Synergy in B-Cell Activation between Toll-Like Receptor 9 and Transmembrane Activator and Calcium-Modulating Cyclophilin Ligand Interactor (TACI) in A181E/C104R Compound Heterozygous Siblings

Purpose. Approximately 9% of common variable immunodeficiency (CVID) patients harbor variants in the transmembrane activator and CAML interactor gene, TACI, which contribute to CVID development. We found identical compound heterozygous TACI variants (C104R and A181E) in kindred of which one sibling had severe CVID with refractory auto immunity, and a second sibling remained asymptomatic. This study investigated possible differences in B-cell phenotype and function that could explain this divergent clinical expression. Methods. C104R and A181E TACI variants were identified through Sanger sequencing. Phenotypic evaluation of the lymphocyte compartment was performed by flow cytometry analyses. Lymphoblastoid cell lines (LCL) from the index patient, asymptomatic sibling, and controls were generated. Intracellular TACI expression was determined, and activation-associated calcium flux capacity was measured. In vitro stimulation assays and RT PCR were performed. Results. Both intracellular levels and surface expressed TACI protein were higher in the asymptomatic sibling than the CVID patient as were TACI-triggering-induced mRNA expression AID and production of Ig class-switched antibodies. In analogy, the asymptomatic sibling displayed enhanced Toll-like receptor 9 expression and signaling, suggesting a compensatory immune mechanism. Conclusions. Posttranscriptional regulation of TACI protein and cross-talk with TLR9 signaling may contribute to phenotypic diversity between individuals with TACI variants.

Possible Perforin-Munc13-4 Gene-Gene Interaction in Familial Hemophagocytic Lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive immune dysregulation disorder associated with Perforin, Munc13-4 and Syntaxin 11 genes. The mutations in the Perforin gene are the most common cause of the disease. Among these mutations, the role of the Alanin91Valin (A91V) alteration in the pathogenesis of the disease has long been controversial. Even though this alteration can be considered as a polymorphism based on its high frequency in normal population (>3.7%) and homozygous existence in some healthy individuals, it is also considered as a genetic risk factor depending on its much higher frequency observed in FHL patients (22.7%) and compound heterozygous existence with other disease causing mutations in the Perforin gene in some FHL patients. Contrary to the previous publications concerning with the co-existence of heterozygous A91V with homozygous mutations in other described FHL genes, there has been no reports on homozygous co-existence of A91V up until this communication where we present the interesting results of a study which shed light not only on the role of A91V in development of FHL, but also on the etiopathogenesis of genetic diseases in general. The subject of the study was a 12 year old female patient who was a product of a first degree consanguineous marriage. Initial diagnosis was lymphomatoid granulomatosis due to the presence of symptoms associated solely with central nervous system. The correct diagnosis could be made 1 year later upon development of systemic findings of FHL. There was no history of similar disorder in the family. Linkage analysis in the family revealed homozygosity for both Perforin and Munc13-4 genes in the patient and for only Munc13-4 gene in one of the asymptomatic sibling who was heterozygous for Perforin gene. Syntaxin 11 gene was excluded in this analysis. Detected merely in the patient was a homozygous A91V substitution (272C>T) in the sequencing of the Perforin gene. Sequencing of the complete coding (32 exons) and the flanking sequences, on the other hand, led to the identification of a homozygous three nucleotide in-frame deletion (2135-2137delTCG) in exon 23 of Munc13-4 gene. This novel mutation resulted in the replacement of nonpolar two aminoacids (Ile-Gly) at positions 712-713 with a polar single aminoacid (Ser). It is plausible that the substitution of highly conserved two aminoacids, especially one (Ile) playing important role in the stability of proteins, with a hydrophilic one would alter the three dimensional structure and the stability of the protein, and would lead to FHL. Ironically, however, an asymptomatic sibling who is currently 22 year old was also homozygous for the mutation. This finding led to the assumption that the Munc13-4 mutation alone may not be sufficient for the development of the disease, but may be a genetic risk factor requiring co-existence of additional homozygous genetic risk factor situated in another FHL gene. If this is the case, it is reasonable to state that homozigosity for A91V in Perforin as well as homozygosity for the 2135-2137del mutation in Munc13-4 is a strong genetic susceptibility factor contributing significantly to the pathogenesis of the disease when they are co-existed. However, this notion could be valid as long as the sibling with homozygous Munc13-4 mutation stays asymptomatic. On the other hand, late onset and atypical presentation in the propositus may indicate that the homozygous co-existence of both alterations is not associated with serious clinical course of the disease as far as the presenting age of the disease is concerned.

Abnormal Erythrocyte Endothelial Adherence in Hereditary Stomatocytosis

Hereditary stomatocytosis is a red cell membrane protein disorder, which results in hemolytic anemia. Some patients with hereditary stomatocytosis experience dyspnea, chest pain, and abdominal pain, particularly after splenectomy. These symptoms may represent vaso-occlusion secondary to adherence of an abnormal erythrocyte membrane to vascular endothelium. We studied three members of a family with varying clinical expression of hereditary stomatocytosis. Adherence of red cells to endothelium was quantified by measuring the shear force required to separate individual cells from endothelial monolayers using a micropipette technique. Two patients with symptoms of in situ thromboses had a higher percentage of adherent cells compared with their asymptomatic sibling and normal controls. Correlation between this in vitro phenomenon and the clinical course suggests that flow abnormalities in the microcirculation attributable to erythrocyte endothelial adherence may play an important pathogenetic role in the illness. When the proportion of adherent red cells was reduced by a chronic transfusion program in one patient and pentoxifyllin therapy in another, the vaso-occlusive complications were eliminated.

B19 parvovirus replicates in circulating cells of acutely infected patients

B19 parvovirus is the etiologic agent of fifth disease and transient aplastic crisis. In natural infections, B19 antigen and DNA have been detected in sera early in the course of aplastic crisis and only rarely in fifth disease. We have found B19 DNA in circulating cells of infected patients by DNA dot blot with a virus-specific probe: in four of four sickle cell patients with aplastic crisis, in one asymptomatic sibling, and in one normal adult with fifth disease. Only two of the sera showed B19 DNA. High-molecular weight intermediate forms were detected by Southern analysis of DNA extracted from cells, thus indicating active replication of virus in cells rather than passive adsorption to their surface membranes. Separation of cells into high- and low-density fractions resulted in a concentration of the virus DNA in the granulocytic fraction.

B19 parvovirus replicates in circulating cells of acutely infected patients

B19 parvovirus is the etiologic agent of fifth disease and transient aplastic crisis. In natural infections, B19 antigen and DNA have been detected in sera early in the course of aplastic crisis and only rarely in fifth disease. We have found B19 DNA in circulating cells of infected patients by DNA dot blot with a virus-specific probe: in four of four sickle cell patients with aplastic crisis, in one asymptomatic sibling, and in one normal adult with fifth disease. Only two of the sera showed B19 DNA. High-molecular weight intermediate forms were detected by Southern analysis of DNA extracted from cells, thus indicating active replication of virus in cells rather than passive adsorption to their surface membranes. Separation of cells into high- and low-density fractions resulted in a concentration of the virus DNA in the granulocytic fraction.