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Haemophilia B (HB) is a rare inherited bleeding disorder caused by the deficiency of coagulation factor IX (FIX). The major clinical issues are bleedings, often targeting the joints, and the development of neutralising antibodies, i.e. inhibitors, to the FIX replacement therapy. Historically HB has been seen as identical to the more common haemophilia A (HA), i.e. deficiency of coagulation factor VIII (FVIII), but important differences between the two diseases exist. As a result of the rarity of HB, much of our knowledge of HB has been extrapolated from what is known about HA. To improve the care for persons with HB (PwHB), studies focusing on HB are of importance. The aim of this thesis was to characterise HB regarding its diagnostic challenges, treatment, clinical outcomes, and the quality of life of PwHB, and to compare some of these aspects to those of HA.Paper I describes the comparison of the one-stage and the chromogenic assays in measuring the FIX activity level. In HA, a discrepancy between the two methods in measuring FVIII has been reported in approximately one-third of persons with non-severe HA; however, this has not previously been evaluated in HB. We found that 25% of persons with non-severe HB had discrepant results between the two methods, with higher values recorded when the chromogenic method was used. All but one of these persons had the same FIX gene (F9) mutated amino acid. This was the first study to show that assay discrepancy occurs in HB and we concluded that both the one-stage and the chromogenic assays are needed for the correct diagnosis and classification of HB.Papers II-IV describe a cohort of 79 persons with severe HB from the Nordic countries, and 79 matched controls with HA. In Paper II, joint assessment using ultrasound and haemophilia joint health score (HJHS) was conducted and showed that despite the fact that 95% of PwHB were treated with prophylaxis, 37% reported joint bleedings during the prior year. Ultrasound scores were ove

Introduction: The development of inhibitory antibodies (inhibitors) in persons with hemophilia B (PwHB) causes significant morbidity. Data on the impact of the F9 variant and immune tolerance induction (ITI) outcome are limited. The aim of this study was to investigate the presence of neutralizing and non-neutralizing antibodies (NNA) in severe hemophilia B (HB) and to evaluate ITI outcome and complications in relation to the pathogenic F9 variant. Materials and methods: Persons with severe HB in the Nordic countries were enrolled and information on F9 variants, inhibitors, ITI and complications were collected. Analyses of anti-FIX antibodies with a fluorescence-immunoassay (xFLI) and an ELISA method were conducted. Results: Seventy-nine PwHB were enrolled. Null variants were seen in 33 (42 %) PwHB and 12 (15 %) had a current or former inhibitor. Eleven (92 %) of the inhibitor patients had experienced allergic manifestations and three (25 %) nephrotic syndrome. Of 10 PwHB with at least one ITI attempt, eight (80 %) were considered tolerant at enrolment. Immunosuppression was included in seven of eight successful or partially successful attempts. Five PwHB had at least one ITI failure before a successful or partially successful ITI. No NNA could be identified. Conclusion: A high proportion of severe F9 gene defects among persons with severe HB in the Nordic countries may explain the observed relatively high prevalence of inhibitors. ITI success was independent of the F9 variant and attained despite allergic manifestations and previous ITI failures. Inclusion of immunosuppression tentatively enhances the chances of ITI success. No NNA were observed.

Introduction: The development of inhibitory antibodies (inhibitors) in persons with hemophilia B (PwHB) causes significant morbidity. Data on the impact of the F9 variant and immune tolerance induction (ITI) outcome are limited. The aim of this study was to investigate the presence of neutralizing and non-neutralizing antibodies (NNA) in severe hemophilia B (HB) and to evaluate ITI outcome and complications in relation to the pathogenic F9 variant. Materials and methods: Persons with severe HB in the Nordic countries were enrolled and information on F9 variants, inhibitors, ITI and complications were collected. Analyses of anti-FIX antibodies with a fluorescence-immunoassay (xFLI) and an ELISA method were conducted. Results: Seventy-nine PwHB were enrolled. Null variants were seen in 33 (42 %) PwHB and 12 (15 %) had a current or former inhibitor. Eleven (92 %) of the inhibitor patients had experienced allergic manifestations and three (25 %) nephrotic syndrome. Of 10 PwHB with at least one ITI attempt, eight (80 %) were considered tolerant at enrolment. Immunosuppression was included in seven of eight successful or partially successful attempts. Five PwHB had at least one ITI failure before a successful or partially successful ITI. No NNA could be identified. Conclusion: A high proportion of severe F9 gene defects among persons with severe HB in the Nordic countries may explain the observed relatively high prevalence of inhibitors. ITI success was independent of the F9 variant and attained despite allergic manifestations and previous ITI failures. Inclusion of immunosuppression tentatively enhances the chances of ITI success. No NNA were observed.

Classic infantile Pompe disease (IPD) is a rare lysosomal storage disorder characterized by severe hypertrophic cardiomyopathy and profound muscle weakness. Without treatment, death occurs within the first 2 years of life. Although enzyme replacement therapy (ERT) with alglucosidase alfa has improved survival, treatment outcome is not good in many cases and is largely dependent on age at initiation. The objective of the study was (a) to analyse the different stages in the diagnosis and specific treatment initiation procedure in IPD patients, and (b) to compare clinical and biochemical outcomes depending on age at ERT initiation (<1 month of age vs. <3 months of age). Here, we show satisfactory clinical and biochemical outcomes in two IPD patients after early treatment initiation before 3 months of life with immunomodulatory therapy in the ERT-naïve setting, with a high ERT dose from the beginning. Despite the overall good evolution, the patient who initiated treatment <1 month of life presented even better outcomes than the patient who started treatment <3 months of life, with an earlier normalization of hypertrophic cardiomyopathy, along with CK normalization, highlighting the importance of early treatment initiation in this progressive disease before irreversible muscle damage has occurred.

Classic infantile Pompe disease (IPD) is a rare lysosomal storage disorder characterized by severe hypertrophic cardiomyopathy and profound muscle weakness. Without treatment, death occurs within the first 2 years of life. Although enzyme replacement therapy (ERT) with alglucosidase alfa has improved survival, treatment outcome is not good in many cases and is largely dependent on age at initiation. The objective of the study was (a) to analyse the different stages in the diagnosis and specific treatment initiation procedure in IPD patients, and (b) to compare clinical and biochemical outcomes depending on age at ERT initiation (<1 month of age vs. <3 months of age). Here, we show satisfactory clinical and biochemical outcomes in two IPD patients after early treatment initiation before 3 months of life with immunomodulatory therapy in the ERT-naïve setting, with a high ERT dose from the beginning. Despite the overall good evolution, the patient who initiated treatment <1 month of life presented even better outcomes than the patient who started treatment <3 months of life, with an earlier normalization of hypertrophic cardiomyopathy, along with CK normalization, highlighting the importance of early treatment initiation in this progressive disease before irreversible muscle damage has occurred.

Human serum albumin (HSA) is the most abundant protein in plasma and has an exceptionally long circulatory half-life of around three weeks in humans. The enhanced half-life properties of HSA result from the selective interaction with the neonatal Fc receptor (FcRn) in acidic endosomes, which protects endocytosed albumin from lysosomal degradation and mediates recycling back to the plasma membrane. Endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about FcRn-albumin cell biology in physiologically relevant primary cells and the spatiotemporal aspects of the FcRn-albumin interaction within intracellular endosomes. My studies have used cell biological and biophysical approaches to examine FcRn-albumin interactions and trafficking in primary macrophages and endothelial cells. Here, I used two independent biophysical approaches to visualise the intracellular receptor-ligand interactions within globular endosomes and tubular transport carriers of primary macrophages. Firstly, fluorescence lifetime imaging microscopy (FLIM) of Foerster resonance energy transfer (FRET) and secondly, raster image correlation spectroscopy (RICS) to monitor the diffusion kinetics of single fluorescent-labelled HSA molecules. Based on these analyses, I identified an interaction between FcRn and albumin within intracellular endosomes, and emerging tubules, in human FcRn-expressing macrophages. Furthermore, I detected a higher population of immobile, FcRn-bound wildtype HSA molecules within the lumen of endosomal structures compared to the non-FcRn binding rHSAH464Q mutant. My findings revealed the kinetics of FcRn-albumin binding within endosomal structures for recruitment into transport carriers for recycling. To investigate FcRn-albumin cell biology in physiologically relevant primary endothelial cells, I established cell lines of primary human vascular endothelial cells from the outgrowth in cultu

Classic infantile Pompe disease (IPD) is a rare lysosomal storage disorder characterized by severe hypertrophic cardiomyopathy and profound muscle weakness. Without treatment, death occurs within the first 2 years of life. Although enzyme replacement therapy (ERT) with alglucosidase alfa has improved survival, treatment outcome is not good in many cases and is largely dependent on age at initiation. The objective of the study was (a) to analyse the different stages in the diagnosis and specific treatment initiation procedure in IPD patients, and (b) to compare clinical and biochemical outcomes depending on age at ERT initiation (<1 month of age vs. <3 months of age). Here, we show satisfactory clinical and biochemical outcomes in two IPD patients after early treatment initiation before 3 months of life with immunomodulatory therapy in the ERT-naïve setting, with a high ERT dose from the beginning. Despite the overall good evolution, the patient who initiated treatment <1 month of life presented even better outcomes than the patient who started treatment <3 months of life, with an earlier normalization of hypertrophic cardiomyopathy, along with CK normalization, highlighting the importance of early treatment initiation in this progressive disease before irreversible muscle damage has occurred.

Introduction: Inhibitors develop less frequently in haemophilia B (HB) than haemophilia A (HA). However, when present, the success of tolerization by immune tolerance induction (ITI) therapy is lower and the risk of complications higher. Aim: To evaluate the use and outcome of ITI in patients with HB and inhibitors. Methods: Subjects include singletons or siblings with a current/history of inhibitors enrolled in B-Natural—an observational study designed to increase understanding of clinical management of patients with HB. Patients were followed for 6 months and information on demographics, medical and social history, and treatment were recorded. Results: Twenty-nine patients with severe HB and inhibitors were enrolled in 24 centres. Twenty-two underwent one or more courses of ITI with or without immune suppression. Eight patients (36.4%) were successfully tolerized after the first course of ITI. One of these successes (12.5%) experienced allergic manifestations, whereas the corresponding number for the 10 treatment failures was five (50%). One of seven (14.2%) patients with large deletions and three of eight (37.5%) with nonsense mutations were tolerized at the first attempt, and all patients experiencing nephrosis either failed or were on-going. At study end, 11 (50%) were considered successfully tolerized after one or more ITI courses, three were unsuccessful, and eight were still undergoing treatment. Conclusion: Our data underscore the possibilities and difficulties of achieving tolerization in patients with HB with inhibitors. The type of mutation and complications appear to correlate with ITI outcome, but more accurate definitions of successful ITI are warranted.

Introduction: Inhibitors develop less frequently in haemophilia B (HB) than haemophilia A (HA). However, when present, the success of tolerization by immune tolerance induction (ITI) therapy is lower and the risk of complications higher. Aim: To evaluate the use and outcome of ITI in patients with HB and inhibitors. Methods: Subjects include singletons or siblings with a current/history of inhibitors enrolled in B-Natural—an observational study designed to increase understanding of clinical management of patients with HB. Patients were followed for 6 months and information on demographics, medical and social history, and treatment were recorded. Results: Twenty-nine patients with severe HB and inhibitors were enrolled in 24 centres. Twenty-two underwent one or more courses of ITI with or without immune suppression. Eight patients (36.4%) were successfully tolerized after the first course of ITI. One of these successes (12.5%) experienced allergic manifestations, whereas the corresponding number for the 10 treatment failures was five (50%). One of seven (14.2%) patients with large deletions and three of eight (37.5%) with nonsense mutations were tolerized at the first attempt, and all patients experiencing nephrosis either failed or were on-going. At study end, 11 (50%) were considered successfully tolerized after one or more ITI courses, three were unsuccessful, and eight were still undergoing treatment. Conclusion: Our data underscore the possibilities and difficulties of achieving tolerization in patients with HB with inhibitors. The type of mutation and complications appear to correlate with ITI outcome, but more accurate definitions of successful ITI are warranted.