To determine whether human X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome (IPEX; MIM 304930) is the genetic equivalent of the scurfy (sf) mouse, we sequenced the human ortholog (FOXP3) of the gene mutated in scurfy mice (Foxp3), in IPEX patients. We found four non-polymorphic mutations. Each mutation affects the forkhead/winged-helix domain of the scurfin protein, indicating that the mutations may disrupt critical DNA interactions.

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Acidified glycerol lysis test (AGLT) is a screening procedure which has been developed for spherocytosis. AGLT was found positive in 100% of 48 patients suffering from hereditary spherocytosis, 100% of nine couples of affected parents, and 86% of 14 couples of clinically healthy parents. The test was positive in acquired spherocytosis and negative in normal controls. AGLT appears to have a predictive value higher than the entire battery of conventional tests. It is simple, rapid, inexpensive, gives clear-cut results and requires minute amounts of blood. It can be performed on blood stored for up to 24 h.

Mutations of forkhead box p3 (FOXP3), the master gene for naturally occurring regulatory T cells (nTregs), are responsible for the impaired function of nTregs, resulting in an autoimmune disease known as the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. The relevance of other peripheral tolerance mechanisms, such as the presence and function of type 1 regulatory T (Tr1) cells, the major adaptive IL-10-producing Treg subset, in patients with IPEX syndrome remains to be clarified. FOXP3mutated Tr1-polarized cells, differentiated in vitro from CD4+ T cells of four IPEX patients, were enriched in IL-10+IL-4−IFN-γ+ T cells, a cytokine production profile specific for Tr1 cells, and expressed low levels of FOXP3 and high levels of Granzyme-B. IPEX Tr1 cells were hypoproliferative and suppressive, thus indicating that FOXP3 mutations did not impair their function. Furthermore, we isolated Tr1 cell clones from the peripheral blood of one FOXP3null patient, demonstrating that Tr1 cells are present in vivo and they can be expanded in vitro in the absence of WT FOXP3. Overall, our results (i) show that functional Tr1 cells differentiate independently of FOXP3, (ii) confirm that human Tr1 and nTregs are distinct T-cell lineages, and (iii) suggest that under favorable conditions Tr1 cells could exert regulatory functions in IPEX patients.

Forkhead box P3 (FOXP3) is constitutively expressed by CD4 ؉ CD25 hi regulatory T cells (nTregs). IntroductionNaturally occurring CD4 ϩ CD25 hi regulatory T cells (nTregs) play a central role in immune tolerance. nTregs are generated in the thymus, comprise 5% to 10% of peripheral CD4 ϩ T cells, and constitutively express forkhead box P3 (FOXP3), a transcription factor essential for their regulatory activity. 1 Indeed, knockdown of FOXP3 in nTregs reduces suppressive capacity and overexpression of FOXP3 confers suppressive activity to conventional CD4 ϩ T cells. [2][3][4] However, an essential role of FOXP3 in the development and maintenance of nTregs in humans has not yet been shown. Moreover, FOXP3 can be expressed in activated effector T cells, suggesting that it may also be important for cells other than nTregs. 5,6 In male infants, mutations of FOXP3 cause a severe autoimmune disease known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). 7-9 Depending on the mutation, nTregs may be present in IPEX patients, but display an impaired function. 10 The FOXP3 gene (Xp11.23) is subject to X-chromosome inactivation (XCI), 11 a random process by which 1 of the 2 X-chromosomes is methylated during embryogenesis and remains stably inactive in daughter cells. Accordingly, female carriers of FOXP3 mutations are expected to be cell mosaics in which either the wild-type (WT) or the mutated (mut)-FOXP3 allele is active. Therefore, they represent a unique system to investigate the role of WT versus mut-FOXP3 in nTregs and other cell subsets in humans. Because in heterozygous Foxp3 wt/null female mice, only nTregs expressing WT-Foxp3 have the nTreg phenotype and are maintained in the periphery, 12 we investigated whether expression of human WT-FOXP3 is necessary for the development and/or homeostasis of nTregs, by analyzing the pattern of XCI in T-cell subsets from women carrying FOXP3 mutations. Methods SubjectsCarriers of FOXP3 mutations are healthy adults with no autoimmune symptoms. Peripheral blood was obtained upon informed consent from healthy females and carriers of FOXP3 mutations in accordance with approval from the Internal Ethical Committee of the San Raffaele Scientific Institute (protocol TIGET02) and with the Declaration of Helsinki.For complete materials and methods information, see supplemental methods (available on the Blood website; see the Supplemental Materials link at the top of the online article). Results and discussion Healthy carriers of FOXP3 mutations are mosaics of cells expressing either the WT or the mut-FOXP3 alleleNine healthy female carriers of 5 different FOXP3 mutations, relatives of severely affected IPEX patients, were studied (Figure 1A For personal use only. on April 11, 2019. by guest www.bloodjournal.org From and supplemental methods). The distribution of active WT and mut-FOXP3 alleles in peripheral blood mononuclear cells (PBMCs; n ϭ 9), CD4 ϩ T (n ϭ 8), CD8 ϩ T (n ϭ 3), CD56 ϩ natural killer (n ϭ 2), and CD19 ϩ B (n ϭ 3) cells was evaluated by de...

A total of 137 fragile X and 235 control chromosomes from various regions of Italy were haplotyped by analyzing two neighbouring marker microsatellites, FRAXAC1 and DXS548. The number of CGG repeats at the 5′ end of the FMR1 gene was also assessed in 141 control chromosomes and correlated with their haplotypes. Significant linkage disequilibrium between some “major” haplotypes and fragile X was observed, while other “minor” haplotypes may have originated by subsequent mutation at the marker microsatellite loci and/or recombination between them. Recent evidence suggests that the initial mechanism leading to CGG instability might consist of rare (10 6/‐7) CGG repeat slippage events and/or loss of a stabilizing AGG via A‐to‐C transversion. Also, the apparently high variety of fragile X chromosomes may be partly due to the relatively high mutation rate (10 −4/‐5) of the microsatellite markers used in haplotyping. Our fragile X sample also showed a higher than expected heterozygosity when compared to the control sample and we suggest that this might be explained by the chance occurrence of the few founding events on different chromosomes, irrespective of their actual frequency in the population. Alternatively, a local mechanism could enhance the microsatellite mutation rate only on fragile X chromosomes, or fragile X mutations might occur more frequently on certain background haplotypes. © 1996 Wiley‐Liss, Inc.