Hypomorphic RAG deficiency: impact of disease burden on survival and thymic recovery argues for early diagnosis and HSCT.

Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.

Novel intraoral phenotypes in hyperimmunoglobulin-E syndrome.

AIM: Hyperimmunoglobulin-E syndrome (HIES) is a primary immunodeficiency characterized by eczema, recurrent skin and lung infections with pneumatocoele formation, and extremely elevated serum immunoglobulin-E. The precise immunologic defect and genetic etiology remain unknown. Non-immunologic findings include characteristic facial features (prominent forehead, fleshy nasal tip, and increased interalar distance); skeletal involvement (pathological fractures, scoliosis, and craniosynostosis); and retention of primary teeth. This study aims to characterize intraoral soft tissue findings in HIES patients. METHODS: Sixty HIES patients (4-54 years, 27 males, 33 females) received intraoral and radiographic evaluations. Chronological dental development was also assessed. RESULTS: Lesions of the hard palate and dorsal tongue were found in 55% and 60% of patients, respectively. Palatal lesions ranged from a generalized surface keratosis to a midline sagittal fibrotic bridge. Tongue lesions consisted of multiple fissures and a midline cleft. On the lip and buccal mucosa, keratotic plaques and/or surface fissures were found in 8% and 23% of patients, respectively. Manifested in 76.7% of patients, the intraoral lesions were significantly more prevalent than the characteristic facial traits (P=0.0013). CONCLUSIONS: Alterations in oral mucosa and gingiva were present in the majority of HIES patients. These novel intraoral findings may facilitate the diagnosis of HIES.

Immunophenotypic profiles in families with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) type Ia is caused by inherited defects in apoptosis and is characterized by nonmalignant lymphoaccumulation, autoimmunity, and increased alpha/beta(+) double-negative T cells (alpha/beta(+)-DNT cells). This study reports immunophenotypic findings in 166 members of 31 families with ALPS type Ia, associated with genetic mutations in the TNFRSF6 gene encoding Fas. The ALPS type Ia probands (n = 31) and relatives having both a Fas mutation and clinically proven ALPS (n = 28) showed significant expansion of CD8(+) T cells, alpha/beta(+)-DNT cells, gamma/delta(+)-DNT cells, CD3(+)/ HLA-DR(+) T cells, CD8(+)/CD57(+) T cells, and CD5(+) B cells. Relatives with Fas mutations, but without all the required criteria for ALPS (n = 42), had expansions of CD8(+) T cells, alpha/beta(+)-DNT cells, and gamma/delta(+)-DNT cells. Interestingly, relatives without a Fas mutation and with no features of ALPS (n = 65) demonstrated a small but significant expansion of CD8(+) T cells, both DNT cell subsets, and CD5(+) B cells. As compared to unrelated healthy controls, lymphocyte subset alterations were greatest in the probands, followed by the relatives with mutations and ALPS. Probands and relatives with mutations and ALPS also showed a lower number of CD4(+)/CD25(+) T cells that, in combination with an independent increase in HLA-DR(+) T cells, provided a profile predictive of the presence of clinical ALPS. Because quantitative defects in apoptosis were similar in mutation-positive relatives regardless of the presence of clinical ALPS, factors, other than modifiers of the Fas apoptosis pathway, leading to these distinctive immunophenotypic profiles most likely contribute to disease penetrance in ALPS.

TcR-alpha/beta(+) CD4(-)CD8(-) T cells in humans with the autoimmune lymphoproliferative syndrome express a novel CD45 isoform that is analogous to murine B220 and represents a marker of altered O-glycan biosynthesis.

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor alpha/beta(+) CD4(-)CD8(-) T cells (alpha/beta(+) double-negative T cells [alpha/beta(+)-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The alpha/beta(+)-DNT cells are immunophenotypically and functionally similar to alpha/beta(+)-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, alpha/beta(+)-DNT cells express the B-cell-specific CD45R isoform B220. We show that alpha/beta(+)-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4(+) T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell-cell interactions, and access to alternative apoptosis pathways.

An immune defect causing dominant chronic mucocutaneous candidiasis and thyroid disease maps to chromosome 2p in a single family.

We describe a large family in which a combination of chronic mucocutaneous candidiasis (fungal infections of the skin, nails, and mucous membranes) and thyroid disease segregate as an autosomal dominant trait with reduced penetrance. The family includes (a) four members with both candidiasis and thyroid disease, (b) five members, including one pair of phenotype-concordant MZ twins, with candidiasis only, and (c) three members with thyroid disease only. A whole-genome scan using DNA samples from 20 members of the family identified a candidate linkage region on chromosome 2p. By sampling additional individuals and genotyping supplementary markers, we established linkage to a region of approximately 15 cM bounded by D2S367 and D2S2240 and including seven adjacent markers consistent with linkage. With a penetrance estimate of.8, which was based on pedigree and affected status, the peak two-point LOD score was 3.70 with marker D2S2328, and the peak three-point LOD score was 3.82. This is the first linkage assignment of a dominant locus for mucocutaneous candidiasis.

Sib understanding of genetics and attitudes toward carrier testing for X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (XSCID) is the most common genetic form of SCID, a rare disease with profoundly impaired immunity. SCID was previously fatal but now can be treated by bone marrow transplantation. Mapping of XSCID in 1985 and identification of the disease gene, IL2RG, in 1993 made possible patient and carrier diagnosis. We assessed understanding of the genetics of XSCID in adult sibs recruited from families in which a proband had enrolled in our protocols and had attended an XSCID family workshop. Thirty-seven female and three male sibs completed a questionnaire and semistructured interview. Overall knowledge of genetics of XSCID was excellent. An overwhelming majority of participants (93%) believed that daughters should be tested for XSCID carrier status; 89% would prefer to have their own daughter tested prior to age 18 years (M = 9, median = 12), and 34% would test at birth. Moreover, 89% felt they would disclose carrier results to their daughter before adulthood (M = 12 years, median = 12); 51% would tell prior to adolescence. XSCID sibs were optimistic about medical science and assertive in their search for the latest information. Genetic information should be made available to families over time and should include discussion of reproductive risks for sons surviving with XSCID and daughters as they grow up. We recommend that genetic counseling for XSCID include children in age-appropriate discussions and that counselors help parents weigh benefits of early testing and disclosure versus the potential harm of loss of child autonomy.

Family pictures: growing up with a brother with X-linked severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) is a rare syndrome of profoundly impaired immunity, most often X-linked (XSCID). In past generations male infants with XSCID succumbed to infections during the first year of life, but prompt diagnosis and bone marrow transplantation currently make survival possible for over 80%. This treatment typically requires hospitalization for several months; thus, the burden on the family is considerable. We assessed the psychological impact on sibs of boys with XSCID. Forty adult sibs from families studied by J.M.P. were interviewed by J.H.F., and rating scales developed. The majority expressed distress over prolonged maternal absence during the affected child's hospitalization; 67% believed the mother had unsuccessfully mourned son(s) who died of XSCID. Half of the sibs reported that communication in the family about XSCID had been poor. Families with a spontaneous mutation were significantly more likely to report separation issues (P = 0.05), perhaps due to stronger maternal guilt. Family communication was significantly related to parental mourning (P = 0.001) and to survivor guilt (P = 0.05). Difficulties for daughters included desire to repair the mother's loss of her own child, as well as attempts to undo feelings of being flawed, by heightened wishes to bear a healthy son. In light of these findings we suggest: 1) bone marrow transplantation and the period of isolation places great stress on the family; parents need help balancing needs of well sibs with needs of the affected son; 2) parents need help with mourning the loss of a son so family secrets will not prevail; and 3) sibs, both bone marrow donors and non-donors, face psychological risks and need support.

The development of lymphomas in families with autoimmune lymphoproliferative syndrome with germline Fas mutations and defective lymphocyte apoptosis.

Lymphomas were studied in kindreds with autoimmune lymphoproliferative syndrome (ALPS; Canale-Smith syndrome), a disorder of lymphocyte homeostasis usually associated with germline Fas mutations. Fas (CD95/APO-1) is a cell surface receptor that initiates programmed cell death, or apoptosis, of activated lymphocytes. Lymphoma phenotype was determined by immunohistochemistry, frequency of CD3(+)CD4(-)CD8(-) T-cell-receptor alpha/beta cells by flow cytometry, nucleotide sequences of the gene encoding Fas (APT1, TNFRSF6), and the percentage of lymphocytes undergoing apoptosis in vitro. Of 223 members of 39 families, 130 individuals possessed heterozygous germline Fas mutations. Eleven B-cell and T-cell lymphomas of diverse types developed in 10 individuals with mutations in 8 families, up to 48 years after lymphoproliferation was first documented. Their risk of non-Hodgkin and Hodgkin lymphomas, respectively, was 14 and 51 times greater than expected (each P <.001). Investigation of these 10 patients and their relatives with Fas mutations revealed that all had defective lymphocyte apoptosis and most had other features of ALPS. The tumor cells retained the heterozygous Fas mutations found in the peripheral blood and manifested defective Fas-mediated killing. These data implicate a role for Fas-mediated apoptosis in preventing B-cell and T-cell lymphomas. Inherited defects in receptor-mediated lymphocyte apoptosis represent a newly appreciated risk factor for lymphomas.

Increases in circulating and lymphoid tissue interleukin-10 in autoimmune lymphoproliferative syndrome are associated with disease expression.

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder in which genetic defects in proteins that mediate lymphocyte apoptosis, most often Fas, are associated with enlargement of lymph nodes and the spleen and a variety of autoimmune manifestations. Some patients with ALPS have relatives with these same apoptotic defects, however, who are clinically well. This study showed that the circulating levels of interleukin 10 (IL-10) were significantly higher (P <.001) in 21 patients with ALPS than in healthy controls. Moreover, the peripheral blood mononuclear cells (PBMCs) and lymphoid tissues of these patients with ALPS contained significantly higher levels of IL-10 messenger RNA (mRNA; P <.001 and P <.01, respectively). By fractionating PBMC populations, disproportionately high concentrations of IL-10 mRNA were found in the CD4(-)CD8(-) T-cell population, expansion of which is virtually pathognomonic for ALPS. Immunohistochemical staining showed intense IL-10 protein signals in lymph node regions known to contain CD4(-)CD8(-) T cells. Nonetheless, in vitro studies showed no influence of IL-10 on the survival of CD4(-)CD8(-) T cells. Overexpression of IL-10 in patients with inherited apoptotic defects is strongly associated with the overt manifestations of ALPS.

Comparison of five retrovirus vectors containing the human IL-2 receptor gamma chain gene for their ability to restore T and B lymphocytes in the X-linked severe combined immunodeficiency mouse model.

X-linked severe combined immunodeficiency (XSCID) is caused by mutations in the IL-2 receptor gamma chain (IL2RG) gene, resulting in absent T lymphocytes and nonfunctional B lymphocytes. Recently T lymphocyte production and B lymphocyte function were restored in XSCID patients infused with autologous stem cells transduced with a retrovirus containing the human IL2RG cDNA. To optimize the expression of human IL2RG for future clinical trials, we compared five retroviral vectors expressing human IL2RG from different LTR enhancer-promoter elements in a mouse model. Northern and Southern blot analysis of hematopoietic tissues from repopulated mice revealed that the retroviral vector with the highest expression per copy number was MFG-S-hIL2RG, followed by MND-hIL2RG. All five vectors were capable of restoring lymphopoiesis in irradiated XSCID mice transplanted with transduced IL2RG-deficient hematopoietic stem cells. Transduction of IL2RG-deficient hematopoietic stem cells with all five vectors restored T lymphopoiesis in transplanted stem cell-deficient W/W(v) mouse recipients. However, only XSCID stem cells transduced with the MFG-S-hIL2RG vector generated B lymphocytes in W/W(v) mice. We conclude that the MFG-S-hIL2RG vector provides the best opportunity for in vivo selection and development of B and T lymphocytes for human XSCID gene therapy.

Primary immunodeficiency mutation databases.

Primary immunodeficiencies are intrinsic defects of immune systems. Mutations in a large number of cellular functions can lead to impaired immune responses. More than 80 primary immunodeficiencies are known to date. During the last years genes for several of these disorders have been identified. Here, mutation information for 23 genes affected in 14 immunodefects is presented. The proteins produced are employed in widely diverse functions, such as signal transduction, cell surface receptors, nucleotide metabolism, gene diversification, transcription factors, and phagocytosis. Altogether, the genetic defect of 2,140 families has been determined. Diseases with X-chromosomal origin constitute about 70% of all the cases, presumably due to full penetrance and because the single affected allele causes the phenotype. All types of mutations have been identified; missense mutations are the most common mutation type, and truncation is the most common effect on the protein level. Mutational hotspots in many disorders appear in CPG dinucleotides. The mutation data for the majority of diseases are distributed on the Internet with a special database management system, MUTbase. Despite large numbers of mutations, it has not been possible to make genotype-phenotype correlations for many of the diseases.

Genetic testing and screening in pediatric populations.

It is conceivable that in the near future a family could present themselves to their health care provider and request to be tested for diseases X, Y, and Z, equipped only with a web page listing of disease-causing genes. The testing of children suggests subtle and controversial inherent conflicts, however. Decisions about whether to provide genetic testing become increasingly murky for a health care professional as the requests advance from testing a child for carrier status for an autosomal recessive disorder, to testing a girl for a sex-linked mutation, to testing an asymptomatic child for a susceptibility to a particular disorder. Although no single case can exemplify every variable and circumstance confronting health care professionals today, this case-based discussion of x-linked severe combined immune deficiency can serve as a framework to examine some of the potential dilemmas surrounding the testing of children for genetic disorders.

Lymphoid development and function in X-linked severe combined immunodeficiency mice after stem cell gene therapy.

Mutations of the common gamma chain (gammac) of cytokine receptors cause X-linked severe combined immunodeficiency (XSCID), a candidate disease for gene therapy. Using an XSCID murine model, we have tested the feasibility of stem cell gene correction. XSCID bone marrow (BM) cells were transduced with a retroviral vector expressing the murine gammac (mgammac) and engrafted in irradiated XSCID animals. Transplanted mice developed mature B cells, naive T cells, and mature natural killer (NK) cells, all of which were virtually absent in untreated mice. The mgammac transgene was detected in all treated mice, and we could demonstrate mgammac expression in newly developed lymphocytes at both the RNA and protein level. In addition, treated mice showed T cell proliferation responses to mitogens and production of antigen-specific antibodies upon immunization. Four of seven treated animals showed a clear increase of the transgene positive cells, suggesting in vivo selective advantage for gene-corrected cells. Altogether, these results show that retroviral-mediated gene transfer can improve murine XSCID and suggest that similar strategies may prove beneficial in human clinical trials.

Efficient detection of thirty-seven new IL2RG mutations in human X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (XSCID) is a rare and potentially fatal disease caused by mutations of IL2RG, the gene encoding the interleukin-2 receptor gamma chain, a component of multiple cytokine receptors that are essential for lymphocyte development and function. To date, over 100 different mutations of IL2RG resulting in XSCID have been published. Using nonradioactive, direct DNA sequencing of a single PCR amplicon containing the whole IL2RG gene, we found IL2RG mutations in 78 previously unpublished unrelated cases of XSCID. We report 37 newly identified mutations of IL2RG, including 23 point mutations, 10 small deletions, 3 instances of the same single nucleotide insertion, 1 large deletion, and 2 complex mutations. More than half of the mutations (22 of 37) were predicted to result in unstable IL2RG mRNA. The remaining 14 mutations disrupted conserved functional motifs common to all cytokine receptor family members; changed protein conformation, charge, or hydrophobicity; or altered the intracellular portion of the protein, which is critical for proper interaction with signal-transducing molecules including Janus family tyrosine kinase 3.

Delayed eruption of permanent teeth in hyperimmunoglobulinemia E recurrent infection syndrome.

OBJECTIVE: To determine the incidence of abnormal tooth eruption in patients with hyperimmunoglobulinemia E (hyper-IgE) syndrome. STUDY DESIGN: This study evaluated 34 individuals with hyper-IgE syndrome (age range, 2-40 years). A comprehensive dental history and a head and neck evaluation were performed on all patients. Dental age was assessed in patients younger than 17 years by 2 methods: (1) clinical assessment of tooth eruption and (2) a radiographic method. Relationships between the chronologic age, dental developmental age, and age at tooth eruption were determined. Other oral or dental anomalies were recorded. RESULTS: Of patients older than 7 years, 75% reported problems with permanent tooth eruption, as evidenced by retained primary teeth or the need for elective extractions of primary teeth to allow eruption of permanent teeth. None of the patients experienced problems with eruption of primary teeth. Eruption of the first and second permanent molars also occurred on time. Dental maturity scores were established for 14 patients 17 years of age or younger. In each case, the difference between chronologic age and the estimated dental developmental age was less than 12 months; however, we found a significant discrepancy between the chronologic age and the mean age of tooth eruption in 80% of these patients when using a particular set of standardized values. Persistence of Hertwig's epithelial root sheath was observed on histologic examination. Chronic multifocal oral candidiasis was a consistent feature in patients with hyper-IgE recurrent infection syndrome. Other oral anomalies were also noted. CONCLUSION: We confirmed that a disorder of tooth eruption is part of the hyper-IgE syndrome. This problem occurs because of delayed primary tooth exfoliation rather than a developmental delay in the formation of the permanent dentition. The persistence of Hertwig's epithelial root sheath is unusual and may be associated with the lack of resorption of the primary teeth. Dentists should be aware of this feature of hyper-IgE syndrome because timely intervention will allow normal eruption to occur.

Autoimmune lymphoproliferative syndrome, a disorder of apoptosis.

Autoimmune Lymphoproliferative Syndrome (ALPS) is a recently recognized disease in which a genetic defect in programmed cell death, or apoptosis, leads to breakdown of lymphocyte homeostasis and normal immunologic tolerance. Some authors have referred to ALPS as Canale-Smith syndrome or lymphoproliferative syndrome with autoimmunity. Patients with ALPS have chronic enlargement of the spleen and lymph nodes, various manifestations of autoimmunity, and elevation of a normally rare population of "double negative T cells" (DNTs), T lymphocytes bearing alpha beta T cell receptors and expressing neither cluster differentiation (CD)4 nor CD8 surface antigens. When lymphocytes from patients with ALPS are cultured in vitro, they are resistant to apoptosis as compared to cells from healthy controls. Most patients with ALPS have mutations in a gene now named TNFRSF6 (tumor necrosis factor receptor gene superfamily member 6). This gene, previously known as apoptosis antigen 1 (APT1), encodes the cell surface receptor for the major apoptosis pathway in mature lymphocytes; this receptor has also had many names, including Fas (to be used here), CD95, and APO-1. ALPS is subdivided into: 1) Type Ia, ALPS with mutant Fas; 2) Type Ib, lymphadenopathy and mutation in the ligand for Fas in one patient with systemic lupus erythematosus; 3) Type II, ALPS with mutant caspase 10; and 4) Type III, ALPS as yet without any defined genetic cause.