David Halsall

Predicting Hla Class Ii Alloantigen Immunogenicity From the Number and Physiochemical Properties of Amino Acid Polymorphisms

Background. We have shown previously that human leukocyte antigen (HLA) class I immunogenicity can be predicted by the number, position, and physiochemical differences of polymorphic amino acids (AAs). We have now modeled the structural and physiochemical polymorphisms of HLA class II alloantigens and correlated these with humoral alloimmunity in sensitized patients awaiting kidney transplantation. Methods. Sera obtained from 30 patients with high levels of IgG HLA-specific antibodies were screened using single-antigen HLA antibody detection beads. A computer program was developed to determine the number of AA mismatches (after interlocus and intralocus subtraction) and their hydrophobicity and electrostatic mismatch score for each mismatched HLA-DR and -DQ specificity. Regression methods were used to compare these variables with the occurrence and magnitude of alloantibody responses. Results. HLA-specific antibody was detected against 879 (55%) of 1604 mismatched HLA specificities evaluated. There was a strong correlation between increasing number of AA mismatches and the occurrence (P<0.001, odds ratio 3.85 per AA) and magnitude of alloantibody responses (P<0.001); only 6% of alloantigens with 0 to 2 mismatched AA-induced alloantibody (median fluorescence intensity 37) compared with 82% of alloantigens with more than or equal to 20 mismatched AAs (median fluorescence intensity 9969). Hydrophobicity and electrostatic mismatch scores also correlated closely with alloantibody response (P<0.001), but neither variable had independent predictive value over the number of AA mismatches alone. Conclusion. Differences in the number of polymorphic AA mismatches and their physiochemical properties for a given recipient HLA type are strong predictors of class II alloantigen immunogenicity and alloantibody response before kidney transplantation.

High-resolution, three-dimensional modeling of human leukocyte antigen class I structure and surface electrostatic potential reveals the molecular basis for alloantibody binding epitopes

The potential of human leukocyte antigens (HLA) to stimulate humoral alloimmunity depends on the orientation, accessibility and physiochemical properties of polymorphic amino acids. We have generated high-resolution structural and physiochemical models of all common HLA class I alleles and analyzed the impact of amino acid polymorphisms on surface electrostatic potential. Atomic resolution three-dimensional structural models of HLA class I molecules were generated using the MODELLER computer algorithm. The molecular surface electrostatic potential was calculated using the DelPhi program. To confirm that electrostatic surface topography reflects known HLA B cell epitopes, we examined Bw4 and Bw6 and ascertained the impact of amino acid polymorphisms on their tertiary and physiochemical composition. The HLA protein structures generated performed well when subjected to stereochemical and energy-based testing for structural integrity. The electrostatic pattern and conformation of Bw4 and Bw6 epitopes are maintained among HLA molecules even when expressed in a different structural context. Importantly, variation in epitope amino acid composition does not always translate into a different electrostatic motif, providing an explanation for serologic cross-reactivity. Mutations of critical amino acids that abrogate antibody binding also induce distinct changes in epitope electrostatic properties. In conclusion, high-resolution structural modeling provides a physiochemical explanation for serologic patterns of antibody binding and provides novel insights into HLA immunogenicity.

Identification of Mutations in SLC40A1 That Affect Ferroportin Function and Phenotype of Human Ferroportin Iron Overload

BACKGROUND & AIMSnPatients with ferroportin iron overload due to loss-of-function mutations in SLC40A1 have macrophage iron overload, hyperferritinemia, and normal transferrin saturation. In contrast, hepatocellular iron storage, hyperferritinemia, and increased saturation of transferrin are a distinct clinical presentation of ferroportin iron overload that results from SLC40A1 mutations that confer resistance of ferroportin to hepcidin-mediated inactivation.nnnMETHODSnSLC40A1 was sequenced in patients from 2 independent pedigrees affected by hepatic iron overload unrelated to HFE. Functions of the ferroportin variants were tested in vitro.nnnRESULTSnA patient heterozygous for the variant p.W158C in SLC40A1 presented with macrophage iron overload, hyperferritinemia, and normal transferrin saturation. A patient with hepatocellular iron storage, hyperferritinemia, and increased transferrin saturation was heterozygous for p.H507R. Expression of the p.W158C form of ferroportin in 293T cells resulted in defective trafficking to the plasma membrane and reduced iron export activity; the iron export activity of cells that expressed the p.H507R form of ferroportin did not differ from cells that expressed ferroportin without this mutation. The p.H507R of ferroportin localizes normally to the plasma membrane but is resistant to hepcidin-mediated inactivation. Addition of a synthetic peptide derived from ferroportin without these mutations (amino acids 500-518) decreased the inhibitory activity of hepcidin in cells, whereas a peptide from the same region, with p.H507R, had no effect on hepcidin activity.nnnCONCLUSIONSnThe variant p.W158C in SLC40A1 impairs intracellular trafficking of ferroportin, resulting in reduced iron export. The variant p.H507R does not bind hepcidin in vitro and results in apparent hepcidin resistance.

Clinical Presentation and Molecular Pathophysiology of Autosomal Dominant Hemochromatosis Caused by a Novel Ferroportin Mutation

Mutations in the SLC40A1 gene, which encodes ferroportin, are associated with autosomal dominant hemochromatosis. Ferroportin is inhibited directly by hepcidin, a key iron‐regulatory peptide, and functional consequences of SLC40A1 mutations account for observed phenotypic differences in patients with ferroportin disease. We describe a large pedigree with a novel SLC40A1 mutation and, through in vitro analysis, elucidate the associated molecular mechanism of iron overload. The entire coding sequence of the SLC40A1 gene was sequenced in a pedigree, presenting with autosomal dominant hyperferritinemia. The functional effects of a novel SLC40A1 mutation were studied by overexpression of wild‐type and mutant ferroportin fusion proteins in human embryonic kidney cells. Iron export was studied in these cells using 59Fe transport assays; subcellular localization of ferroportin was examined by way of confocal microscopy. A novel SLC40A1 mutation p.R489K segregated with iron overload in a family with clinical and histopathological signs of macrophage‐type ferroportin disease. Human embryonic kidney cells overexpressing p.R489K ferroportin showed decreased iron export capacity when compared with wild‐type ferroportin overexpressing cells. Subcellular localization studies demonstrated that p.R489K ferroportin was retained abnormally within an intracellular compartment. Conclusion: We report a novel pathological SLC40A1 variant associated with abnormal cell surface expression of ferroportin due to intracellular retention of the mutant protein. These findings predict macrophage‐type ferroportin disease, the phenotype observed in this kindred. Study of the molecular cell biology of ferroportin and its mutants is key to understanding the pathogenesis of this increasingly recognized form of hemochromatosis, which responds poorly to conventional therapy. (HEPATOLOGY 2009.)

HLA class I amino acid sequence-based matching after interlocus subtraction and long-term outcome after deceased donor kidney transplantation

We have shown previously that human leukocyte antigen (HLA) immunogenicity, defined by the physiochemical properties of mismatched amino acids, predicts humoral alloimmunity, and now report the effect on long-term graft survival after kidney transplantation. The influence of HLA-A and -B mismatch, number of amino acid mismatches (after interlocus subtraction) and their physiochemical (electrostatic and hydrophobic) disparity on the outcome of fully HLA matched and single HLA-A or -B mismatched deceased donor kidney transplants undertaken in the United Kingdom (1990-2005) were analyzed (n = 5,247). Grafts with a single HLA-A or -B mismatch had significantly lower survival than fully matched transplants (81.9% vs 84.2% at 5 years, p = 0.004). However, single HLA-A or -B mismatched grafts with no or one amino acid mismatch had better survival than grafts with two or more amino acid mismatches (89.3% vs 81.8% at 5 years, HR 1.5, p = 0.03). The number of mismatched amino acids was an independent predictor of transplant survival after adjusting for the underlying HLA matching effect (p = 0.02). Physiochemical disparity scores correlated closely with amino acid mismatches and provided no additional predictive value. The immunogenicity of HLA class I alloantigens defined at the level of amino acid sequence correlates more closely with outcome after renal transplantation than conventional serologic HLA matching.

Assessment and Management of Anti-insulin Autoantibodies in Varying Presentations of Insulin Autoimmune Syndrome.

Abstract Context Insulin autoimmune syndrome (IAS), spontaneous hyperinsulinemic hypoglycemia due to insulin-binding autoantibodies, may be difficult to distinguish from tumoral or other forms of hyperinsulinemic hypoglycemia, including surreptitious insulin administration. No standardized treatment regimen exists. Objectives To evaluate an analytic approach to IAS and responses to different treatments. Design and Setting Observational study in the UK Severe Insulin Resistance Service. Patients Six patients with hyperinsulinemic hypoglycemia and detectable circulating anti–insulin antibody (IA). Main Outcome Measures Glycemia, plasma insulin, and C-peptide concentrations by immunoassay or mass spectrometry (MS). Immunoreactive insulin was determined in the context of polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC). IA quantification using ELISA and RIA, and IA were further characterized using radioligand binding studies. Results All patients were diagnosed with IAS (five IgG, one IgA) based on a high insulin/C-peptide ratio, low insulin recovery after PEG precipitation, and GFC evidence of antibody-bound insulin. Neither ELISA nor RIA result proved diagnostic for every case. MS provided a more robust quantification of insulin in the context of IA. One patient was managed conservatively, four were treated with diazoxide without sustained benefit, and four were treated with immunosuppression with highly variable responses. IA affinity did not appear to influence presentation or prognosis. Conclusions IAS should be considered in patients with hyperinsulinemic hypoglycemia and a high insulin/C-peptide ratio. Low insulin recovery on PEG precipitation supports the presence of insulin-binding antibodies, with GFC providing definitive confirmation. Immunomodulatory therapy should be customized according to individual needs and clinical response.

A novel IGSF1 mutation in a large Irish kindred highlights the need for familial screening in the IGSF1 deficiency syndrome

Loss‐of‐function mutations in IGSF1 result in X‐linked central congenital hypothyroidism (CeCH), occurring in isolation or associated with additional pituitary hormone deficits. Intrafamilial penetrance is highly variable and a minority of heterozygous females are also affected. We identified and characterized a novel IGSF1 mutation and investigated its associated phenotypes in a large Irish kindred.

High-dose biotin in infants mimics biochemical hyperthyroidism with some commercial assays

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