Cristina Woellner

A homozygous CARD9 mutation in a family with susceptibility to fungal infections.

BACKGROUND Chronic mucocutaneous candidiasis may be manifested as a primary immunodeficiency characterized by persistent or recurrent infections of the mucosa or the skin with candida species. Most cases are sporadic, but both autosomal dominant inheritance and autosomal recessive inheritance have been described. METHODS We performed genetic studies in 36 members of a large, consanguineous five-generation family, in which 4 members had recurrent fungal infections and an additional 3 members died during adolescence, 2 after invasive infection of the brain with candida species. All 36 family members were enrolled in the study, and 22 had blood samples taken for DNA analysis. Homozygosity mapping was used to locate the mutated gene. In the 4 affected family members (patients) and the 18 unaffected members we sequenced CARD9, the gene encoding the caspase recruitment domain-containing protein 9, carried out T-cell phenotyping, and performed functional studies, with the use of either leukocytes from the patients or a reconstituted murine model of the genetic defect. RESULTS We found linkage (lod score, 3.6) to a genomic interval on chromosome 9q, including CARD9. All four patients had a homozygous point mutation in CARD9, resulting in a premature termination codon (Q295X). Healthy family members had wild-type expression of the CARD9 protein; the four patients lacked wild-type expression, which was associated with low numbers of Th17 cells (helper T cells producing interleukin-17). Functional studies based on genetic reconstitution of myeloid cells from Card9(-/-) mice showed that the Q295X mutation impairs innate signaling from the antifungal pattern-recognition receptor dectin-1. CONCLUSIONS An autosomal recessive form of susceptibility to chronic mucocutaneous candidiasis is associated with homozygous mutations in CARD9.

Mutations in STAT3 and diagnostic guidelines for hyper-IgE syndrome

BACKGROUND The hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by infections of the lung and skin, elevated serum IgE, and involvement of the soft and bony tissues. Recently, HIES has been associated with heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3) and severe reductions of T(H)17 cells. OBJECTIVE To determine whether there is a correlation between the genotype and the phenotype of patients with HIES and to establish diagnostic criteria to distinguish between STAT3 mutated and STAT3 wild-type patients. METHODS We collected clinical data, determined T(H)17 cell numbers, and sequenced STAT3 in 100 patients with a strong clinical suspicion of HIES and serum IgE >1000 IU/mL. We explored diagnostic criteria by using a machine-learning approach to identify which features best predict a STAT3 mutation. RESULTS In 64 patients, we identified 31 different STAT3 mutations, 18 of which were novel. These included mutations at splice sites and outside the previously implicated DNA-binding and Src homology 2 domains. A combination of 5 clinical features predicted STAT3 mutations with 85% accuracy. T(H)17 cells were profoundly reduced in patients harboring STAT3 mutations, whereas 10 of 13 patients without mutations had low (<1%) T(H)17 cells but were distinct by markedly reduced IFN-gamma-producing CD4(+)T cells. CONCLUSION We propose the following diagnostic guidelines for STAT3-deficient HIES. Possible: IgE >1000IU/mL plus a weighted score of clinical features >30 based on recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic face, and high palate. Probable: These characteristics plus lack of T(H)17 cells or a family history for definitive HIES. Definitive: These characteristics plus a dominant-negative heterozygous mutation in STAT3.

Reduced memory B cells in patients with hyper IgE syndrome

Dominant-negative mutations in STAT-3 have recently been found in the majority of patients with sporadic or autosomal-dominant hyper IgE syndrome (HIES). Since STAT-3 plays a role in B cell development and differentiation, we analyzed memory B cells in 20 patients with HIES, 17 of which had STAT-3 mutations. All but four patients had reduced non-switched and/or class-switched memory B cells. No reduction in these B cell populations was found in 16 atopic dermatitis patients with IgE levels above 1000 KU/L. There was no correlation between the reduction of memory B cells and the ability to produce specific antibodies. Moreover, there was no correlation between the percentage of memory B cells and the infection history. Analysis of memory B cells can be useful in distinguishing patients with suspected HIES from patients with atopic disease, but probably fails to identify patients who are at high risk of infection.

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.

Screening of functional and positional candidate genes in families with common variable immunodeficiency

BackgroundCommon variable immunodeficiency (CVID) comprises a heterogeneous group of primary antibody deficiencies with complex clinical and immunological phenotypes. The recent discovery that some CVID patients show monogenic defects in the genes encoding ICOS, TACI or CD19 prompted us to investigate several functional candidate genes in individuals with CVID.ResultsThe exonic, protein coding regions of the genes encoding: APRIL, BCMA, IL10, IL10Rα, IL10Rβ, IL21, IL21R, and CCL18, were analyzed primarily in familial CVID cases, who showed evidence of genetic linkage to the respective candidate gene loci and CVID families with a recessive pattern of inheritance. Two novel SNPs were identified in exon 5 and exon 8 of the IL21R gene, which segregated with the disease phenotype in one CVID family. Eleven additional SNPs in the genes encoding BCMA, APRIL, IL10, IL10Rα, IL21 and IL21R were observed at similar frequencies as in healthy donors.ConclusionWe were unable to identify obvious disease causing mutations in the protein coding regions of the analyzed genes in the studied cohort.

Toll-Like Receptor Stimulation Induces Higher TNF-α Secretion in Peripheral Blood Mononuclear Cells from Patients with Hyper IgE Syndrome

Hyper IgE syndromes (HIES) are primary immunodeficiency disorders of unknown pathogenesis. Patients are typically affected with ‘cold’ abscesses of the skin, recurrent cyst-forming pneumonia, chronic mucocutaneous candidiasis and other less frequent features such as progressive skeletal abnormalities. Defective signaling in the Toll-like receptor (TLR) pathways has been suggested as a responsible pathologic mechanism, however, in previous reports, 10 patients revealed no defect in inflammatory cytokine responses to different TLR ligands. Here, we report the increase in pro-inflammatory cytokines TNF-α and IL-8, following TLR2 and TLR4 stimulation in a larger cohort of 25 additional patients with HIES, and provide a meta-analysis of the TLR data in HIES.

Pneumocystis jirovecii pneumonia in a baby with hyper-IgE syndrome

A 4-month-old baby with a family history of hyper-IgE syndrome acquired Pneumocystis jirovecii pneumonia. The patient’s hyper-IgE syndrome score was low, but a genetic study yielded a STAT3 mutation. P. jirovecii pneumonia can be added to the infections associated with hyper-IgE syndrome. In some cases, it may be the presenting manifestation of this immunodeficiency.

The hyper-IgE syndrome is not caused by a microdeletion syndrome

The hyper-immunoglobulin E syndrome (HIES) is a rare primary immunodeficiency characterized by recurrent infections, elevated serum IgE-levels, and involvement of the soft- and bony tissues. We speculated that this complex disease may be caused by a microdeletion syndrome. We therefore analyzed 30 sporadic HIES patients for the presence of chromosomal imbalances using Affymetrix 50k XbaI and 23 of the 30 patients with the higher-resolution 250k StyI SNP mapping arrays. We detected only eight different copy number alterations in six patients with the 50k approach, and seven of these presented known polymorphic regions not associated with disease. However, one patient showed a unique gain on chromosome 20p. 250k array analysis identified this gain as a rare polymorphism segregating in the patient’s family, but not associated with the HIES phenotype. In addition, 265 known and novel copy number variants (CNVs) were identified with the 250k arrays, but no recurrent imbalances reminescent of a microdeletion syndrome were found. We aligned the identified CNVs with loci that have been associated with HIES or phenotypically overlapping syndromes. Doing so, a 2-Mb deletion spanning the PEPD gene on 19q13.11 was identified on one allele of one patient. Homozygous mutations in PEPD are responsible for the autosomal-recessive prolidase deficiency which resembles HIES in some aspects. Sequencing of the healthy allele, however, revealed a wild-type sequence. In summary, our results suggest that HIES is not likely to be a microdeletion syndrome.

Community acquired Staphylococcus aureus meningitis and cerebral abscesses in a patient with a Hyper-IgE and a Dubowitz-like syndrome

The Hyper-IgE syndrome (HIES) is a rare primary immunodeficiency which recently has been associated with heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3). Although HIES is characterized by recurrent staphylococcal infections, the microbial invasion of the central nervous system (CNS) is definitively uncommon. We here report on Staphylococcus aureus meningitis and cerebral abscesses acquired in the community in a 31-year-old female patient with a de novo heterozygous mutation of STAT3 and a Dubowitz-like syndrome characterized by growth retardation, microcephaly and eczema. The patient presented with a relative paucity of clinical symptoms despite severe cerebrospinal fluid pathology and multiple cerebral abscesses. Antimicrobial as well as treatment with intravenous immunoglobulin was well tolerated and led to a slow recovery over a 6 months period. Our observation adds community acquired S. aureus meningitis to the list of life-threatening infections in STAT3-deficient HIES and should also raise awareness for the unusual clinical presentation of severe neuroinfection in this syndrome. Whether the association of HIES with Dubowitz-like syndrome was purely coincidental, possibly supportive of the CNS infection, or suggests a genetic overlap of these syndromes, awaits clarification.

Early-onset inflammatory bowel disease caused by mutant IL10 receptor

Methods We performed genetic linkage analysis and candidate gene sequencing in two unrelated consanguineous families with children affected by early-onset IBD. We screened six additional patients for mutations in two candidate genes and carried out functional assays in patients’ peripheral blood mononuclear cells. We treated one patient with an allogeneic hematopoietic stem cell transplant (HSCT).