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Objective: The aim of this study was to refine the information regarding the quantitative and spatial dynamics of infiltrating lymphocytes and remaining beta-cell volume during the progression of type 1 diabetes in the NOD mouse model of the disease.

Research design and methods: Using an ex vivo technique, optical projection tomography (OPT), we quantified and assessed the 3D spatial development and progression of insulitis and beta-cell destruction in pancreas from diabetes prone NOD and non-diabetes prone congenic NOD.H-2b mice between 3 and 16 weeks of age.

Results: Together with results showing the spatial dynamics of the insulitis process we provide data of beta-cell volume distributions down to the level of the individual islets and throughout the pancreas during the development and progression of type 1 diabetes. Our data provide evidence for a compensatory growth potential of the larger insulin(+) islets during the later stages of the disease around the time point for development of clinical diabetes. This is in contrast to smaller islets, which appear less resistant to the autoimmune attack. We also provide new information on the spatial dynamics of the insulitis process itself, including its apparently random distribution at onset, the local variations during its further development, and the formation of structures resembling tertiary lymphoid organs at later phases of insulitis progression.

Conclusions: Our data provides a powerful tool for phenotypic analysis of genetic and environmental effects on type 1 diabetes etiology as well as for evaluating the potential effect of therapeutic regimes.

A predicament when assessing the mechanisms underlying the pathogenesis of type-1 diabetes (T1D) has been to maintain simultaneous global and regional information on the loss of insulin-cell mass and the progression of insulitis. We present a procedure for high-resolution 3-D analyses of regions of interest (ROIs), defined on the basis of global assessments of the 3-D distribution, size, and shape of molecularly labeled structures within the full volume of the intact mouse pancreas. We apply a refined protocol for optical projection tomography (OPT)-aided whole pancreas imaging in combination with confocal laser scanning microscopy of site-directed pancreatic microbiopsies. As such, the methodology provides a useful tool for detailed cellular and molecular assessments of the autoimmune insulitis in T1D. It is anticipated that the same approach could be applied to other areas of research where 3-D molecular distributions of both global and regional character is required.

Wilms tumor gene 1 (WT1) expression has been suggested as an applicable minimal residual disease marker in acute myeloid leukemia (AML). We evaluated the use of this marker in 43 adult AML patients. Quantitative assessment of WT1 gene transcripts was performed using real-time quantitative-polymerase chain reaction assay. Samples from both the peripheral blood and the bone marrow were analyzed at diagnosis and during follow-up. A strong correlation was observed between WT1 normalized with 2 different control genes (beta-actin and ABL1, P < 0.001). WT1 mRNA level at diagnosis was of no prognostic relevance (P > 0.05). A >= 1-log reduction in WT1 expression in bone marrow samples taken < 1 month after diagnosis significantly correlated with an improved overall survival (P = 0.004) and freedom from relapse (P = 0.010) when beta-actin was used as control gene. Furthermore, a reduction in WT1 expression by >= 2 logs in peripheral blood samples taken at a later time point significantly correlated with a better outcome for overall survival (P = 0.004) and freedom from relapse (P = 0.012). This result was achieved when normalizing against both b-actin and ABL1. These results therefore suggest that WT1 gene expression can provide useful information for minimal residual disease detection in adult AML patients and that combined use of control genes can give more informative results.

Background: Tumour necrosis factor receptor associated factor 6 (TRAF6) promotes inflammation in response to various cytokines. Aberrant Wnt3a signals promotes cancer progression through accumulation of β-Catenin. Here we investigated a potential role for TRAF6 in Wnt signaling.

Methods: TRAF6 expression was silenced by siRNA in human prostate cancer (PC3U) and human colorectal SW480 cells and by CRISPR/Cas9 in zebrafish. Several biochemical methods and analyses of mutant phenotype in zebrafish were used to analyse the function of TRAF6 in Wnt signaling.

Findings: Wnt3a-treatment promoted binding of TRAF6 to the Wnt co-receptors LRP5/LRP6 in PC3U and LNCaP cells in vitro. TRAF6 positively regulated mRNA expression of β-Catenin and subsequent activation of Wnt target genes in PC3U cells. Wnt3a-induced invasion of PC3U and SW480 cells were significantly reduced when TRAF6 was silenced by siRNA. Database analysis revealed a correlation between TRAF6 mRNA and Wnt target genes in patients with prostate cancer, and high expression of LRP5, TRAF6 and c-Myc correlated with poor prognosis. By using CRISPR/Cas9 to silence TRAF6 in zebrafish, we confirm TRAF6 as a key molecule in Wnt3a signaling for expression of Wnt target genes.

Interpretation: We identify TRAF6 as an important component in Wnt3a signaling to promote activation of Wnt target genes, a finding important for understanding mechanisms driving prostate cancer progression.

Background: Whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS), with the ability to provide comprehensive genomic information, have become the focal point of research interest as novel techniques that can support precision diagnostics in routine clinical care of patients with various cancer types, including hematological malignancies. This national multi-center study, led by Genomic Medicine Sweden, aims to evaluate whether combined application of WGS and WTS (WGTS) is technically feasible and can be implemented as an efficient diagnostic tool in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In addition to clinical impact assessment, a health-economic evaluation of such strategy will be performed.

Methods and Analysis: The study comprises four phases (i.e., retrospective, prospective, real-time validation, and follow-up) including approximately 700 adult and pediatric Swedish AML and ALL patients. Results of WGS for tumor (90×) and normal/germline (30×) samples as well as WTS for tumors only will be compared to current standard of care diagnostics. Primary study endpoints are diagnostic efficiency and improved diagnostic yield. Secondary endpoints are technical and clinical feasibility for routine implementation, clinical utility, and health-economic impact.

Discussion: Data from this national multi-center study will be used to evaluate clinical performance of the integrated WGTS diagnostic workflow compared with standard of care. The study will also elucidate clinical and health-economic impacts of a combined WGTS strategy when implemented in routine clinical care.

Clinical Trial Registration: [https://doi.org/10.1186/ISRCTN66987142], identifier [ISRCTN66987142].

Background: Target enrichment and resequencing is a widely used approach for identification of cancer genes and genetic variants associated with diseases. Although cost effective compared to whole genome sequencing, analysis of many samples constitutes a significant cost, which could be reduced by pooling samples before capture. Another limitation to the number of cancer samples that can be analyzed is often the amount of available tumor DNA. We evaluated the performance of whole genome amplified DNA and the power to detect subclonal somatic single nucleotide variants in non-indexed pools of cancer samples using the HaloPlex technology for target enrichment and next generation sequencing. Results: We captured a set of 1528 putative somatic single nucleotide variants and germline SNPs, which were identified by whole genome sequencing, with the HaloPlex technology and sequenced to a depth of 792-1752. We found that the allele fractions of the analyzed variants are well preserved during whole genome amplification and that capture specificity or variant calling is not affected. We detected a large majority of the known single nucleotide variants present uniquely in one sample with allele fractions as low as 0.1 in non-indexed pools of up to ten samples. We also identified and experimentally validated six novel variants in the samples included in the pools. Conclusion: Our work demonstrates that whole genome amplified DNA can be used for target enrichment equally well as genomic DNA and that accurate variant detection is possible in non-indexed pools of cancer samples. These findings show that analysis of a large number of samples is feasible at low cost, even when only small amounts of DNA is available, and thereby significantly increases the chances of indentifying recurrent mutations in cancer samples.

Approximately half of all cases of Hoyeraal-Hreidarsson syndrome (HHS), a multisystem disorder characterized by bone marrow failure, developmental defects and very short telomeres, are caused by germline mutations in genes related to telomere biology. However, the varying symptoms and severity of the disease indicate that additional mechanisms are involved. Here, a 3-year-old boy with HHS was found to carry biallelic germline mutations in WRAP53 (WD40 encoding RNA antisense to p53), that altered two highly conserved amino acids (L283F and R398W) in the WD40 scaffold domain of the protein encoded. WRAP53 beta (also known as TCAB1 or WDR79) is involved in intracellular trafficking of telomerase, Cajal body functions and DNA repair. We found that both mutations cause destabilization, mislocalization and faulty interactions of WRAP53 beta, defects linked to misfolding by the TRiC chaperonin complex. Consequently, WRAP53 beta HHS mutants cannot elongate telomeres, maintain Cajal bodies or repair DNA double-strand breaks. These findings provide a molecular explanation for the pathogenesis underlying WRAP53 beta-associated HHS and highlight the potential contribution of DNA damage and/or defects in Cajal bodies to the early onset and/or severity of this disease.

Myeloid leukemia of Down syndrome has a better prognosis than sporadic pediatric acute myeloid leukemia. Most cases of myeloid leukemia of Down syndrome are characterized by additional cytogenetic changes besides the constitutional trisomy 21, but their potential prognostic impact is not known. We, therefore, conducted an international retrospective study of clinical characteristics, cytogenetics, treatment, and outcome of 451 children with myeloid leukemia of Down syndrome. All karyotypes were centrally reviewed before assigning patients to subgroups. The overall 7-year event-free survival for the entire cohort was 78% (± 2%), with the overall survival rate being 79% (± 2%), the cumulative incidence of relapse 12% (± 2%), and the cumulative incidence of toxic death 7% (± 1%). Outcome estimates showed large differences across the different cytogenetic subgroups. Based on the cumulative incidence of relapse, we could risk-stratify patients into two groups: cases with a normal karyotype (n=103) with a higher cumulative incidence of relapse (21%± 4%) than cases with an aberrant karyotype (n=255) with a cumulative incidence of relapse of 9% (± 2%) (P=0.004). Multivariate analyses revealed that white blood cell count ≥ 20 × 10(9)/L and age >3 years were independent predictors for poor event-free survival, while normal karyotype independently predicted inferior overall survival, event-free survival, and relapse-free survival. In conclusion, this study showed large differences in outcome within patients with myeloid leukemia of Down syndrome and identified novel prognostic groups that predicted clinical outcome and hence may be used for stratification in future treatment protocols.

To evaluate pallidal DBS in a non-DYT1 form of hereditary dystonia. We present the results of pallidal DBS in a family with non-DYT1 dystonia where DYT5 to 17 was excluded. The dystonia is following an autosomal dominant pattern. Ten members had definite dystonia and five had dystonia with minor symptoms. Four patients received bilateral pallidal DBS. Mean age was 47 years. The patients were evaluated before surgery, and "on" stimulation after a mean of 2.5 years (range 1-3) using the Burke-Fahn-Marsden scale (BFM). Mean BFM score decreased by 79 % on stimulation, from 42.5 +/- 24 to 9 +/- 6.5 at the last evaluation. Cervical involvement improved by 89%. The 2 patients with oromandibular dystonia and blepharospasm demonstrated a reduction of 95% regarding these symptoms. The present study confirms the effectiveness of pallidal DBS in a new family with hereditary primary segmental and generalized dystonia.

Background. Despite increased knowledge about genetic aberrations in pediatric T-cell acute lymphoblastic leukemia (T-ALL), no clinically feasible treatment-stratifying marker exists at diagnosis. Instead patients are enrolled in intensive induction therapies with substantial side effects. In modern protocols, therapy response is monitored by minimal residual disease (MRD) analysis and used for postinduction risk group stratification. DNA methylation profiling is a candidate for subtype discrimination at diagnosis and we investigated its role as a prognostic marker in pediatric T-ALL. Procedure. Sixty-five diagnostic T-ALL samples from Nordic pediatric patients treated according to the Nordic Society of Pediatric Hematology and Oncology ALL 2008 (NOPHO ALL 2008) protocol were analyzed by HumMeth450K genome wide DNA methylation arrays. Methylation status was analyzed in relation to clinical data and early T-cell precursor (ETP) phenotype. Results. Two distinct CpG island methylator phenotype (CIMP) groups were identified. Patients with a CIMP-negative profile had an inferior response to treatment compared to CIMP-positive patients (3-year cumulative incidence of relapse (CIR3y) rate: 29% vs. 6%, P = 0.01). Most importantly, CIMP classification at diagnosis allowed subgrouping of high-risk T-ALL patients (MRD >= 0.1% at day 29) into two groups with significant differences in outcome (CIR3y rates: CIMP negative 50% vs. CIMP positive 12%; P = 0.02). These groups did not differ regarding ETP phenotype, but the CIMP-negative group was younger (P = 0.02) and had higher white blood cell count at diagnosis (P = 0.004) compared with the CIMP-positive group. Conclusions. CIMP classification at diagnosis in combination with MRD during induction therapy is a strong candidate for further risk classification and could confer important information in treatment decision making.

Background: Few biological markers are associated with survival after relapse of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In pediatric T-cell ALL, we have identified promoter-associated methylation alterations that correlate with prognosis. Here, the prognostic relevance of CpG island methylation phenotype (CIMP) classification was investigated in pediatric BCP-ALL patients.

Methods: Six hundred and one BCP-ALL samples from Nordic pediatric patients (age 1-18) were CIMP classified at initial diagnosis and analyzed in relation to clinical data.

Results: Among the 137 patients that later relapsed, patients with a CIMP-profile (n = 42) at initial diagnosis had an inferior overall survival (pOS(5years) 33%) compared to CIMP+ patients (n = 95, pOS(5years) 65%) (p = 0.001), which remained significant in a Cox proportional hazards model including previously defined risk factors.

Conclusion: CIMP classification is a strong candidate for improved risk stratification of relapsed BCP-ALL.

Fuchs endothelial corneal dystrophy (FECD) is caused by a corneal endothelial cell loss, leading to corneal edema and visual impairment. The most significant genetic risk factor for FECD is an expansion of the CTG18.1 locus in transcription factor 4 (TCF4). The current treatment for severe FECD is corneal transplantation, with Descemet stripping automated keratoplasty (DSAEK) as a common surgical method. Although successful in most cases, the risk for transplant failure due to diverse causes must be considered. In this study, we investigated if presence of TCF4 CTG18.1 expansion with more than 31 (n ≥ 31) repeats in donated corneal grafts could be a reason for corneal transplant failure after DSAEK. For this, nine consecutively failed DSAEK corneal grafts were genotyped for CTG18.1 repeat length. One-sided Mann–Whitney U test was performed to evaluate if failed DSAEK corneal grafts had longer CTG18.1 repeats than healthy controls from the same population. All failed corneal grafts had CTG18.1 n ≤ 27 with a median of 18 (IQR 8.0) repeats for the longest allele. There was no statistical difference in CTG18.1 repeat lengths between failed corneal grafts and the geographically matched healthy control group. In conclusion, none of the nine failed corneal grafts in our material had CTG18.1 repeat lengths ≥ 31, a cut-off known to have a biological relevance in FECD. Thus, our results suggest that the assessment of donors and inspection of the corneal tissue before the decision for procurement is sufficient, in terms of recognizing FECD in the donor.

Little is known about target organ-infiltrating NK cells in type 1 diabetes and other autoimmune diseases. In this study, we identified NK cells with a unique phenotype in the pancreas of NOD mice. Pancreatic NK cells, localized to the endocrine and exocrine parts, were present before T cells during disease development and did not require T cells for their infiltration. Furthermore, NK cells, or NK cell precursors, from the spleen could traffic to the pancreas, where they displayed the pancreatic phenotype. Pancreatic NK cells from other mouse strains shared phenotypic characteristics with pancreatic NK cells from NOD mice, but displayed less surface killer cell lectin-like receptor G1, a marker for mature NK cells that have undergone proliferation, and also did not proliferate to the same extent. A subset of NOD mouse pancreatic NK cells produced IFN-gamma spontaneously, suggesting ongoing effector responses. However, most NOD mouse pancreatic NK cells were hyporesponsive compared with spleen NK cells, as reflected by diminished cytokine secretion and a lower capacity to degranulate. Interestingly, such hyporesponsiveness was not seen in pancreatic NK cells from the nonautoimmune strain C57BL/6, suggesting that this feature is not a general property of pancreatic NK cells. Based on our data, we propose that NK cells are sentinel cells in a normal pancreas. We further speculate that during inflammation, pancreatic NK cells initially mediate proinflammatory effector functions, potentially contributing to organ-specific autoimmunity, but later become hyporesponsive because of exhaustion or regulation.

Genetic polymorphism is by definition the occurrence in the same population of two or more alleles at one locus,each with a frequency high enough not to be maintained by recurrent mutation only (1). Among the human plasmaproteins two major categories of polymorphism have been described, allotypic and electrophoretic heterogeneity. Allotypy is defined by Oudin as individual antigenic differences among proteins within a species (2). The first discovered polymorphism of this category was the Gm system of immunoglobulin G by Grubb (3). The first described electrophoretic heterogeneity in plasma proteins was theHp (haptoglobin) system discovered by Smithies (4). Sincethen genetic variants of several other human plasma proteins have been found. This dissertation is concerned with thegenetic and immunological aspects of the polymorphism of the third component of human complement, C3.

Children with Down syndrome (DS) have an increased risk of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). The prognostic factors and outcome of DS-ALL patients treated in contemporary protocols are uncertain. We studied 653 DS-ALL patients enrolled in 16 international trials from 1995 to 2004. Non-DS BCP-ALL patients from the Dutch Child Oncology Group and Berlin-Frankfurt-Munster were reference cohorts. DS-ALL patients had a higher 8-year cumulative incidence of relapse (26% +/- 2% vs 15% +/- 1%, P < .001) and 2-year treatment-related mortality (TRM) (7% +/- 1% vs 2.0% +/- < 1%, P < .0001) than non-DS patients, resulting in lower 8-year event-free survival (EFS) (64% +/- 2% vs 81% +/- 2%, P < .0001) and overall survival (74% +/- 2% vs 89% +/- 1%, P < .0001). Independent favorable prognostic factors include age <6 years (hazard ratio [HR] = 0.58, P = .002), white blood cell (WBC) count <10 x 10(9)/L (HR = 0.60, P = .005), and ETV6-RUNX1 (HR = 0.14, P = .006) for EFS and age (HR = 0.48, P < .001), ETV6-RUNX1 (HR = 0.1, P = .016) and high hyperdiploidy (HeH) (HR = 0.29, P = .04) for relapse-free survival. TRM was the major cause of death in ETV6-RUNX1 and HeH DS-ALLs. Thus, while relapse is the main contributor to poorer survival in DS-ALL, infection-associated TRM was increased in all protocol elements, unrelated to treatment phase or regimen. Future strategies to improve outcome in DS-ALL should include improved supportive care throughout therapy and reduction of therapy in newly identified good-prognosis subgroups.

Objective: To describe the central retinal findings early in the course of Bothnia dystrophy caused by the homozygous missense R234W sequence variation in the RLBP1 gene. Methods: In 8 young patients with Bothnia dystrophy (aged 9-34 years), high- and low-contrast distance visual acuity and visual fields were measured with Humphrey central (24-2) threshold testing and Goldmann perimetry. Central retinal thickness was measured with optical coherence tomography. Cross-sectional images were analyzed and a linear scanning protocol was applied to examine retinitis punctata albescence in the posterior pole. Results: Affected visual acuity (4 of 8 cases) and poor low-contrast visual acuity (8 of 8 cases) were found. Significant foveal depression and visual field loss were evident with Humphrey threshold testing at all ages, and paracentral and central scotomata in the second decade of life advanced in adulthood as verified with Goldmann perimetry. Optical coherence tomography showed generalized retinal thinning in the central foveal, foveal (innermost ring diameter [empty set], 1 mm), and inner ring (empty set, 3 mm) areas in all ages, and early retinal thinning was found in the inferior areas of the outer macula (empty set, 6 mm). Foveal and extrafoveal thinning of the retinal layers and outer nuclear layer were found. Homogeneous retinitis punctata albescence changes were visualized in and/or adjacent to the retinal pigment epithelium choriocapillaris complex with high reflectance. Conclusions: In the RLBP1 Bothnia dystrophy phenotype, a loss of function and thinning of the central macula are found, indicating early damage of the cone photoreceptors in this disease of the visual cycle. Retinitis punctata albescence spots in the posterior pole are situated close to or in the retinal pigment epithelium-choriocapillaris complex.