The phenotypical implications of immune dysregulation in fragile X syndrome.

BACKGROUND AND PURPOSE: Immune system dysfunction and inflammatory dysregulation have been shown in several animal models of fragile X syndrome (FXS). However, the phenotypical implications of this dysregulation have not been systematically evaluated in a large patient cohort. METHODS: Five thousand seven hundred thirty-six FXS patients from a nationwide health insurance database were identified and compared to 573 600 age- and sex-matched controls. The phenome-wide association studies codes of FXS patients and those without FXS were compared and the false discovery rate was controlled at 0.05 using the Benjamini-Hochberg procedure. RESULTS: In addition to the commonly reported comorbidities of FXS, an over-representation of infectious diseases, including otitis media, cellulitis and abscess of fingers or toes, viral enteritis, candidiasis and pneumonia, was discovered. In addition, there was an under-representation of autoimmune disorders in FXS patients. CONCLUSIONS: Our systematic comorbidity analyses identified immunologically-based phenotypes associated with FXS. Our findings align with previous observations of compromised immunity and phagocytic defects in animal models of FXS. These results suggest the importance of immune-related pathways in FXS patients and their relevance to the FMR1 gene.

Fragile phagocytes: FMRP positively regulates engulfment activity.

Defective immune system function is implicated in autism spectrum disorders, including Fragile X syndrome. In this issue, O'Connor et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607093) demonstrate that phagocytic activity of systemic immune cells is compromised in a Drosophila melanogaster model of Fragile X, highlighting intriguing new mechanistic connections between FMRP, innate immunity, and abnormal development.

A Drosophila model of Fragile X syndrome exhibits defects in phagocytosis by innate immune cells.

Fragile X syndrome, the most common known monogenic cause of autism, results from the loss of FMR1, a conserved, ubiquitously expressed RNA-binding protein. Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. We found that Drosophila melanogaster Fmr1 mutants exhibit increased sensitivity to bacterial infection and decreased phagocytosis of bacteria by systemic immune cells. Using tissue-specific RNAi-mediated knockdown, we showed that Fmr1 plays a cell-autonomous role in the phagocytosis of bacteria. Fmr1 mutants also exhibit delays in two processes that require phagocytosis by glial cells, the immune cells in the brain: neuronal clearance after injury in adults and the development of the mushroom body, a brain structure required for learning and memory. Delayed neuronal clearance is associated with reduced recruitment of activated glia to the site of injury. These results suggest a previously unrecognized role for Fmr1 in regulating the activation of phagocytic immune cells both in the body and the brain.

Anti-neuronal antibodies in patients with fragile X syndrome: is there a role of autoimmunity in its pathogenesis?

BACKGROUND: Fragile X syndrome (FXS) is a single-gene disorder with a broad spectrum of involvement, including cognitive and behavioural impairments of varying degrees with specific physical features and a strong association with autism. OBJECTIVES: In this study, the frequency of serum anti-neural antibodies was investigated in FXS patients who did and those who did not manifest autism spectrum disorders (ASD) in comparison to typically developing controls. METHODS: The study involved 23 males (mean age, 19.78 ± 6.56 years) who harboured a full mutation in the FMR1 gene. The control group comprised 19 healthy students (mean age 24.63 ± 1.89 years). Serum anti-neuronal antibodies were analyzed using Western blotting. RESULTS: Serum anti-neuronal antibodies were present in 10/23 (43.48%) FXS males. CONCLUSION: Serum anti-neuronal antibodies were found in a subgroup of FXS patients. Autistic symptoms in FXS may, in part, be caused by auto-immune factors. Further studies in larger patient and control groups are necessary to elucidate the aetiopathogenic role of anti-neuronal antibodies in FXS patients.

Immune dysregulation as a cause of autoinflammation in fragile X premutation carriers: link between FMRI CGG repeat number and decreased cytokine responses.

BACKGROUND: Increased rates of autoinflammatory and autoimmune disorders have been observed in female premutation carriers of CGG repeat expansion alleles of between 55-200 repeats in the fragile X mental retardation 1 (FMR1) gene. To determine whether an abnormal immune profile was present at a cellular level that may predispose female carriers to autoinflammatory conditions, we investigated dynamic cytokine production following stimulation of blood cells. In addition, splenocyte responses were examined in an FMR1 CGG knock-in mouse model of the fragile X premutation. METHODS: Human monocyte and peripheral blood leukocytes (PBLs) were isolated from the blood of 36 female FMR1 premutation carriers and 15 age-matched controls. Cells were cultured with media alone, LPS or PHA. In the animal model, splenocytes were isolated from 32 CGG knock-in mice and 32 wild type littermates. Splenocytes were cultured with media alone or LPS or PMA/Ionomycin. Concentrations of cytokines (GM-CSF, IL-1ß, IL-6, IL-10, IL-13, IL-17, IFNγ, TNFα, and MCP-1) were determined from the supernatants of cellular cultures via Luminex multiplex assay. Additionally, phenotypic cellular markers were assessed on cells isolated from human subjects via flow cytometry. RESULTS: We found decreases in cytokine production in human premutation carriers as well as in the FMR1 knock-in mice when compared with controls. Levels of cytokines were found to be associated with CGG repeat length in both human and mouse. Furthermore, T cells from human premutation carriers showed decreases in cell surface markers of activation when compared with controls. CONCLUSIONS: In this study, FMR1 CGG repeat expansions are associated with decreased immune responses and immune dysregulation in both humans and mice. Deficits in immune responses in female premutation carriers may lead to increased susceptibility to autoimmunity and further research is warranted to determine the link between FMR1 CGG repeat lengths and onset of autoinflammatory conditions.

Experiencias técnicas en el uso de la prueba inmunohistoquímica para el diagnóstico del síndrome de frágil X / Technical experiences in the use of the immunohistochemical test for diagnosing Fragile X syndrome

Se evaluó un método inmunohistoquímico diseñado por Willemsen y otros en 1995, para detectar la presencia o ausencia de la proteína FMRP, involucrada en el síndrome de frágil X. Este proceder se fundamenta en una secuencia de reacciones antígeno-anticuerpo, que garantiza la inmunodetección de la proteína, que resulta fácil de realizar con un costo menor a las demás técnicas descritas para el diagnóstico de la afección. No obstante, se hizo una exhaustiva valoración de los posibles errores técnicos, partiendo de la experiencia acumulada durante el pesquisaje inmunohistoquímico que se aplicó a 6 615 recién nacidos y a 658 individuos varones con retraso mental de etiología no precisada. Se expusieron algunas fotos para ilustrar los errores más frecuentes(AU)

Experiencias técnicas en el uso de la prueba inmunohistoquímica para el diagnóstico del síndrome de frágil X / Technical experiences in the use of the immunohistochemical test for diagnosing Fragile X syndrome

Se evaluó un método inmunohistoquímico diseñado por Willemsen y otros en 1995, para detectar la presencia o ausencia de la proteína FMRP, involucrada en el síndrome de frágil X. Este proceder se fundamenta en una secuencia de reacciones antígeno-anticuerpo, que garantiza la inmunodetección de la proteína, que resulta fácil de realizar con un costo menor a las demás técnicas descritas para el diagnóstico de la afección. No obstante, se hizo una exhaustiva valoración de los posibles errores técnicos, partiendo de la experiencia acumulada durante el pesquisaje inmunohistoquímico que se aplicó a 6 615 recién nacidos y a 658 individuos varones con retraso mental de etiología no precisada. Se expusieron algunas fotos para ilustrar los errores más frecuentes

[FMRP immunodetection on hair roots: application to the diagnosis of fragile X syndrome]. / Estudio de la proteína FMRP en la raíz de cabello: aplicación al diagnóstico del síndrome del cromosoma X frágil.

INTRODUCTION: Fragile X syndrome is the most common inherited form of mental retardation. The absence of FMRP protein, codified by the FMR1 gene, results in fragile X phenotype. DNA-based diagnostic methods determine the length of the CGG repeat within the FMR1 gene, the main mutation causing the syndrome. Immunohistochemical diagnostic tests detect all mutations leading to the absence of FMRP expression. Results of the antibody test on hair roots correlate with intellectual quotient in affected men and women. PATIENTS AND METHODS: Immunohistochemical techniques were used to study FMRP expression in hair roots in a control group to establish the correlation with the length of the CGG repeat. Subsequently, 65 girls and boys with mental retardation attending special schools were screened by using the FMRP test on hair roots. RESULTS: Males and females molecularly characterized as within the normal and premutated range expressed FMRP in more than 70 % of hair roots. Full mutation carriers expressed FMRP in less than 70 % of hair roots. Immunohistochemical studies in males and females with mental retardation led to the identification of one affected male. CONCLUSIONS: Fragile X syndrome detection by immunohistochemical testing of hair roots is a valid method of population screening because of the relative noninvasiveness of obtaining samples, and the ease and rapidness of the technique, which can be applied to routine clinical practice.

Prenatal fragile X detection using cytoplasmic and nuclear-specific monoclonal antibodies.

We have been carrying out studies aimed at improving prenatal detection of the fragile X chromosome/mutation. Our current protocol requires a turnaround time (TAT) of several days. In an attempt to reduce the TAT, we have turned to the use of monoclonal antibodies (mAbs). Monoclonal antibody 1A1 (provided by Dr. Mandel of INSERM) immunostaining was performed according to a modified three-step immunocytochemical procedure. We found that cytoplasmic staining intensities, using mAb 1A1/avidin biotinylated complex/diaminobenzidine, varied from light to heavy within each sample, with controls exhibiting a majority of heavily stained cells in both chorionic villus (CV) sample and amniotic fluid cultured cells. Using mAb 1A1 and a new nuclear-specific antibody, mAb 3F11, we found that CV cultured cells harboring the FMR1 full mutation could be distinguished from controls as early as 10 weeks of gestation in both male and female specimens. Western blot analysis showed that the antibodies have similar staining patterns but that mAb 3F11 has fewer background/nonspecific bands. Our results demonstrate that it is feasible to detect fragile X full mutations within one day after obtaining cells from CV specimens taken as early as 10 weeks of gestation.