Mutations in Activation-Induced Cytidine Deaminase in Patients with Hyper IgM Syndrome

Clinical Immunology Vol. 97, No. 3, December, pp. 203–210, 2000 doi:10.1006/clim.2000.4956, available online at http://www.idealibrary.com on

Mutations in Activation-Induced Cytidine Deaminase in Patients with Hyper IgM Syndrome Yoshiyuki Minegishi,* Aubert Lavoie,† Charlotte Cunningham-Rundles,‡ Pierre-Michel Be´dard,† Jacques He´bert,† Louise Coˆte´,† Kazuo Dan,§ Debra Sedlak, ¶ Rebecca H. Buckley, ¶ Alain Fischer,㛳 Anne Durandy,㛳 and Mary Ellen Conley* ,** *Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38101-0318; †Departement de Me´decine, Universite´ Laval, Quebec G1S 4W1, Canada; ‡Department of Medicine, Mount Sinai Medical Center, New York, New York 10029; §Third Department of Internal Medicine, Nippon Medical School, Tokyo, Japan; ¶Department of Pediatrics, Duke University School of Medicine, Durham, North Carolina 27710; 㛳Inserm U429, Hoˆpital Necker-Enfants Malades, Paris 75015, France; and **Department of Pediatrics, University of Tennessee College of Medicine, Memphis, Tennessee 38105

Recent studies have shown that mutations in a newly described RNA editing enzyme, activation-induced cytidine deaminase (AID), can cause an autosomal recessive form of hyper IgM syndrome. To determine the relative frequency of mutations in AID, we evaluated a group of 27 patients with hyper IgM syndrome who did not have defects in CD40 ligand and 23 patients with common variable immunodeficiency. Three different mutations in AID were identified in 18 patients with hyper IgM syndrome, including 14 French Canadians, 2 Lumbee Indians, and a brother and sister from Okinawa. No mutations were found in the remaining 32 patients. In the group of patients with hyper IgM syndrome, the patients with mutations in AID were older at the age of diagnosis, were more likely to have positive isohemagglutinins, and were less likely to have anemia, neutropenia, or thrombocytopenia. Lymphoid hyperplasia was seen in patients with hyper IgM syndrome and normal AID as well as the patients with hyper IgM syndrome and defects in AID. © 2000 Academic Press Key Words: immunodeficiency; IgM; lymphoid hyperplasia; somatic mutation; class switch recombination; RNA editing; B cells.

INTRODUCTION

Hyper IgM syndrome is a heterogeneous group of disorders characterized by normal or elevated serum IgM but low serum IgG, IgA, and IgE (1). The majority of affected patients have the X-linked form of the disease which is due to mutations in the gene for CD40 ligand (CD154) (2– 6). CD40 ligand, a member of the TNF family, is expressed transiently on the surface of activated T cells (7, 8). Its cognate receptor, CD40, is constitutively expressed on the surface of B cells and is also found on monocytes, dendritic cells, epithelial cells, endothelial cells, and some carcinomas (9). Stim-

ulation of B cells through CD40 enhances cell proliferation and increases expression of adhesion and activation markers. Cross-linking of CD40 protects B cells from apoptosis at some stages of differentiation but induces Fas expression with increased susceptibility to apoptosis at other stages of maturation (10). The CD40 –CD40 ligand interaction plays a critical role in isotype switching. Stimulation of B cells through CD40 in the presence of cytokines can induce the production of IgG, IgA, and IgE (11, 12). Our group and others have shown that at least some of the patients with hyper IgM syndrome who do not have defects in CD40 ligand have decreased or absent responses to B cell stimulation through CD40 (13–15). Revy et al. have recently shown that some of these patients have mutations in a gene called AID (16). AID (Activation-Induced cytidine Deaminase) is a 198-amino-acid protein identified in 1999 by Muramatsu et al. in their search for genes that are upregulated after stimulation of B cells with IL-4, TGF-␤, and CD40 ligand (17). Each of those three activators independently increased AID transcription in the murine B cell line CH12F3-2, but they were synergistic when used together. The function of AID in isotype switch and B cell maturation is not yet understood; however, AID has greatest the homology with APOBEC-1, an mRNA editing enzyme that deaminates a cytidine within the transcript for apoB mRNA (18). This deamination, which replaces a CAA codon with a UAA premature stop codon, allows a full-length protein and a truncated protein to be produced from the same transcript. Revy et al. have identified 10 different mutations in AID in 18 patients with autosomal recessive hyper IgM syndrome (16). These mutations included amino acid substitutions, premature stop codons, and deletions. The affected patients developed recurrent sinopulmonary and gastrointestinal infections in childhood and

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TABLE 1 Primer Paris Used to Screen Genomic DNA for Mutation in AID Exon

Sense primer

Antisense primer

Fragment size (bp)

1 2 3 4 5

5⬘-ACTAAGACAGAGAACCATCA-3⬘ 5⬘-AATCCAGAGTGACCAGATTA-3⬘ 5⬘-AGTGAATTTTAGCGTGGTC-3⬘ 5⬘-TTTGGAACCTGAACTGTCTT-3⬘ 5⬘-ACCTCGTTTTGAAGCCAT-3⬘

5⬘-CTCATTACTCAGATTTGCTC-3⬘ 5⬘-TTGACCATTAAACTGCTTGC-3⬘ 5⬘-AACTCATTCATTCCGCATC-3⬘ 5⬘-CTCCAAAAAAGACTGTAGAG-3⬘ 5⬘-CTGTTTTTTATCCACTGTC-3⬘

161 252 379 238 168

15 of the 18 patients had at least one episode of lymphoid hyperplasia. Laboratory studies demonstrated normal numbers of CD19 ⫹ peripheral blood B cells with normal expression of the memory marker CD27. However, cultured cells from the patients did not produce IgE in response to stimulation with CD40 ligand and IL-4. In addition, V regions genes from the CD19 ⫹, CD27 ⫹ cells from these patient did not contain the expected somatic mutations typical of memory cells. To determine the frequency of AID mutations in patients with hyper IgM syndrome and normal CD40 ligand, we analyzed DNA from affected patients. The clinical characteristics of the patients with and without mutations in AID were compared. MATERIALS AND METHODS

Patients. The patients included in this evaluation were enrolled in a study to examine the genetic etiology of immunodeficiencies. Clinical and laboratory information on each patient was obtained from the referring physician using a standard survey form. All of the patients except patients 16, 17, and 18 were receiving gammaglobulin replacement therapy at the time of the evaluation. Patients 15, 19, 25, 26, and 27 have been previously reported (13). Mutation detection. Genomic DNA from patients and controls was analyzed by SSCP using previously described techniques (19, 20). The PCR conditions were 95°C for 5 min, followed by 30 cycles of 95°C for 45 s, 56°C for 30 s, 72°C for 30 s, with a final 5-min extension at 72°C. The primers for the five exons of AID are shown in Table 1. Fragments demonstrating altered migration were amplified in two separate PCR reactions. At least one clone from each reaction was sequenced. Haplotype analysis. Genomic DNA from the 14 French Canadians with mutations in AID, from 10 unaffected relatives of these individuals, and from 5 healthy unrelated French Canadians was amplified in the presence of 32P-labeled ␣-dCTP using primers that amplify highly polymorphic short tandem repeats at D12S99, D12S1623, GATA151H05, D12S1695, D12S77, D12S1697, and D12S358. PCR products were separated on a 6% denaturing polyacrylamide gel.

B cell activation. Peripheral blood lymphocytes were cultured with IL-4 and/or 626.1, a murine IgG1 monoclonal antibody to human CD40, as previously described (13). RESULTS

Twenty-seven patients with hyper IgM syndrome and normal CD40 ligand were analyzed for mutations in AID. Fourteen of these patients had a family history of immunodeficiency and 16 of the patients were French Canadians or Lumbee Indians, populations that have an increased incidence of hyper IgM syndrome (21). There was no known history of consanguinity in any of the patients. To facilitate genetic screening, PCR primers were designed to flank each of the five exons of AID and genomic DNA from each patient was examined by single strand conformational polymorphism (SSCP). All 14 of the DNA samples from the French Canadians demonstrated the same abnormal SSCP pattern in exon 3 of AID (Fig. 1). The absence of the normal bands in this pattern suggested that the alteration was homozygous in each patient. Cloning and sequencing of this region of the gene revealed a C to T transition in codon 112. This change results in the substitution of the wild-type arginine with cysteine. Haplotype analysis using seven highly polymorphic probes flanking the AID locus at 12p13 showed that all of the affected individuals were homozygous for the same alleles in a 5 cM region surrounding the gene. This indicates that

FIG. 1. SSCP analysis of exon 3 of AID. Genomic DNA from patients with hyper IgM syndrome and from controls was examined by PCR using primers specific for exon 3. Healthy controls are shown in lanes 1 and 2; patients 3, 4, and 11 are shown in lanes 4, 5, and 6; patient 15 is shown in lane 9; and patient 17 is shown in lane 13.

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MUTATIONS IN AID IN PATIENTS WITH HYPER IgM SYNDROME

TABLE 2 Patients with Hyper IgM Syndrome Serum immunoglobulins in mg/dl Patient number

Ethnic group

Sex

Age at diagnosis

Current age

IgM

IgA

IgG

Mutation in AID

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian French Canadian Lumbee Indian Lumbee Indian Japanese Japanese European American European American European American European American European American European American European American European American African American

F M F F F M M M F F M F M F M F F M M M M M M M M M M

11 (19) a 12 (28) 5 (7) 11 6 27 (24) 25 17 (26) 6 2 7 28 (25) 0.9 4 2 0.6 (0.6) 0.9 0.9 (0.9) 1.5

44 46 38 51 16 13 39 39 53 50 41 49 47 b 18 32 10 35 32 18 13 13 7 7 19 18 b 14 20

554 599 915 699 318 864 522 1130 1490 1210 1410 528 652 535 896 1384 4114 2164 934 1160 453 116 163 521 402 723 2590

⬍15 ⬍15 24 ⬍7 ⬍7 ⬍15 9 41 ⬍30 ⬍30 ⬍7 ⬍15 7 ⬍15 ⬍7 ⬍7 40 22 ⬍7 27 ⬍7 ⬍8 14 ⬍7 8 87 ⬍8

1370 1850 1049 1180 2280 673 1460 1010 331 737 1420 1620 962 1490 571 ⬍30 40 91 570 465 1730 1190 865 829 1010 603 259

R112C R112C R112C R112C R112C R112C R112C R112C R112C R112C R112C R112C R112C R112C W84X W84X R112H R112H — — — — — — — — —

a If the age at diagnosis is shown in parenthesis, the patient was related to the preceding patient and was diagnosed after that patient. Patients 1 and 2 were cousins; patients 3 and 4 were sisters; patients 5 and 6 were siblings; patients 9 and 10 were sisters; patients 12 and 13 were siblings; patients 17 and 18 were siblings; patients 22 and 23 were fraternal twins; patients 25 and 26 were maternal half-brothers. b Patient 13 died in 1997 at 47 years of age; patient 24 died in 1998 at 18 years of age.

all of the affected French Canadian patients shared a common ancestor. Both of the Lumbee Indians were also homozygous for an altered pattern in exon 3. These patients had a homozygous G to A base pair substitution in codon 84 resulting in the replacement of tryptophan with a premature stop codon within the highly conserved catalytic domain of the cytidine deaminase. DNA from a brother and sister from a Japanese family demonstrated a homozygous G to A base pair substitution in codon 112 in exon 3, resulting in the replacement of the wild-type arginine with histidine. The mutations in this family and the French Canadian families occurred at different nucleotides within the same CpG dinucleotide. The arginine at codon 112 is conserved not only in murine AID, but also in murine APOBEC-1 (17). The three mutations identified in this study have not been previously reported. To ensure that the alterations in exon 3 did not represent polymorphic variants, DNA from 100 normal controls was analyzed by SSCP. None of these samples

exhibited the patterns seen in the patients; however, 3 of the 100 controls were heterozygous for an altered pattern due to an A to G substitution at the ⫹36 position in intron 3. Two common, functionally insignificant polymorphisms in AID were also identified. The first is a G to A at position ⫹16 in intron 2 and the other is a C to T at the third position in codon 145 in exon 4. This base pair change does not alter the amino acid sequence at this site. The DNA samples from the remaining 9 patients with hyper IgM syndrome were identical to those of the controls. Because patients with atypical forms of single gene defects of the immune system are sometimes diagnosed as having common variable immunodeficiency, we screened genomic DNA from 23 patients with this diagnosis for mutations in AID. Three of these patients had serum IgM concentrations within the normal range. No defects in AID were found in this group of patients. The patients with hyper IgM syndrome are described in Tables 2 and 3. All of the patients with mutations in

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TABLE 3 Clinical Findings in Patients with Hyper IgM Syndrome Patient number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Pneumonia

Bronchiectasis

Lymphoid hyperplasia X X

X X X X X X X X X X X

Meningitis

X X

X X X X X

X X X X X

X X X X

Breast cancer Giardiasis; arthralgias; mastoiditis Mastoiditis; staphylococcal sacroileitis; chronic diarrhea

X

X X X X X

X

Eczema H. influenzae epiglottitis; eczema Thyroid nodule; urinary tract infections Giardiasis; urinary tract infections Arthralgias Poliomyelitis; thyroid nodule Giardiasis Mastoiditis Died of pulmonary hemorrhage Arthralgias; urinary tract infections Recurrent herpes simplex Septic arthritis

X

X

20 21

X X

X X

22 23 24 25

X

X

X X

X

26 27

X X

X X

a

Other

X X

Pseudomonas sepsis; E. coli sepsis; colitis; growth failure; neutropenia Eczema; giardiasis; thrombocytopenia Salmonella gastroenteritis; AIHA a; growth failure; neutropenia Eczema; colitis; abnormal liver function tests Eczema; abnormal liver function tests Pseudomonas sepsis; giardiasis; neutropenia Osteomyelitis; ectodermal dysplasia; died after lung transplant H. influenzae meningitis; ectodermal dysplasia E. coli sepsis; pyelonephritis

AIHA, autoimmune hemolytic anemia.

AID had the onset of recurrent otitis and sinusitis in the first 2 years of life; however, a surprising number of these patients were not recognized as having immunodeficiency until the second or third decade of life. The majority of the AID-deficient patients had at least one episode of pneumonia and many of those greater than 35 years of age had developed bronchiectasis. The availability of intravenous gammaglobulin since 1985 has decreased the incidence of chronic lung disease in the younger patients. Revy et al. reported that the majority of their patients with defects in AID had lymphoid hyperplasia (16). Lymphoid hyperplasia was also seen in 9 of our 18 patients with mutations in AID. Several of the patients had troublesome diarrhea; however, none had growth failure or problems maintaining a normal weight. Seven of the French Canadian patients with mutations in AID had a history of meningitis; patients 4 and 9 had two episodes of meningococcal meningitis and patient 3 had four episodes of meningococcal meningitis. The meningitis occurred when the patients were receiving inadequate doses of intramuscular gammaglobulin or no gammaglobulin replacement therapy. The patients with mutations in

AID did not have cytopenias, autoimmune disease, or opportunistic infections. Most were full-time students or had full-time jobs. The nine patients with hyper IgM syndrome and normal AID were all males who had been evaluated for mutations in CD40 ligand by both immunofluorescence and genetic screening (13, 22). This group of patients was heterogeneous but tended to be sicker than the patients with mutations in AID. All were recognized as having immunodeficiency at less than 5 years of age. Like the patients with defects in AID, these patients had the onset of recurrent otitis and sinusitis at less than 2 years of age. Most also had at least one episode of pneumonia and five of the nine had lymphoid hyperplasia. Patients 19 and 21 had had their spleens removed because of severe splenomegaly. These two patients also had growth failure. Cytopenias were common in this group of patients. Five patients had neutropenia and one patient had thrombocytopenia. Transiently or persistently reversed CD4/CD8 ratios were seen in both patients with and patients without mutations in AID. There were no routine laboratory studies that would always distinguish the patients with and without mu-

MUTATIONS IN AID IN PATIENTS WITH HYPER IgM SYNDROME

tations in AID. The serum IgM was markedly elevated in almost all of the patients. Patients 22 and 23 had minimally elevated concentrations of serum IgM in the first few years of life; they were included in this study because they were among the patients evaluated for X-linked hyper IgM syndrome. Some of the patients without mutations in AID (patients 19, 20, 24, and 27) did not have elevated serum IgM during the first few years of life but the serum IgM became markedly elevated by 5 years of age. IgA could be detected in the serum of some of the patients with mutations in AID and some of the patients without mutations in AID. Isohemagglutinins were evaluated in 8 of the 14 French Canadian patients and in all cases they were within normal range. By contrast, in the group of patients without mutations in AID, patients 19, 21, 22, 24, 26, and 27, had negative isohemagglutinins but isohemagglutinins were normal in patients 23 and 25. All of the patients with mutations in AID and most of the patients without mutations in AID had normal numbers of B cells. Fraternal twin brothers without mutations in AID (patients 22 and 23) had markedly elevated percentages and absolute numbers of CD19 ⫹ cells in the first few years of life (40 –50% CD19 ⫹ cells with absolute numbers greater than 5000/mm 3). At 7 years of age, these patients had 29 –34% CD19 ⫹ cells, with absolute numbers of 1500 –2000/mm 3). Patient 21 had 6% CD19 ⫹ cells at 9 years of age but had less than 1% CD19 ⫹ cells at 13 years of age. In the normal individual 15–25% of CD19 ⫹ cells are surface IgG or IgA positive and surface IgM negative. This surface IgM negative population was absent in the patients with mutations in AID (Fig. 2) but it was also absent in the patients with mutations in CD40 ligand and the majority of patients with hyper IgM syndrome who did not have mutations in AID or CD40 ligand. In other respects, the B cells from the patients with mutations in AID demonstrated a normal phenotype. A normal percentage of cells expressed the memory marker CD27 (Fig. 2). The majority of B cells were positive for CD21 and there was reduced expression of CD38. By contrast, two of the patients with hyper IgM syndrome of unknown etiology had an immature B cell phenotype characterized by poor expression of CD21 and increased expression of CD38 (Table 4). Our previous study evaluated B cell activation in response to stimulation with CD40 and IL-4 in four patients with hyper IgM syndrome and normal CD40 ligand (13). That study included one patient with a mutation in AID (patient 15) and three patients with hyper IgM syndrome of unknown etiology (patients 19, 25, and 27 ). B cells from all four of these patients and three additional patients with mutations in AID (patients 3, 4, and 13) demonstrated an increase in expression of CD23 and CD25 in response to IL-4 but addition of anti-CD40 to the cultures did not result in a syner-

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FIG. 2. Peripheral blood lymphocytes from two patients with mutations in AID and two controls were stained with phycoerythrinlabeled CD19 and FITC-labeled anti-human IgM (left) or CD27 (right).

gistic increase in CD23 and CD25, as was seen in normal controls. B cells from two patients with hyper IgM syndrome of unknown etiology (patients 20 and 21) did not increase expression of CD23 and CD25 in response to IL-4 alone, CD40 antibody alone, or the two stimuli together. These two patients (who also had an immature B cell phenotype) appear to have a more global B cell defect. Peripheral blood lymphocytes from four patients with mutations in AID and four patients without mutations in AID were stimulated with IL-4 plus antibody to CD40 and secretion of IgE was evaluated. Cells from patients 11, 13, and 21 made no detectable IgE; cells from patients 3 and 4 made a detectable response that was less than 0.1% of the control; and cells from patients 19, 25, and 26 secreted less than 2% of the control. DISCUSSION

We have identified a group of 18 patients with hyper IgM syndrome and mutations in AID. Although none of these patients had a family history of consanguinity, all of the affected patients were from relatively isolated populations and all were homozygous for their genetic defect. The 14 French Canadian patients with mutations in AID were from nine unrelated families. The majority of these families were from the northeastern part of the province of Quebec, a region that was set-

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TABLE 4 B Cell Phenotype in Patients with Hyper IgM Syndrome Percentage of CD19 ⫹ cells expressing

Patients 3 4 13 20 21 Controls CVID XHIM Healthy controls (6)

Percentage of CD19 ⫹

CD21

CD38

IgM

IgD

IgG

12.0 5.0 13.3 7.5 6.3

69.0 74.1 72.7 8.1 6.3

22.4 18.0 29.1 70.7 89.3

96.8 89.3 99.2 93.5 83.7

90.4 95.8 92.0 34.1 76.6

⬍1 ⬍1 ⬍1 ⬍1 ⬍1

13.8 16.0 (4.6–12.5)

45.4 92.0 (73.2–90.4)

14.9 50.8 (12.3–30.2)

89.0 98.9 (72.5–84.6)

92.2 97.9 (74.9–84.5)

tled by those of French descent in the mid 1600s (23). Haplotype analysis demonstrated that this group of patients inherited their genetic defect from a common founder. The second population with a high incidence of defects in AID were the Lumbee Indians (21). The Lumbee tribe, which currently consists of approximately 50,000 individuals, has maintained an ancestral homeland in North Carolina for over 300 years. The Japanese bother and sister were from Okinawa in southern Japan. As is true with all rare genetic disorders, it is difficult to estimate the frequency of mutations in AID. Comparisons with other immunodeficiencies may be useful. It has been estimated that X-linked agammaglobulinemia (XLA) occurs in 3– 6 individuals per million (24, 25). By comparison, population studies that attempt to identify all patients with immunodeficiency consistently find that hyper IgM syndrome of any type occurs two to five times less frequently than XLA (26 – 29). Further, 55– 65% of patients with hyper IgM syndrome are males with mutations in CD40 ligand (1, 22). The remaining patients with hyper IgM syndrome include not only patients with mutations in AID but also patients with other autosomal recessive disorders resulting in a similar immunodeficiency and patients with acquired or autosomal dominant hyper IgM syndrome (30 –33). Although our study could be interpreted as suggesting that more than half of patients with hyper IgM syndrome who do not have defects in CD40 ligand will have defects in AID, the fact that the 14 French Canadians were recruited to the study because they were assumed to have a shared autosomal recessive disorder biases the data. We would estimate that AID deficiency occurs in the North American population at a frequency of no greater than 2 per 10 million. Mutations in AID were not seen in nine patients with early onset hyper IgM syndrome of unknown etiology included in this study. This very heterogeneous

1.8 ⬍1 (4.7–16.7)

group included two half-brothers with mild ectodermal dysplasia and two fraternal twin brothers with neutropenia and elevated numbers of B cells. These four patients are highly likely to have single gene defects of the immune system. It may be that some of the remaining patients do not have single gene defects but instead have disorders that are due to a combination of susceptibility genes and environmental factors. On the whole, this group of patients was sicker than the patients with mutations in AID. They were recognized to have immunodeficiency at an earlier age and they were more likely to have significant infections in spite of adequate gammaglobulin therapy. Some had failure to thrive; others had neutropenia. The majority of these patients did not make isohemagglutinins and some had an immature B cell phenotype. Of interest, lymphoid hyperplasia was common in patients both with and without mutations in AID. Most patients with single gene defects of the immune system are recognized to have immunodeficiency in early childhood. Although the patients with mutations in AID did have the onset of recurrent otitis and sinusitis within the first few years of life, in most cases the infections were not severe or frequent enough to elicit an evaluation of immune function until later. Many of the patients survived more than 30 years without the benefit of what we now consider adequate gammaglobulin replacement therapy. As the mutations in the French Canadians and the Japanese siblings result in an amino acid substitution at the same site, it is possible that the mutant proteins in these patients retain some function and are associated with a milder phenotype. The possibility that there might be variation in severity of phenotype based on the specific mutation in the gene is at least partially supported by the observations of Revy et al., who found that patients with null mutations in AID had fewer somatic mutations in ␮ heavy chain VH regions than patients with amino acid substitutions (16).

MUTATIONS IN AID IN PATIENTS WITH HYPER IgM SYNDROME

The mechanism by which mutations in AID result in defects in class switch recombination and somatic mutation of immunoglobulin variable regions is not yet known. Because both of these events result in DNA alterations that are likely to be dependent on complex interactions between DNA, RNA, and protein (34 –36), it is possible that AID is directly involved both of these processes. However, at this point, one cannot rule out the alternative possibility, the possibility that AID is required to induce a step in B cell maturation that renders the cell susceptible to both isotype switch and somatic mutation. Molecular studies have suggested that AID expression is transient and limited to mature B cells (17). Thus, whereas many immunodeficiencies involve multiple hematopoietic cell lineages, defects in AID are highly focused. The significant infections in patients with mutations in AID highlight the importance of switched isotypes and somatic mutation in providing immune protection.

8.

9. 10.

11.

12.

ACKNOWLEDGMENTS We appreciate the willingness of the patients and their families to participate in these research studies. We thank Lisa Rapalus and Krista Kaul for technical assistance and Susan Saucier for secretarial assistance. These studies were supported by grants from the National Institute of Health, AI25129, National Cancer Institute Grant P30 CA21765, the Assisi Foundation, March of Dimes FY970384, American Lebanese Syrian Associated Charities, and by funds from the Federal Express Chair of Excellence.

13.

14.

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