|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
IMMUNOBIOLOGY
From the Department of Pathology and Microbiology,
Center for Human Molecular Genetics, Eppley Institute for Research in
Cancer and Allied Diseases; the Departments of Pathology and
Microbiology and Pediatrics, University of Nebraska Medical Center,
Omaha, NE; the Center for Microbiology and Tumorbiology, Karolinska
Institute, Stockholm, Sweden; the Laboratorio di Genetica Molecolare,
Instituto Giannina Gaslini, Genoa, Italy; the Department of Pediatrics,
Kochi Medical School, Nankoku, Japan; and the Division of Hematology
and Oncology, Children's Hospital Medical Center, Cincinnati, OH.
The purposes of this study were to determine the frequency of
mutations in SH2D1A in X-linked lymphoproliferative disease (XLP) and
the role of SH2D1A mutations and Epstein-Barr virus (EBV) infection in
determining the phenotype and outcome of patients with XLP. Analysis of
35 families from the XLP Registry revealed 28 different mutations in 34 families X-linked lymphoproliferative disease (XLP) (Duncan
disease; MIM 308 240) is a congenital immunodeficiency disease
estimated to affect approximately 1 in 1 × 106
males.1 Although more than 98% of persons in the general
population experience little or no clinical manifestations after
Epstein-Barr virus (EBV) infection, XLP has been characterized by
exquisite vulnerability to EBV infection. The spectrum of phenotypes
seen in XLP is varied. The most common phenotype is fulminant
infectious mononucleosis (FIM), which is often fatal and
clinicopathologically identical to the virus-associated hemophagocytic
syndrome. Lymphoproliferative disorders (LPD) are the next most often
observed. These may manifest as polyclonal or monoclonal proliferations
and may range from lymphoid vasculitides to malignant lymphoma. Both
B-cell and T-cell non-Hodgkin lymphoma and Hodgkin disease have been
observed. Dysgammaglobulinemia (dys- The gene mutated in XLP has been mapped to the interval between DXS1001
and DXS8057.5 This chromosomal region contains the
tenascin-m gene, a human homologue of the Drosophila
extracellular protein coding gene and SH2D1A.6 The latter
was found to be mutated in 9 of 16 unrelated patients with
XLP.6 The gene spans 40 kb of genomic sequence and
contains 4 exons. The human SH2D1A gene gives rise to a transcript of
2.5 kb expressed in CD4+ and CD8+ T
lymphocytes, the thymus, fetal liver, colon, and spleen, which encodes
a protein with a src homology 2 (SH2) domain.6-8 The SH2
domain is a highly conserved structure containing approximately 100 amino acid residues and is found in cytosolic nonreceptor tyrosine
kinases and in a number of proteins that play key roles in signal
transduction that modulate enzyme activity or target proteins to
certain cellular locations. The core element of the structure is an
antiparallel This study is a comprehensive analysis of the correlation of SH2D1A
mutations and EBV infection with clinical phenotype and outcome in XLP.
Data and material from the David T. Purtilo International XLP Registry
were used for these studies. SH2D1A mutational analysis was performed
on 35 families with a confirmed X-linked inheritance (ie, 2 or more
maternally related affected males from the XLP Registry). We also
studied 25 unrelated patients with sporadic XLP, a phenotype
reminiscent of XLP but without the corroborating family history and 9 persons with chronic, active EBV (CAEBV) syndrome.12 Using
data from the XLP Registry, the EBV status of boys with XLP was
determined at the time of their first clinical manifestation to
determine what effect EBV infection has on clinical phenotype and
outcome. We provide evidence that mutations in the SH2D1A gene are
responsible for XLP. However, this study illustrates that EBV infection
is not the only trigger for the clinical manifestations of XLP, and
that neither EBV infections nor mutations in the SH2D1A gene are
predictive of clinical phenotype or outcome.
Patients
Linkage analysis
Haplotype analysis.
Haplotypes for the RFLP markers DXS37, DXS6, DXS100, and DXS42 were
constructed, as described earlier.13 Labeled
microsatellite markers DXS8088, DXS1220, DXS8055, DXS8053, DXS8081,
DXS8064, DXS8067, DXS1001, DXS8057, AFM240xb10, DXS8093, DXS8009,
DXS1206, DXS8078, DXS1046, and DXS1047 were also used in some cases.
Primers and annealing temperatures used to amplify the microsatellite markers were as previously described.14
Mutation detection.
Peripheral blood lymphocytes from family members with XLP, patients
with so-called sporadic XLP, and patients with CAEBV were collected
throughout North and South America, Europe, the Middle East, Australia,
and Japan. Genomic DNA was isolated from blood samples using
standard techniques. When lymphoblasts were available, they were grown
in RPMI 1640 supplemented with 10% fetal bovine serum under standard
culture conditions. DNA and RNA were isolated using standard techniques
and Qiagen (Valencia, CA) RNA isolation kits, respectively. Coding
sequences, 5' regulatory region ( Reverse transcriptase-PCR
EBV status Males who underwent EBV testing at time of their first clinical manifestation were analyzed for age, phenotype of first manifestation, and survival. Evidence of EBV infection included one or more of the following: positive heterophile antibody test result, positive EBV titer to any EBV antigen, or evidence of EBV by in situ hybridization in tissue or by PCR in tissue or blood. Patients who did not undergo testing or who previously received gamma-globulin supplementation or blood products before serologic testing of EBV status were considered indeterminable for EBV status.Statistical analysis Median age of first clinical presentation and survival calculated for the EBV+ and EBV groups were
compared using the Mann-Whitney U test. Differences in
phenotypes between the groups were compared by the
 2 test. Statistical analysis and calculations were
performed using software from Statview SE+Graphics, version 1.02 (Abacus Concepts, Berkeley, CA).
We previously reported that a definitive diagnosis of XLP can be
made if 2 or more maternally related males manifest an XLP phenotype.2 To determine the frequency of mutations of the SH2D1A gene in XLP, 35 families with definitive diagnoses of XLP were analyzed. Thirty-three of the 35 underwent previous RFLP linkage
analysis studies that demonstrated a strong association with the SH2D1A
locus. The families are of diverse ethnic origins, from the United
States, Canada, the United Kingdom, Australia, The Netherlands, New
Zealand, Israel, Finland, Turkey, and Argentina. Twenty-five males with
sporadic XLP who manifested a phenotype consistent with XLP (FIM, LPD
or dys- To identify the molecular alterations in the SH2D1A gene responsible
for the pathologic phenotype(s) of XLP, we designed 5 primer pair sets
based on the genomic sequence of the SH2D1A gene to amplify the 4 exons, adjacent intron regions, and 300 nucleotides of the 5'
regulatory region of the SH2D1A gene (Table
1). This analysis revealed 3 different
types of mutations in 34 of 35 (97%) families with a definitive
diagnosis of XLP: (1) deletion of the entire SH2D1A gene or part of the
coding region, (2) splice-site mutations, and (3) nucleotide
substitutions, including nonsense and missense mutations. In the 35 families surveyed for mutations, 28 different mutations were found.
Table 2 summarizes the mutations observed. Codon numbering starts with the first in-frame methionine of
the SH2D1A gene.
When analyzed under the same conditions, no mutations in the SH2D1A gene were found in any of the 25 males with sporadic XLP, suggesting that they do not have XLP. Similarly, no mutations of the SH2D1A gene were observed among the 9 patients with CAEBV (data not shown). Deletion mutations The single most common mutation was represented by deletions ranging from several megabase pairs of DNA in families K043, K063, and K073 to 47 nucleotides in family K061, leading to the complete or partial loss of the SH2D1A gene (Table 2). The small interstitial deletion of 47 nucleotides in the second exon of family K061 caused a frameshift mutation and premature termination of translation at the TGA stop codon 11 nucleotides downstream of the 3' breakpoint of the deletion. In family K055, a deletion of 89 nucleotides was detected. The deletion involved the splice acceptor site in intron 1 and the splice donor site in intron 2 and led to skipping exon 2 (Figure 1).
Splice-site mutations Splice-site mutations accounted for 11% of this series. Two mutations were found at the 5' donor splice site, and one was found at the 3' acceptor splice site. An A-C transversion in the conserved AG of the intronic splice-acceptor region preceding exon 2 was observed in family K008 (Table 2). Family K065 carried a G-A transition in the conserved GT dinucleotide sequence (Table 2).In family K048, for whom lymphoblasts were available for RNA
extraction, we performed RT-PCR analysis to determine the consequences of the genomic mutation on the splicing process. The mutation changed the ctGTGA
Nonsense mutations Nine families with XLP showed 3 different nonsense mutations 462C T (Arg55stop), 471C T (Gln58stop), and
490G A Trp64stop), resulting in the premature termination of
polypeptide synthesis. Nonsense mutations were found solely in the
second exon. The nonsense mutation 462C T (Arg55stop) was
detected in 6 unrelated families (Figure 2).
Missense mutations Nine of the 28 mutations were missense mutations found in 9 XLP families. We screened 100 control X-chromosomes for the presence of missense substitutions identified in patients with XLP to exclude the possibility that such mutations represent rare polymorphisms. None of the missense mutations was found in the control chromosomes. All missense mutations resulted in the substitution of a conserved amino acid residue identical in the human and murine SH2D1A proteins.Missense mutations affected highly conserved amino acid residues in the
SH2 domain, changing them to residues that are never found at this
position in related SH2 domains (Figure
3). The 319A
Specificity and sensitivity of SH2D1A mutation for XLP In the 35 families with definitive diagnoses of XLP, we found 34 of 35 (97%) to have mutations in the SH2DA1 gene that should result in the abnormal expression of the protein. The one kindred in whom no mutation was found also did not undergo RFLP analysis. In 25 males with sporadic XLP and 9 with CAEBV, no mutation was found. Although we cannot exclude the possibility that mutations in the SH2D1A gene are involved in the pathogenesis of sporadic FIM and CAEBV, our failure to identify mutations in these patients makes it unlikely that a defect in the SH2D1A gene is the cause of sporadic XLP and CAEBV. Because there is no biochemical or laboratory test that is diagnostic for the defect in XLP, the gold standard must be stringent clinical criteria. Using the 35 families with definitive diagnoses as true positive and the other patients as true negatives for XLP, mutations in SH2D1A have a sensitivity of 97%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 97%.Genotype-phenotype correlation It was postulated that mutations that remove or truncate the SH2D1A protein are more often associated with a severe phenotype, whereas missense mutations occur preferentially in mildly affected patients.6 Not uncommonly, identical mutations manifest different phenotypes within the same family (Figure 5). To determine the functional significance of the mutations found in the SH2D1A gene, we undertook a careful evaluation of phenotype, age at first clinical manifestation, and survival (Table 3). No significant differences were observed in phenotypes or in severity of disease based on type (missense, nonsense, truncating) or localization of mutations (Table 3). The age of onset of clinical disease varied considerably, from younger than 1 year of age to 40 years, as did survival, but there was no correlation with the type of mutation. Indeed, such a variation was often seen between affected members of the same kindred, or even brothers. In addition, the large genomic deletion of 3.5 Mb in families K043 and K063 involving SH2D1A did not appear to have an impact on phenotype or severity of disease.
EBV-negative patients with XLP Currently, there are 309 affected males in 89 kindreds in the XLP Registry. Five have been genetically diagnosed but, to date, have not had any clinical manifestations. Of the 304 in whom symptoms have developed, 38 (12.5%) had no evidence of EBV infection at time of their first clinical manifestation. The number may be higher than this because EBV status could not be determined at the time of first manifestation in 152 males. To determine the effect of EBV infection on clinical outcome, the 114 males with evidence of EBV infection at time of first clinical manifestation were compared with those without EBV infection (Table 4). FIM is the most common manifestation and is decidedly more frequent in the EBV+ group (P = .0001). However, when these patients are excluded, there was no statistical difference in the frequency of antecedent EBV infection when dys- and LPD were the
presenting phenotypes (Table 4). AA appears to be more common in the
EBV+ group, and this is not surprising because autoimmune
cytopenias are a known complication of EBV infection, even in
immunocompetent persons. An interesting finding is that 44 of 114 affected males had evidence of present or past EBV infection but that
FIM never developed, suggesting factors other than EBV. In addition,
the clinical outcomes of EBV+ and EBV
patients were evaluated. There was no difference in median time to
first manifestation, but, again, the time of onset varied greatly for
both groups and for all phenotypes, including FIM (ie, younger than 1 to 40 years of age). Survival for the EBV group as a
whole was significantly better (P = .0001), but this is
entirely accounted for by the poor survival with FIM (Table 5).
This report provides a comprehensive analysis of the effects of
SH2DA1 mutations and EBV infection on the clinical outcome of XLP.
Previous studies demonstrated a relatively low frequency (approximately
62%) of mutations in a mixed population of familial (definitive) XLP
and sporadic XLP (males with clinical phenotypes reminiscent of XLP but
without a family history of the disease).6-8 It was
postulated that XLP may be a polygenic disorder. In this study, we
evaluated 35 families that met strict criteria for X-linked inheritance
and symptoms consistent with XLP. We found 34 of 35 (97%) to have
mutations that should result in the abnormal expression of the SH2D1A
protein. In 25 males in whom sporadic XLP was diagnosed because FIM,
dys- Before this report, 18 mutations in the SH2D1A gene had been reported in 29 unrelated patients with XLP disease or in a clinical phenotype reminiscent of XLP.4,6-8 In this study, 26 of 28 mutations have not been previously reported. The range of mutations identified spanned nearly all types of mechanisms by which genetic change can disrupt gene function. The most common changes consisted of deletions resulting in the complete loss of the SH2D1A gene and shorter intragenic deletions resulting in frameshifts and single-base changes leading to splice-site (3), nonsense (9), and missense (9) mutations. Of the nonsense mutations, more than two thirds were C-T transitions, which occurred in CpG dinucleotides. The mutations were distributed over the gene, though the number of mutations in the second exon appeared to be more frequent. One hot spot of mutations was detected at nucleotide position 462, resulting in an Arg55stop transition. The same mutation was also observed in unrelated families by Coffey et al6 and Nichols et al.8 Results from the current study, in addition to data previously reported, bring the total of different mutations to 56 in the SH2D1A gene.4,6-8 Most consist of point mutations, with only 10 occurring in CpG dinucleotides in the coding region of the gene; 10 amino acid substitutions may be of interest in functional studies. Mutations are evenly distributed throughout the gene, but there appears to be a clustering in the second exon. The mutations described herein and in 4 previous communications could shed light on the nature of the SH2D1A protein.20 Large genomic deletions result in the complete loss of the protein, whereas small intragenic deletions and splice-site mutations result in a nonfunctional protein and reveal only limited information about the protein. In contrast, missense mutations that lead to disease can reveal significant information about the functionally important amino acid residues. Of the 56 mutations known, 11 are missense mutations that result in amino acid substitutions. As shown in Figure 5, all mutations leading to an amino acid change are located in the SH2 domain, but no mutations have been detected in the amino-terminal and carboxyl-terminal flanking sequences. Coffey et al6 and Nichols et al8 have speculated about alternating mutations in SH2D1A and tenascin-m genes, accounting for the diverse XLP phenotypes. Yet, we could find no significant phenotypic differences among families with the loss of several megabase pairs of DNA (including SH2D1A and the adjacent tenascin-m gene) and families with missense mutations in the coding region of the SH2D1A gene (Tables 3, 4). The fact that identical mutations have been described in patients with greatly varying clinical phenotype suggests that additional factors are involved. It has been a long-held doctrine that males affected with XLP will not manifest symptoms before EBV infection. Now we and others4 provide data to the contrary. Although EBV infection is the most common trigger of symptoms and FIM is the most common phenotype, at least 12% of affected males have symptoms without evidence of a past or current EBV infection. We also demonstrate that when FIM is excluded, EBV infection is not a predictive factor for phenotype, age of onset of symptoms, or severity of disease (defined by survival). Therefore, there must be additional factors along with aberrant SH2D1A expression that contribute to the pathogenesis of XLP. Other elements that could be involved in shaping the clinical phenotypes include environmental factors, such as other viral triggers, or cellular factors, such as modifier genes. Modifier genes have been postulated for other diseases21 and are a particularly attractive hypothesis, given the structure and function of SH2D1A. Should such a gene (or genes) be found to be implicated, this could provide a mechanism to explain the diverse clinical phenotypes of XLP.
We thank Carolyn D. Hall for the excellent editorial assistance in the preparation of the manuscript and Joseph S. Edwards for the artwork.
Submitted March 16, 2000; accepted July 6, 2000.
Supported by the National Institutes of Health (grant 1 RO1 AI33532-OIA3), the Nebraska Department of Health LB506 (grant 93-07R), the William C. Havens Foundation, and the Lymphoproliferative Research Fund.
David T. Purtilo is deceased.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Janos Sumegi, Department of Pathology and Microbiology, University of Nebraska Medical Center, 985454 Nebraska Medical Center, Omaha, NE 68198-5454; e-mail: jsumegi{at}unmc.edu.
1. Purtilo DT, Cassel CK, Yang JP, Harper R. X-linked recessive progressive combined variable immunodeficiency (Duncan's disease). Lancet. 1975;7913:935-940. 2. Seemayer TA, Gross TG, Egeler RM, et al. X-linked lymphoproliferative disease: twenty-five years after the discovery. Pediatr Res. 1995;38:471-478[Medline] [Order article via Infotrieve]. 3. Gross TG, Kelly CM, Davis JR, Pirruccello SJ, Sumegi J, Seemayer TA. Manifestations of X-linked lymphoproliferative disease (XLP) without evidence of Epstein-Barr virus (EBV) infection. Clin Immunol Immunopathol. 1995;75:281.
4.
Brandau O, Schuster V, Weiss M, et al.
Epstein-Barr virus-negative boys with non-Hodgkin lymphoma are mutated in the SH2D1A gene, as are patients with X-linked lymphoproliferative disease.
Hum Mol Genet.
1999;8:2407-2413
5.
Skare JC, Milunsky A, Byron KS, Sullivan JL.
Mapping the X-linked lymphoproliferative syndrome.
Proc Natl Acad Sci U S A.
1987;84:2015-2018 6. Coffey AJ, Brooksbank RA, Brandau O, et al. Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene. Nat Genet. 1998;20:129-135[Medline] [Order article via Infotrieve]. 7. Sayos J, Wu C, Morra M, et al. The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM. Nature. 1998;395:462-469[Medline] [Order article via Infotrieve].
8.
Nichols KE, Harkin DP, Leveitz S, et al.
Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome.
Proc Natl Acad Sci U S A.
1998;95:13765-13770
9.
Koch CA, Anderson D, Ellis C, Pawson T.
SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.
Science.
1991;252:668-674 10. Cocks BG, Chang CC, Carballido JM, Yssel H, de Vries JE, Aversa G. A novel receptor involved in T-cell activation. Nature. 1995;376:260-263[Medline] [Order article via Infotrieve].
11.
Tangye SG, Lazetic S, Woollatt E, Sutherland GR, Lanier LL, Phillips JH.
Cutting edge: human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein SAP.
J Immunol.
1999;162:6981-6985 12. Maeda A, Wakigushi H, Yokoyama W, Hisakawa H, Tomoda T, Kurashinge T. Persistently high Epstein-Barr virus (EBV) loads in peripheral blood lymphocytes from patients with chronic active EBV infection. J Infect Dis. 1999;179:1012-1015[Medline] [Order article via Infotrieve]. 13. Skare JC, Grierson HL, Sullivan JL, et al. Linkage analysis of seven kindreds with the X-linked lymphoproliferative syndrome (XLP) confirms that the XLP locus is near DXS42 and DXS37. Hum Genet. 1989;82:354-358[Medline] [Order article via Infotrieve]. 14. Bolino A, Yin L, Seri M, et al. A new candidate region for the positional cloning of the XLP. Eur J Hum Genet. 1998;6:509-517[Medline] [Order article via Infotrieve].
15.
Bibbins KB, Boeuf H, Varmus HE.
Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.
Mol Cell Biol.
1993;13:7278-7287 16. Eck MJ, Shoelson S, Harrison SC. Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck. Nature. 1993;362:87-91[Medline] [Order article via Infotrieve]. 17. Katzav S. Single point mutations in the SH2 domain impair the transforming potential of vav and fail to activate proto-vav. Oncogene. 1993;8:1757-1763[Medline] [Order article via Infotrieve]. 18. Booker GW, Breeze AL, Downing AK, et al. Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase. Nature. 1992;358:684-687[Medline] [Order article via Infotrieve].
19.
Yoakim M, Hou W, Songyang Z, Liu Y, Cantley L, Schaffhausen B.
Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.
Mol Cell Biol.
1994;14:5929-5938 20. Poy F, Yaffe MB, Sayos J, et al. Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition. Mol Cell. 1999;4:555-561[Medline] [Order article via Infotrieve]. 21. Houlston RS, Tomlison IP. Modifier genes in humans: strategies for identification. Eur J Hum Genet. 1998;7:1537-1545.
© 2000 by The American Society of Hematology.
| ||||||||||
 |
|
 |
|
  W.-C. Hsieh, Y. Chang, M.-C. Hsu, B.-S. Lan, G.-C. Hsiao, H.-C. Chuang, and I.-J. Su Emergence of Anti-Red Blood Cell Antibodies Triggers Red Cell Phagocytosis by Activated Macrophages in a Rabbit Model of Epstein-Barr Virus-Associated Hemophagocytic Syndrome Am. J. Pathol., May 1, 2007; 170(5): 1629 - 1639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mischler, G. M. Fleming, T. P. Shanley, L. Madden, J. Levine, V. Castle, A. H. Filipovich, and T. T. Cornell Epstein-Barr Virus-Induced Hemophagocytic Lymphohistiocytosis and X-Linked Lymphoproliferative Disease: A Mimicker of Sepsis in the Pediatric Intensive Care Unit Pediatrics, May 1, 2007; 119(5): e1212 - e1218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Crotty, M. M. McCausland, R. D. Aubert, E. J. Wherry, and R. Ahmed Hypogammaglobulinemia and exacerbated CD8 T-cell-mediated immunopathology in SAP-deficient mice with chronic LCMV infection mimics human XLP disease Blood, November 1, 2006; 108(9): 3085 - 3093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 |
Top
Abstract
Introduction
large genomic deletions (n = 3), small intragenic deletions
(n = 10), splice-site (n = 3), nonsense (n = 3), and missense
(n = 9) mutations. No mutations were found in 25 males, so-called
sporadic XLP (males with an XLP phenotype after EBV infection but
no family history of XLP) or in 9 patients with chronic active EBV
syndrome. Of 304 symptomatic males in the XLP Registry, 38 had no
evidence of EBV infection at first clinical manifestation. When
fulminant infectious mononucleosis (FIM) was excluded, there was no
statistical difference in the frequency of EBV infectivity in the other
XLP phenotypes. Furthermore, there was no difference at age of first
clinical manifestation between EBV+ and EBV
-sheet that is sandwiched between 2 
-helices.
300 nucleotides from the
transcription initiation site), and intronic splice-site sequences were
amplified by the polymerase chain reaction (PCR). From each XLP family,
at least 2 affected members, 2 carriers, and 2 normal members were
analyzed for mutations in the SH2D1A gene. Amplified exons from
patients and control subjects were purified by the Qiaquick PCR
purification kit (Qiagen) and sequenced using appropriate end-labeled
primers. Labeled products were separated by a LI-COR 4000L automated
DNA sequencer (Li-Cor; Lincoln, NE). Exon deletions, initially detected
by a failure of the exons to amplify in PCR assays, were confirmed by
Southern blotting and hybridization with DNA probes derived from the
cDNA fragments of the SH2D1A gene. To verify that similar changes were
not present in healthy persons (ie, they represent polymorphic
variants), the 5' regulatory, coding, and splice-site sequences were
also analyzed in 100 additional normal X-chromosomes.
