did not manifest recombination with the XLID loci, the lod scores for linkage with the XLID gene at 0 = 0-0 are given below the linkage map. C, centromeric. The given recombination fractions are derived from Keats, Ott & Connealy (1989), Skare et al. (1989b), and Kwan et al. (1990). At the XLP locus the localization of DXS12 in relation to DXS42 is unknown, as DXS12 showed no recombination with either DXS42 or XLP.

X-CHROMOSOME INACTIVATION ANALYSIS Early in embryogenesis one of the two X chromosomes in somatic cells of females is inactivated. This phenomenon occurs randomly, but once established, the same X chromosome is inactivated in all progeny cells (Lyon, 1988). In principle, each X chromosome is active in about half of the cells within each female somatic cell lineage. In female XLID carriers, those cells that have the defective XLID gene on the active X chromosome will be arrested in differentiation, as is found in their XLID sons. This phenomenon only refers to primary affected cell lineages. As a result of the intrinsic differentiation arrest, female XLID carriers manifest a unilateral X-chromosome inactivation in one or more haematopoietic cell lineages. Cell lineages that are secondarily involved in the disease exhibit random X-chromosome inactivation. X-chromosome inactivation patterns in female cell populations were determined by the expression of glucose-6-phosphate dehydrogenase (G-6-PD) isoenzymes (Prchal et al., 1980; Conley et al., 1986). This method is limited by the incidence of this polymorphism in less than 1% of Caucasian females. Somatic cell hybrids can be obtained by fusion of human cells with the Chinese hamster fibroblast cell line RJK88 or the murine myeloma X63-Ag8.653, which are both deficient for the

X-encoded hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene (Puck, Nussbaum & Conley, 1987; Hendriks et al., 1989). After fusion, only those hybrids that contain the active human X chromosome express HGPRT and will survive in hypoxanthine-aminopterin-thymidine (HAT) medium. Subsequently the active X chromosome can be identified by any informative X-chromosomal RFLP, which does not have to be linked to the XLID gene in question. The analysis requires the production of 10-20 somatic cell hybrids to ensure a random sample survey. One-hundred and fifty-two out of 167 hybrids derived from fusions of human B or T lymphocytes with the X63-Ag8.653 myeloma were found to contain one or two human immunoglobulin heavy chain alleles as well as the T cell receptor (TCR) beta chain alleles (unpublished observation), both of which are encoded on chromosome 14 in humans. This high incidence of co-transfer of the X chromosome with chromosome 14 provides a method to confirm the polyclonal origin of the B and T lymphocytes utilized for fusion: different human immunoglobulin heavy chain or TCR rearrangements are to be exposed by Southern blotting (Hendriks et al., 1989). The X-chromosome inactivation status can also be determined from the differences in methylation between active and inactive X chromosomes. Hypomethylation of cytosine residues within CpG dinucleotide clusters flanking the X-chromosomal HGPRT, phosphoglycerate kinase (PGK) or G-6-PD loci is correlated with expression of the gene on active X chromosomes, whereas these CpG clusters are methylated on inactive X chromosomes (Wolf et al., 1984a, 1984b; Keith, Singer-Sam & Riggs, 1986). Methylation is determined by alternate digestions using the restriction endonucleases MspI and its methylation-sensitive isoschizomer, HpaII, which recognizes CCGG sequences only if the cytosine residues of CpG dinucleotides are unmethylated. Parallel to this analysis, the paternal and maternal X chromosomes are distinguished by RFLPs of the PGK, HGPRT or DXS255 loci (Vogelstein et al., 1987; Boyd & Fraser, 1990; Hendriks et al., 1991a). The 5' region of the PGK gene (Fig. 2) contains a polymorphic BglI site, which is informative in about 30% of females (Vogelstein et al., 1987). This segment also contains a CpG-rich region with eight CCGG sites, which are all methylated on inactive X chromosomes and unmethylated on active X chromosomes (Keith et al., 1986). HpaII therefore does not cleave any of these sites on inactive X chromosomes, leaving the polymorphic 1-3-kbp or 1-7-kbp fragments (Fig. 2) unchanged. On active X chromosomes these allelic fragments are cleaved

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into small segments by HpaII. A unilateral X-chromosome inactivation pattern is therefore identified by the absence of one of the two allelic fragments after HpaII digestion (Fig. 3). The remaining fragment is derived from the inactive X chromosome. A random X-chromosome inactivation pattern is characterized by the presence of both allelic fragments, each with approximately 50% reduction of the density after Hpall digestion, which results from the inactivated state of about half of the paternal and half of the maternal X chromosomes (Fig. 3). A similar DNA methylation distribution at the 5' region of the HGPRT gene can be linked to a BamHI polymorphism which is informative in almost 30% of females (Vogelstein et al., 1987). The DXS255 locus, recognized by the M27B probe, is hypervariable due to a 26-bp variable number tandem repeat (VNTR) motive with a heterozygosity rate of over 90% (Fraser, Boyd & Craig, 1989). The sizes of the PstI restriction fragments containing the VNTR region range from 4 bp to 10 kb (Fig. 4). The 5' PstI site flanking this VNTR region is 500 bp removed from an MspI site (Fig. 4). This MspI site is found methylated on active X chromosomes (Boyd & Fraser, 1990; Hendriks et al., 1991 a). Methylation results in the presence of PstI-PstI fragments only, after PstI/HpaII digestion. These PstI-PstI fragments are identified as being 500 bp larger than MspI-PstI fragments. The 5' MspI site on inactive X chromosomes is found to be unmethylated or up to 60% methylated (Hendriks et al., 1991 a). Digesion of inactive X chromosomal DNA that is unmethylated at the 5' MspI site results in the same fragments in PstI/HpaII digests as found in PstI/MspI digests. If the 5' MspI on the inactive X chromosome is partially methylated, the same fragments are exposed together with 500 bp larger PstI-PstI fragments. A unilateral X-chromosome inactivation pattern is identified by the presence in PstI/HpaII digests of only one of the two bands that are observed in PstI/MspI digests (Fig. 5). This band represents the inactive X chromosomes, on which the 5' MspI sites are unmethylated. The DXS255 allele of the active X chromosome is present as a 500 bp larger PstI-PstI fragment (Fig. 5). In a random X-chromosome inactivation pattern both bands exposed in PstI/MspI digests are also found in PstI/HpaII digests (Fig. 5; Hendriks et al., 1991a).

WISKOTT-ALDRICH SYNDROME WAS is characterized by a combined cellular and humoral immunodeficiency, thrombocytopenia and eczema (Wiskott, 1937; Aldrich, Steinberg & Campbell, 1954; Cooper etal., 1968; Perry et al., 1980). The affected males usually die in the first decade of life from recurrent and protracted bacterial and viral infections and/or persistent bleeding (Perry et al., 1980). The disease has an increased incidence of lymphoreticular malignancy. T lymphocytes progressively decline in number and function. WAS lymphocytes manifest a defect in the o-glycosylation of the membrane sialoglycoprotein CD43 (Greer et al., 1989a) and have abnormal microvilli (Kenney et al., 1986). The patients have a dysgammaglobulinaemia characterized by high IgA and IgE, low IgM and normal or elevated IgG levels and lack a response to polysaccharide antigens. WAS platelets have a decreased mean platelet size with abnormal granules and a reduced expression of the highly o-glycosylated membrane glycoproteins Ia and Ib (Aldrich et al., 1954; Cooper et al., 1968;

(a)

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( b)

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Fig. 3. Analysis of X-chromosome inactivation patterns by methylation differences of the PGK alleles in T cell populations, showing a random X-chromosome inactivation (a), and a unilateral X-chromosome inactivation in an obligate carrier of XSCID (b). -, DNA digested with

EcoRI, BglI and BgIII; +, DNA digested also with HpalI.

M27B

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Fig. 4. Restriction map of the hypervariable DXS255 locus. P, Pstl; H, HpaII/MspI. Fragment sizes are in kbp. The position ofthe M27B probe is indicated. VNTR, variable number of tandem repeats.

Perry et al., 1980; Ochs et al., 1980; Parkman et al., 1981). WAS granulocytes appear to be deficient in adherence, whereas phagocytosis and intracellular killing were found normal (Cooper et al., 1968; Ochs et al., 1980). In several families, X-linked thrombocytopenia has been reported with chronic eczema or with a high serum IgA level or low isohaemagglutinin titres, but no further disordered immunity (Ata, Fisher & Holman, 1965; Canales & Mauer, 1967; Donner et al., 1988). In one family with isolated X-linked thrombocytopenia linkage was found to the same chromosomal region (Xpl 1.4-Xp 1.21) as the WAS locus (Donner et al., 1988). These data indicate that X-linked thrombocytopenia and WAS are closely related and may involve different mutations within the same locus, this being known to give rise to phenotypic heterogeneity (Monaco et al., 1988). Studies in peripheral blood haematopoietic cell populations of two obligate WAS carriers who were G-6-PD heterozygotes, exposed a unilateral X-chromosome inactivation in T lymphocytes and platelets (Gealy, Dwyer & Hanley, 1980; Prchal et al., 1980). Monocytes, granulocytes and B lymphocytes manifested unilateral or severely skewed X-chromosome inactivation. Random X-chromosome inactivation was found in fibroblasts. Using the X chromosomal methylation analysis method, in

Genetics of X-linked immunodeficiency diseases ( a)

M

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(b) M

glycosylation pathway involved in the membrane expression of several glycoproteins.

H

kbp I- 5-3 -5*I

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.H...0u0;~~~~~~~~~~~~~~ ..... .S .... Fig. 5. Analysis of X-chromosome inactivation by methylation differat the 5' MspI sites of the DXS255 alleles in the T cell populations of two females. The DXS255 polymorphism in these two sisters entailed a 4 6-kbp and a 4-8-kbp MspI-PstI fragment (M lanes). After digestion with PstI and HpaII the 4 6-kbp and 4-8-kbp fragments reflect the HpaII-PstI fragments on the inactive X chromosomes, in which the 5' MspI sites are unmethylated. In addition, 5 1- and 5-3-kbp fragments are detected which reflect PstI-PstI fragments of both active and inactive X chromosomes. In the non-carrier female (a) a random X-chromosome inactivation was observed. In an obligate carrier of Wiskott-Aldrich syndrome (b) a unilateral X-chromosome inactivation was observed: the 4-8-kbp fragment but not the 4-6-kbp fragment was found in the PstI/ HpaII digest (H lane). The 5' MspI site on the active X chromosome was completely methylated, resulting in the absence of the 4-6-kbp HpaIIPstI fragment and the presence of the 5. l-kbp PstI-Pstl fragment only. The 5' MspI site on the inactive X chromosome was partly methylated, resulting in the presence of both a 4-8-kbp HpaII-PstI fragment and a 5 3-kbp PstI-PstI fragment. ences

more

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than 15 females unilateral X chromosome inactivation

patterns were found in B and T lymphocytes, granulocytes and monocytes (Fearon et al., 1988; Greer et al., 1989b; Hendriks et

al., 1991a). The unilateral X-chromosome inactivation in the platelets, granulocytes, monocytes, B and T lymphocytes of carriers implies that within these populations the cells that express the WAS defect are arrested in differentiation, as they do not emerge in the circulation. This situation contrasts with normal numbers of monocytes, granulocytes and B lymphocytes found in affected males (Cooper et al., 1968; Ochs et al., 1980). The genotypic and phenotypic data on haematopoietic cell functions and developments in obligate carriers and their affected sons provide some specifications on the WAS gene. The function of the gene appears to be confined to the haematopoietic cell lineages, possibly with the exception of erythrocytes in which the results were inconclusive (Prchal et al., 1980). Most cell lineages exhibit functional defects that seem to result in selective advantages for functional intact cells. The defect is predominant in platelets and T lymphocytes, more so than in B lymphocytes and granulocytes. As disordered adherence and membrane defects have been observed in more than one cell lineage, WAS may entail a general haematopoietic cell membrane defect. Inferred from the defects in the lymphocyte membrane sialoglycoprotein CD43 and the platelet membrane glycoproteins Ia and Ib, the WAS gene has a function in an o-

X-LINKED SEVERE COMBINED IMMUNODEFICIENCY

SCID is not a single disease entity, but a heterogeneous group of genetic disorders characterized by defects in both T and B lymphocytes. Generally, recurrent infections start a few months after birth and patients die before reaching 2 years of age. SCID is usually associated with malabsorption, growth retardation and failure to thrive (Gelfand & Dosch, 1983; Rosen et al., 1984). Most SCID patients have markedly reduced numbers of circulating T cells with deficient responses to antigens and mitogens. Patients have hypogammaglobulinaemia, often with normal or increased peripheral blood B cell numbers (Gelfand & Dosch, 1983; Conley et al., 1990a; Gougeon et al., 1990). B cells from SCID patients were shown to have an impairment in their responses to stimuli that induce in vitro B cell proliferation, such as IL-2 and IL-6 (Gougeon et al., 1990), but could be transformed into B lymphoblastoid cell lines by Epstein-Barr virus (Thompson et al., 1990; and our unpublished observation). In the presence of normal T cells, B cells from several SCID patients exhibited responses to pokeweed mitogen or antigens (Buckley et al., 1976; Pahwa et al., 1980; Dosch, Schuurman & Gelfand, 1980; Gelfand & Dosch, 1982). SCID patients who had received bone marrow transplants and manifested engraftment of donor T cells only, commenced antibody synthesis, apparently by the host SCID B cells (Buckley et al., 1986). These data argue for a B cell maturation defect due to a T helper cell deficiency, but do not exclude that the B cell population may also be intrinsically affected. Some SCID patients manifest a complete absence of B lymphocytes or even pre-B cells (Hayward, 1978; Thompson et al., 1990). This type of SCID appears to be expressed predominantly in the B cell lineage, where immunoglobulin heavy or light chain rearrangements were found absent, whereas in T lymphocytes the af TCR loci were rearranged (Thompson et al., 1990). Defects in the purine salvage pathway enzymes adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) account for about 50% of the autosomal recessive cases (Hirschhorn, 1986). SCID can also result from defective transcription of IL-2 or multiple T cell lymphokine genes (Weinberg & Parkman, 1990; Chatila et al., 1990), from defects in signal transduction in T lymphocytes (Chatila et al., 1989), in the expression of MHC determinants (Schuurman et al., 1979) or from an abnormal capping of cell surface molecules (Gelfand et al., 1979). Apart from these specified deficiencies SCID can originate from as yet obscure gene defects (Gelfand & Dosch, 1983; Thompson et al., 1990), one of which is localized on the X chromosome. Seventy to eighty per cent of infants with SCID are males (Gelfand & Dosch, 1983; Rosen et al., 1984). Many affected males do not present with a family history of the disease, representing the first expression of an XSCID mutation (Haldane, 1935) or an autosomal form of SCID. XSCID patients usually have increased numbers of B cells (Conley et al., 1990a). If the disease is not due to one of the characterized autosomal defects, patients with XSCID or autosomal SCID with B cells

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cannot be distinguished by their clinical or immunological features. In the T lymphocyte populations of at least 14 XSCID carriers, a unilateral X-chromosome inactivation was found (Puck et al., 1987; Conley et al., 1988; Goodship et al., 1988; Conley et al., 1990a; Goodship et al., 1991). Three XSCID carriers manifested a skewed X-chromosome inactivation pattern, as 7-25% of the T cell-derived somatic cell hybrids contained the X chromosome with the XSCID defect (Conley et al., 1988). In the T cell populations of two obligate carriers that belonged to one pedigree (and thus had an identical mutation in the XSCID gene), a unilateral X-chromosome inactivation was found in one and a skewed X-chromosome inactivation in the other (Conley et al., 1988). The data demonstrate that XSCID involves a severe intrinsic T cell differentiation arrest. Finding a minor fraction of XSCID T lymphocytes in some carriers is consistent with the very low but often detectable numbers of T lymphocytes found in patients. These T lymphocytes most likely reflect an immature T cell subpopulation. In order to investigate whether in XSCID the failure of the B cells to mature to plasma cells originates from the absence of T cell help only, X chromosome inactivation patterns were determined in B lymphocyte populations. In most XSCID carriers a unilateral X-chromosome inactivation was observed (Conley et al., 1988; Goodship et al., 1991). These data are striking, as they indicate an intrinsic defect in B cell development. The normal or elevated numbers of B lymphocytes found in the patients (Gelfand & Dosch, 1982; Conley et al., 1990a), demonstrate that this intrinsic B cell defect can be circumvented. In some XSCID carriers a random X-chromosome inactivation was observed in the B cell population (Goodship et al., 1991). Female carriers that belonged to one pedigree manifested both random and unilateral X-chromosome inactivation, excluding different mutations in the XSCID gene as an explanation for the presence or absence ofXSCID B lymphocytes in the carriers. The occurrence of random X-chromosome inactivation in peripheral B lymphocyte populations of carriers indicate that additional factors can overrule a selective disadvantage for XSCID B cells. Similar variability in the X-chromosome inactivation patterns was also found in the granulocyte and monocyte populations (Conley et al., 1988; Goodship et al., 1991). The presence of unilateral X-chromosome inactivation in diverse cell populations indicates that the XSCID gene is expressed in T cells, B cells, granulocytes and monocytes. The XSCID gene therefore may be involved in a metabolic pathway common to several haematopoietic cell lineages. This metabolic pathway apparently has a predominant function in T lymphocyte differentiation with a less crucial involvement in other haematopoietic cell lineages. X-LINKED AGAMMAGLOBULINAEMIA Characterization of the B tymphocyte defect in XLA XLA is characterized by protracted and recurrent bacterial infections, in most cases confined to the upper and lower airways (Rosen et al., 1983, 1984; Schuurman et al., 1987a; Timmers et al., 1991). XLA patients have very low but detectable serum immunoglobulin levels. Peripheral blood B cell numbers are severely decreased and plasma cells are virtually lacking (Conley, 1985). The bone marrow contains normal

numbers of B cell precursors expressing terminal deoxynucleotidyl transferase, membrane CDl9 and the common acute lymphoblastic leukaemia antigen CD10 (Campana et al., 1990). The numbers of pre-B lymphocytes identified by cytoplasmic immunoglobulin heavy chain mu constant region (Cp) expression have been reported to be normal or decreased (Pearl et al., 1978; Landreth et al., 1985; Campana et al., 1990). The cytoplasmic Cy pre-B lymphocytes that are present in XLA appear to express IgM heavy chain molecules lacking the variable region segment (Schwaber et al., 1983). Based on the absence of rearrangements of immunoglobulin heavy chain variable region gene segments, it was proposed that XLA could be involved in recombinations of these gene segments (Schwaber et al., 1983). A defect in the presently known recombinases would not only lead to deficient expression of immunoglobulin but also of the TCRs, as these recombinases mediate both the immunoglobulin and TCR rearrangements (Timmers et al., 1991). In XLA the T lymphocytes differentiate to antigenrecognizing stages bearing aB or yc TCRs (Campana et al., 1990). Moreover, B lymphoblastoid cell lines established from XLA patients manifest normal immunoglobulin gene rearrangements with a large junctional diversity of immunoglobulin heavy and light chain V, (D) and J gene segments including D-to-D fusions and addition of N segments (Mensink et al., 1986a; Anker, Conley & Pollok, 1989; Timmers et al., 1991). These data refute a defective recombinase in XLA. As the fraction of immunoglobulin-producing B lymphocytes is severely decreased, XLA appears to entail a defect in the clonal expansion of B lymphocytes, possibly due to deficient expression of a B cell lineage specific growth factor receptor. Unilateral X-chromosome inactivation in the peripheral blood B lymphocyte population of obligate XLA carriers was exposed by the expression of only one of the G-6-PD isoenzymes (Conley et al., 1986), by DNA methylation studies (Fearon et al., 1987), and by RFLP analysis of somatic cell hybrids (Conley & Puck, 1988; Hendriks et al., 1989). In 115 somatic cell hybrids only the X chromosome that carried the intact XLA gene was found (Conley et al., 1988). T lymphocytes, monocytes or granulocytes of XLA carriers always manifested a random X chromosomal inactivation pattern which precluded a primary involvement of these cell populations in XLA. In conclusion, the XLA gene is crucial in the differentiation of precursor B lymphocytes, its function being intrinsic and restricted to the B cell lineage. Genetic heterogeneity of XLA By RFLP linkage analyses in more than 30 pedigrees, the XLA gene was localized at Xq22. In three pedigrees the statistical genetic analysis suggested a different location of the gene segregating in these pedigrees. In one pedigree the analysis was inconclusive, and in another the patients were found to have XHM instead of XLA (Lau et al., 1988; Timmers et al., 1991). The third XLA pedigree manifested such a high frequency of recombination between the agammaglobulinaemia locus and the Xq22 RFLP markers, that a separate agammaglobulinaemia gene located elsewhere was postulated (Mensink et al., 1986b; Ott et al., 1986). This analysis was based on the assumption that the XLA defect was transmitted by a female with two daughters who each had an affected son. In the B lymphocyte populations of these daughters a unilateral X inactivation was found, thus providing evidence for the X-

Genetics of X-linked immunodeficiency diseases linked segregation of this B cell differentiation defect (Hendriks et al., 1989). However, the unilateral X-chromosome inactivation involved the paternal X chromosome instead of the maternal X chromosome, which indicated that the agammaglobulinaemia originated from the father of these carriers and not from their mother. Since the father did not have agammaglobulinaemia and XLA has full penetrance, this male was an X chromosomal mosaic: his B lymphocytes contained an X chromosome carrying an intact XLA gene, whereas spermatocytes apparently harboured an X chromosome carrying a defective gene. A paternally derived XLA defect allowed construction of the pedigree without any recombination between an Xq22-located gene and the linked RFLP loci, and thus involved the Xq22 XLA gene (Hendriks et al., 1989). In other XLA pedigrees with a recent origin of the mutation, which is the case in about one-third of the XLA families encountered, X chromosomal RFLP segregation analyses suggested that in about half of the pedigrees the XLA defect was introduced by a healthy male. These analyses also suggested the existence of maternal mosaicism. X chromosomal mosaicisms have also been observed in several other X-linked diseases, including WAS (Hendriks et al., 1989; Arveiler et al., 1990). At present there is no evidence for genetic heterogeneity of XLA, but there is ample evidence for the occurrence of X chromosomal mosaicisms at the introduction of new XLA mutations. XLA with isolated growth hormone deficiency (XLA-GHD) Two families have been described with XLA and growth hormone deficiency (GHD) without other endocrine abnormalities (Fleisher et al., 1980; Conley et al., 1989). The human growth hormone has an autosomal localization (Owerbach et al., 1980). This XLA-GHD syndrome could represent an allelic variant of XLA, a contiguous gene deletion syndrome, or an unrelated gene defect with a similar immunologic phenotype. Analyses of karyotypes in XLA-GHD did not reveal Xchromosome abnormalities (Conley et al., 1989). By RFLP linkage studies in two pedigrees, this disorder was localized at the same X chromosomal region as XLA. In two obligate carriers unilateral X-chromosome inactivation was demonstrated in the B cell lineage but not in T lymphocytes or granulocytes (Conley et al., 1989). XLA-GHD reflects a variant of Xq22-linked XLA, which is accompanied by a closely linked defect in a regulator of growth hormone production.

X-LINKED LYMPHOPROLIFERATIVE SYNDROME

Males affected by XLP develop a large variety of immunodeficiencies, even within the same family, after infection with Epstein-Barr virus (EBV) (Grierson & Purtilo 1987). The XLP syndrome may be characterized by fatal or chronic mononucleosis, hypogammaglobulinaemia, aplastic anaemia and malignant lymphocytic proliferations (Grierson & Purtilo, 1987). XLP patients fail to generate an antibody response to EBV nuclear-associated antigen. The large phenotypic heterogeneity is partly explained by the range of B cell differentiation stages at which the EBV receptor is expressed, i.e. from a precursor B cell stage prior to genomic immunoglobulin

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rearrangements (Thompson et al., 1990) up to the plasma cell stage. Obligate carriers may have abnormal titres to EBV antigens (Grierson & Portilo, 1987). Random X-chromosome inactivation was found in granulocytes, B and T lymphocytes from two unrelated obligate female XLP carriers (Conley et al., 1990b). As it is very likely that these carriers were exposed to EBV, the data suggest that the defect is not intrinsic to EBV infected cells, i.e. the B cell lineage. X-LINKED IMMUNODEFICIENCY WITH HYPERIMMUNOGLOBULINAEMIA M

Patients with immunodeficiency with hyperimmunoglobulinaemia M have frequent or chronic infections, comparable to XLA. XHM patients are susceptible to autoimmune haemolytic anaemia and malignant proliferation of IgM-producing B lymphoblasts (Rosen et al. 1961, 1984; Rosen & Janeway, 1966). Apart from the X-linked form, autosomal recessive and acquired forms of immunodeficiency with hyper-IgM occur (Rosen et al., 1984). The defect originates from an impairment of the immunoglobulin heavy chain class switch of B lymphocytes from IgM to IgG or IgA production. XHM is characterized by elevated IgM levels, normal or elevated IgD and severely decreased IgG, IgA and IgE levels in the serum (Rosen et al., 1961; Schwaber, Lazarus & Rosen, 1981; Geha et al., 1979; Levitt et al., 1983). The proportion of B lymphocytes that express surface IgM and/ or IgD is normal or increased. IgG- or IgA-producing B lymphocytes and plasma cells are lacking in peripheral blood, lymph nodes and bone marrow. B lymphoblastoid cell lines established from peripheral blood fail to secrete IgG or IgA (Schwaber et al., 1981). T lymphocyte (sub)populations and T cell responses to antigens, mitogens and allogeneic cells are normal (Geha et al., 1979; Schwaber et al., 1981; Levitt et al., 1983). The defect was proposed to be intrinsic to the B lymphocytes, since allogeneic T lymphocytes did not correct the defect (Geha et al., 1979; Schwaber et al., 1981; Levitt et al., 1983). However, in vitro addition of T lymphoblasts obtained from a patient with a Sezary-like syndrome induced IgG and IgA production by hyper-IgM B lymphoblasts, suggesting that the immunoglobulin heavy chain class switch system in these B lymphocytes was intact (Mayer et al., 1986). If XHM originates from a switch defect that is intrinsic to the B lymphocytes, obligate female carriers should manifest a switch to IgG and IgA production only in those B cells that express the intact gene on the active X chromosome. In separate IgM-, IgG- and IgA-expressing B cell populations from two obligate female carriers, random X-chromosome inactivation patterns were found (Hendriks et al., 1990). In the IgA- or IgGexpressing B cells that had inactivated the X chromosome that carried the intact XHM gene, the heterogeneity of the immunoglobulin heavy and light chain rearrangements was found to be as extensive as the heterogeneity in the B cells with inactivated homologous X chromosome (Hendriks et al., 1990). The lack of any preference in switch in either population demonstrates that the intrinsic immunoglobulin heavy chain class switch mechanism is intact. XHM may encode a factor that is transferred to the B lymphocytes to induce immunoglobulin heavy chain class switch. This factor could well be produced by T lymphocytes.

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The immunological phenotype ofXHM overlaps with XLA. The distinct X-chromosome inactivation patterns in B cells of the carriers and the different X-chromosome locations of these diseases support the conclusion that XLA and XHM involve distinct genes. The genotype of XHM cannot be distinguished from XLP, as both diseases were found to be linked to the DXS42 RFLP locus and in both XHM and XLP carriers the B lymphocyte population manifest random X-chromosome inactivation.

CLINICAL APPLICATION OF X-CHROMOSOME INACTIVATION The XLIDS entail X-linked recessive disorders in the differentiation of one or more haematopoietic cell lineages. Female carriers do not manifest any haematopoietic abnormality, but manifest a unilateral X inactivation in the haematopoietic cell populations that are intrinsically affected by the XLID defect. Vice versa, X chromosome inactivation analyses in haematopoietic cell populations provide a method to investigate the carrier status of females at risk, i.e. those who belong to an XLID pedigree. In WAS, almost all haematopoietic cell lineages will manifest a unilateral X-chromosome inactivation. In XLA, unilateral X-chromosome inactivation is confined to the B cell lineage. In XSCID, unilateral X-chromosome inactivation in T lymphocytes is conclusive. A significant proportion of XSCID carriers manifested a skewed X-chromosome inactivation in the T cells. This complicates the carrier detection in females at risk, since skewed X-chromosome inactivation patterns have also been found in females without a history of X-linked diseases (Fialkow, 1973; Vogelstein etal., 1987; Boyd & Fraser, 1990). In case of skewed X-chromosome inactivation, an RFLP segregation analysis in the pedigree should be performed to confirm that the preferentially inactivated X chromosome indeed carries the XLID defect. Verification by RFLP analysis also provides the reliability derived from the recombination fractions with the linked RFLPs (Fig. 1). In XHM and XLP, the random X-chromosome inactivation patterns found in the haematopoietic cell (sub)populations preclude carrier detection by X-chromosome inactivation analysis. X-chromosome inactivation analysis can also be applied to differentiate between X-linked and autosomal inheritance of agammaglobulinaemia or SCID. Although autosomal types of agammaglobulinaemia usually present with surface-immunoglobulin-positive B lymphocytes in peripheral blood, occasionally female patients lacking B lymphocytes are observed (Hoffman et al., 1977). About halfofthe SCID patients do not present with one of the defined autosomal defects or with a family history of X-linked segregation. Unilateral X-chromosome inactivation in haematopoietic cell lineages from mothers of patients supports the X-chromosomal inheritance of the immunodeficiency and identifies the X chromosome that carries the defect, the linked RFLP haplotype being verified by segregation analyses. A random X-chromosome inactivation pattern is always found in an autosomal form of the immunodeficiency, but does not exclude X-linked transmission, since maternal X chromosomal mosaicism can be involved. In pedigrees with male patients in one generation only, thus suggesting a recent introduction of the XLID, X-chromosome inactivation analyses should be performed to differentiate

between a paternal or maternal origin of the XLID mutation, to preclude errors in prenatal diagnosis based on RFLP segregation analysis (Hendriks et al., 1989). X-chromosome inactivation analyses are informative in all females. The informative fractions associated with the currently available RFLP loci at which methylation differences assess the X-chromosome inactivation status, leave a chance of about 5% that a female is homozygous for all these loci. In this situation the method of selective transfer of the active X chromosome to somatic cell hybrids can be applied, with more than 200 RFLP markers available to identify the active X chromosome (Kidd et al., 1989). In contrast to carrier detection based on RFLP segregation analysis, the X-chromosome inactivation analysis is not dependent on probabilities of recombination. Prenatal diagnosis for XLIDs is based on segregation analysis of linked RFLP loci (Schuurman et al., 1987b; Lau et al., 1988). The frequencies of recombination with the linked RFLP loci dictate the reliability of the predictions. For all XLIDs except XHM, closely linked RFLP markers at both sides of the gene are available (Fig. 1). If a female carrier is heterozygous for these flanking RFLP markers, genetic counselling can be provided with more than 98% reliability, as the chances for the occurrence of double recombinations between these flanking markers are extremely low. If only one of the RFLP markers is informative, the reliability is dependent on the presently known recombination fractions and their lod scores

(Fig. 1). The DXS255 and PGK loci, which are utilized in the Xchromosome inactivation analysis of XLIDs are closely linked to the WAS and the XSCID gene, respectively. Therefore, the analysis for X-inactivation patterns in the carriers at the same time provides information on which particular RFLP allele is linked to the disease, of use for prenatal diagnosis. The phenotypic heterogeneity of the XLIDs may confuse diagnosis in some patients. This heterogeneity not only occurs between, but also within pedigrees. X-chromosome inactivation analyses in the mother of a patient with atypical clinical and immunological features was shown to be decisive for the diagnosis of WAS (Hendriks et al., 1991a). RFLP linkage analyses have also provided the possibility of an early, presymptomatic diagnosis in XLA, where investigation of the immunoglobulin production in the newborn is precluded because of the presence of maternally derived immunoglobulin (Schuurman et al., 1987b). These examples also established the validity of the genetic investigations. X-chromosome inactivation and RFLP segregation analysis provide relevant methods for prevention of XLA, XSCID and WAS, which involve a major proportion of children with severe inherited immunodeficiencies. CONCLUSIONS The X chromosomal RFLP linkage and deletion analyses established the locations of the X-CGD, PD, WAS, XSCID, XLA, XLP and XHM genes (Table 1). All were found at different chromosomal regions with the exception of XHM and XLP. As the XHM and XLP genes apparently have distinct functions, further RFLP analyses in XHM pedigrees are expected to expose a distinct location, although a distinct mutation in a gene complex cannot be excluded at present.

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Table 1. Genetics of X-linked immunodeficiencies

Location

X-CGD

Xp2l

Cell lineages expressing the gene

Granulocytes, monocytes

PD

Xp21.1-11.23

WAS

Xpl 1.4-pl 1.21

XSCID

Xql2-q21.l

XLA XLP

Xq22 Xq25

XHM

Xq24-q27

Macrophages

Platelets, T lymphocytes, B lymphocytes, granulocytes, monocytes T lymphocytes, B lymphocytes, granulocytes, monocytes B lymphocytes Unknown

Switch inducing T lymphocytes?

Postulated gene function

f-chain of cytochrome b complex Stabilization of the C3 convertase (in the alternative pathway of complement) o-glycosylation of several membrane glycoproteins

Basic metabolic pathway, predominant in T cells

B cell expansion Handling of EBV-infected B lymphocytes Immunoglobulin heavy chain class switch factor, transferred to B lymphocytes

X-CGD, X-linked chronic granulomatous disease; PD, properdin deficiency; WAS, Wiskott-Aldrich syndrome; XSCID, X-linked severe combined immunodeficiency; XLA, X-linked agammaglobulinaemia; XLP, X-linked lymphoproliferative syndrome; XHM, X-linked immunodeficiency with hyper-IgM.

The X-CGD encodes the f-chain of the cytochrome b involved in killing of micro-organisms in granulocytes and monocytes. Properdin stabilizes C3 convertase of the alternative pathway of the complement system. As the murine properdin gene has recently been isolated (Goundis & Reid, 1988), characterization of the human properdin gene is expected to follow soon. The functions of the XLID genes are still obscure. All the XLIDs appear to be involved in the development of the immune response generated by T and/or B lymphocytes. X-chromosome inactivation analyses in carriers of WAS demonstrated that almost all haematopoietic cell lineages were intrinsically affected. These data demonstrate that the WAS gene is expressed in all these cell lineages (Table 1). The WAS gene appears to be critical at different stages of maturation of the various cell lineages. WAS has a vital effect on platelet numbers, morphology and function. The predominant T lymphocyte disorder in the patients does not preclude a development of mature T lymphocytes, although their numbers are decreased. The B lymphocytes appear to be involved only at mature plasma cell stages. Clinical symptoms of an adherence defect in monocytes or granulocytes are hardly manifest but nevertheless evident from the functional studies and X-chromosome inactivation analyses. The WAS gene appears to be involved in o-glycosylation of several membrane proteins expressed by these haematopoietic cell lineages. In XSCID the T cell deficiency is predominant and found to be expressed at an early immature stage of differentiation. The B

lymphocytes appear normal in quantity and in function in the patients, but in many carriers the XSCID B lymphocytes were found to be subject to a selective disadvantage. The random Xchromosome inactivation found in other carriers illustrates that the defect can be circumvented, possibly by the presence of helper T lymphocytes that express the intact gene. The unilateral X chromosomal inactivation in monocytes and granulocytes of a substantial proportion of XSCID carriers demonstrates that the XSCID gene is not only expressed in the lymphoid lineage, but also in monocytes and granulocytes. The proposed defect in a basic metabolic pathway may also be inferred from similar combined immunodeficiency phenotypes originating from autosomal defects in purine metabolism (ADA and PNP deficiency) and in DNA repair (murine SCID mutation) (Fulop & Phillips, 1990). WAS and XSCID are not merely lymphoid immunodeficiencies, but more generalized haematopoietic deficiencies. In XLA, the precursor B lymphocyte differentiation arrest was shown to be dependent on an intrinsic defect. The defect is confined to the B cell lineage and cannot be circumvented by the in vivo presence of intact B lymphocytes, or factors extrinsic to the B lymphocyte compartment, such as other haematopoietic cells or the bone marrow micro-environment. The very few immunoglobulin-expressing lymphocytes found in XLA patients only demonstrate that the immunoglobulin recombinatory mechanism is intact and that the defect is more involved in the expansion of these immature B lymphocytes. In XHM and XLP patients, the disorder appears to be

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confined to the B cell population. The random X-chromosome inactivation patterns in the carriers indicate that the pathogenesis involves other than intrinsic B cell mechanisms. The discrepancies in the development of the affected haematopoietic cell populations found between the XLID carriers and their affected sons demonstrate that the expression of the defect is subject to additional factors. These conclusions stress the probability for larger phenotypic heterogeneity in XLIDs than is presently recognized and provides an explanation for the diagnostic dilemmas which are frequently encountered. In WAS, a few patients reach adulthood with minimal immunodeficiency and no severe symptoms of the platelet disorder (D. Frey, personal communication). Even in XLA the occasional patient may have normal 1gM levels and IgG levels slightly below normal (unpublished observation). Such dilemmas can be resolved by an X-chromosome inactivation analysis in the mothers of the patients and successive demonstration by RFLP analysis that the XLID defect has been transmitted to the patients. These analyses also expose the types of SCID or agammaglobulinaemia, being encoded on the X chromosome as well as on autosomes. Although direct analyses ofthe genes or their functions is not possible, as is the case in X-CGD and PD, the genetic analyses for the XLIDs have provided a better insight into the underlying defects and have contributed significantly to prevention of these severe immunodeficiency diseases in children.

ACKNOWLEDGMENTS These studies were supported in part by the Dutch Prevention Fund (grants 28.954, 28.1102 and 28.1607) and the Dutch Foundation for Science (grants 501.056, 504.102, and 507.113).

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