Familial hemophagocytic lymphohistiocytosis (FHL) type 3 is a life-threatening immune dysregulation syndrome caused by mutations in the UNC13D gene, encoding the munc13–4 protein, which is important ...for function of cytotoxic lymphocytes. FHL3 accounts for 30–40% of FHL cases, and more than 100 mutations in the UNC13D gene have been described to date. We describe the first case of FHL3 carrying an intragenic duplication of UNC13D, apparently mediated by recombination of Alu elements. NK cell degranulation and munc13–4 protein expression assays are useful for early identification of such mutations, which may be missed by analysis of genomic DNA alone.
•The first FHL3 case with intragenic duplication of the UNC13D gene is presented.•The UNC13D gene duplication was caused by an Alu-mediated recombination.•FHL screening by NK degranulation and protein expression assays is important.
The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial ...thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαβ+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.
•The ATO system allows precise definition of developmental blocks in patients with gene defects that cause T-cell lymphopenia.•The ATO system can help distinguish between hematopoietic autonomous and extra-hematopoietic defects that cause T-cell lymphopenia.
Display omitted
Purpose
Anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) is characterized by hypohidrosis, dental abnormalities, sparse hair, and immunodeficiency. Autosomal dominant (AD)-EDA-ID, ...caused by a heterozygous mutation within
NFKBIA
, is very rare and its clinical features remain largely unknown. This study describes a patient with AD-EDA-ID harboring a novel
NFKBIA
mutation who presented with mild EDA and non-infectious systemic inflammation.
Methods
The clinical presentation of an AD-EDA-ID patient was described and immunological, genetic, and biochemical analyses were performed, with a focus on nuclear factor kappa B (NF-κB) activation.
Results
The patient presented with symptoms of mild EDA-ID, namely sparse hair and hypohidrosis, although a skin biopsy confirmed the presence of sweat glands. There were no dental abnormalities. The patient also suffered from non-infectious inflammation, which responded to systemic corticosteroid therapy; however, the patient remained ill. Immunological analyses revealed reduced Toll-like receptor/IL-1 (TLR/IL-1) and tumor necrosis factor (TNF) receptor family responses to various stimuli. Genetic analysis identified a de novo heterozygous missense mutation, p.Ser36Tyr, in NFKBIA, resulting in defective NFKBIA degradation and impaired NF-κB activation. The patient was diagnosed with AD-EDA-ID and underwent hematopoietic stem cell transplantation. Engraftment was successful, with few signs of acute graft versus host disease. However, the patient suffered hemolytic anemia and thrombocytopenia, and died from a brain hemorrhage due to intractable thrombocytopenia.
Conclusion
AD-EDA-ID patients can present with mild ectodermal dysplasia and non-infectious inflammation, rather than with recurrent infections. Also, hematopoietic stem cell transplantation for AD-EDA-ID is still a clinical challenge.
Loss-of-function mutations in filamin A (FLNA) cause an X-linked dominant disorder with multiple organ involvement. Affected females present with periventricular nodular heterotopia (PVNH), ...cardiovascular complications, thrombocytopenia and Ehlers-Danlos syndrome. These mutations are typically lethal to males, and rare male survivors suffer from failure to thrive, PVNH, and severe cardiovascular and gastrointestinal complications. Here we report two surviving male siblings with a loss-of-function mutation in FLNA. They presented with multiple complications, including valvulopathy, intestinal malrotation and chronic intestinal pseudo-obstruction (CIPO). However, these siblings had atypical clinical courses, such as a lack of PVNH and a spontaneous improvement of CIPO. Trio-based whole-exome sequencing revealed a 4-bp deletion in exon 40 that was predicted to cause a lethal premature protein truncation. However, molecular investigations revealed that the mutation induced in-frame skipping of the mutated exon, which led to the translation of a mutant FLNA missing an internal region of 41 amino acids. Functional analyses of the mutant protein suggested that its binding affinity to integrin, as well as its capacity to induce focal adhesions, were comparable to those of the wild-type protein. These results suggested that exon skipping of FLNA partially restored its protein function, which could contribute to amelioration of the siblings' clinical courses. This study expands the diversity of the phenotypes associated with loss-of-function mutations in FLNA.
X-linked anhidrotic ectodermal dysplasia with immunodeficiency (X-EDA-ID) is caused by hypomorphic mutations in the gene encoding nuclear factor-κB essential modulator protein (NEMO). Patients are ...susceptibile to diverse pathogens due to insufficient cytokine and frequently show severe chronic colitis. An 11-year-old boy with X-EDA-ID was hospitalized with autoimmune symptoms and severe chronic colitis which had been refractory to immunosuppressive drugs. Since tumor necrosis factor (TNF) α is responsible for the pathogenesis of NEMO colitis according to intestinal NEMO and additional TNFR1 knockout mice studies, and high levels of TNFα-producing mononuclear cells were detected in the patient due to the unexpected gene reversion mosaicism of NEMO, an anti-TNFα monoclonal antibody was administered to ameliorate his abdominal symptoms. Repeated administrations improved his colonoscopic findings as well as his dry skin along with a reduction of TNFα-expressing T cells. These findings suggest TNF blockade therapy is of value for refractory NEMO colitis with gene reversion.
In vivo imaging of beta -amyloid (A beta ) aggregates in the brain may lead to early detection of Alzheimer's disease (AD) and monitoring of the progression and effectiveness of treatment. The ...purpose of this study was to develop novel 18F-labeled amyloid-imaging probes based on flavones as a core structure. Fluoropegylated (FPEG) flavone derivatives were designed and synthesized. The affinity of the derivatives for A beta aggregates varied from 5 to 321 nM. In brain sections of AD model mice, FPEG flavones with the dimethylamino group intensely stained beta -amyloid plaques. In biodistrubution experiments using normal mice, they displayed high uptake in the brain ranging from 2.9 to 4.2%ID/g at 2 min postinjection. The radioactivity washed out from the brain rapidly (1.3-2.0%ID/g at 30 min), which is highly desirable for beta -amyloid imaging agents. FPEG flavones may be potential PET imaging agents for beta -amyloid plaques in Alzheimer's brains.
Chronic infantile neurological cutaneous and articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory ...disease and is caused by a heterozygous germline gain-of-function mutation in the NLRP3 gene. We recently found a high incidence of NLRP3 somatic mosaicism in apparently mutation-negative CINCA/NOMID patients using subcloning and subsequent capillary DNA sequencing. It is important to rapidly diagnose somatic NLRP3 mosaicism to ensure proper treatment. However, this approach requires large investments of time, cost, and labour that prevent routine genetic diagnosis of low-level somatic NLRP3 mosaicism. We developed a routine pipeline to detect even a low-level allele of NLRP3 with statistical significance using massively parallel DNA sequencing. To address the critical concern of discriminating a low-level allele from sequencing errors, we first constructed error rate maps of 14 polymerase chain reaction products covering the entire coding NLRP3 exons on a Roche 454 GS-FLX sequencer from 50 control samples without mosaicism. Based on these results, we formulated a statistical confidence value for each sequence variation in each strand to discriminate sequencing errors from real genetic variation even in a low-level allele, and thereby detected base substitutions at an allele frequency as low as 1% with 99.9% or higher confidence.
As a first trial for in vivo imaging of β-secretase (BACE1) in Alzheimer's disease brain, we applied a novel non-peptidergic small molecule which has high affinity to the enzyme, ...naphthalene-1-carboxylic acid (3′-chloro-4′-fluoro-4-piperazin-1-yl-biphenyl-3-yl)amide (NCFB) into positron emission tomography (PET) probe. In the current study, N-11C-methylated compound of NCFB, 11CMe-NCFB was synthesized and evaluated for the visualization of BACE1 in brain.
BACE1 inhibitory constant was measured by FRET assay. 11CMe-NCFB was synthesized from NCFB with 11Cmethyl triflate. To evaluate properties of 11CMe-NCFB, log P value, stability in mouse plasma and brain uptake index were measured. The biodistribution in 6-week-old ddY mice was also studied.
BACE1 inhibitory constant showed an affinity of Me-NCFB to the enzyme (IC50=2.3±0.80μM). 11CMe-NCFB was synthesized in a 3.0%±0.55% decay-corrected radiochemical yield. 11CMe-NCFB showed high lipophilicity, high stability in mouse plasma and blood–brain barrier (BBB) permeability. Injected to 6-week-old ddY mice, 11CMe-NCFB penetrated BBB and was retained in the brain (0.79%±0.22% ID/g at 2min and 0.75%±0.08% ID/g at 60min after injection, respectively), moreover, rapid blood clearance was observed.
11CMe-NCFB could have a potential as a PET probe for the imaging of BACE1 in the brain.
PDF
