Inherited thrombocytopenia associated with a variant in the FLI1 binding site in the 5' UTR of ANKRD26.

Variants in the 5' UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26-RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5' UTR. To date, all reported ANKRD26-RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5' UTR of ANKRD26, c.-107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.-140C>G ANKRD26 5' UTR variant is benign and not associated with thrombocytopenia. Identification of the c.-107C>T variant extends the range of the regulatory region in the 5' UTR of ANKRD26 that is associated with ANKRD26-RT.

Discrepancies between two D-dimer assays and impact on clinical decisions; a retrospective analysis of samples tested in community and hospital-based laboratories in Auckland.

AIM: In patients with suspected venous thromboembolism, an elevated D-dimer level provides an important branch-point in the management pathway. This study compared two D-dimer assays, INNOVANCE® DDimer (Innovance) and STA®-Liatest® D-Di Plus (Liatest), to assess potential impact on clinical management. METHOD: Reflecting current practice in Waitemata, Auckland, we compared paired samples from 805 patients referred to hospital following a community D-dimer test. Samples were determined to be positive or negative using a 500µg/L fibrinogen equivalent units (FEU), and age-adjusted cut-offs. RESULTS: In the Innovance assay, 2% of samples had a result <500µg/L FEU. In contrast, by Liatest, 18% were below 500µg/L. This positive bias of Innovance was amplified with use of age-adjusted cut-offs; 23% of samples with an elevated Innovance result showed a normal result by Liatest. On average, the Innovance values were 22% higher than Liatest. Results suggestive of interference from heterophile antibodies were seen in 6% of sample-pairs. CONCLUSION: Innovance D-dimer test yielded higher values than Liatest and experienced interference from suspected heterophile antibodies. Discrepancies in nearly a quarter of patients may be leading to substantial under or over investigation, inefficient use of resources and clinical confusion.

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor-binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

Tuberous sclerosis complex: a complex case.

Tuberous sclerosis complex (TSC) is an inheritable disorder characterized by the formation of benign yet disorganized tumors in multiple organ systems. Germline mutations in the TSC1 (hamartin) or more frequently TSC2 (tuberin) genes are causative for TSC. The malignant manifestations of TSC, pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipoma (AML), may also occur as independent sporadic perivascular epithelial cell tumor (PEComa) characterized by somatic TSC2 mutations. Thus, discerning TSC from the copresentation of sporadic LAM and sporadic AML may be obscured in TSC patients lacking additional features. In this report, we present a case study on a single patient initially reported to have sporadic LAM and a mucinous duodenal adenocarcinoma deficient in DNA mismatch repair proteins. Moreover, the patient had a history of Wilms' tumor, which was reclassified as AML following the LAM diagnosis. Therefore, we investigated the origins and relatedness of these tumors. Using germline whole-genome sequencing, we identified a premature truncation in one of the patient's TSC2 alleles. Using immunohistochemistry, loss of tuberin expression was observed in AML and LAM tissue. However, no evidence of a somatic loss of heterozygosity or DNA methylation epimutations was observed at the TSC2 locus, suggesting alternate mechanisms may contribute to loss of the tumor suppressor protein. In the mucinous duodenal adenocarcinoma, no causative mutations were found in the DNA mismatch repair genes MLH1, MSH2, MSH6, or PMS2 Rather, clonal deconvolution analyses were used to identify mutations contributing to pathogenesis. This report highlights both the utility of using multiple sequencing techniques and the complexity of interpreting the data in a clinical context.

A novel frameshift GP1BB mutation causes autosomal dominant macrothrombocytopenia with decreased vWF receptor expression but normal platelet aggregation.

GP1bß is a component of the von Willebrand factor (vWF) receptor complex that is necessary for platelet formation and activation. A novel frameshift variant in GP1BB has been identified in a family with macrothrombocytopenia. The variant leads to a protein that is 101 amino acids longer than wild type with loss of the transmembrane domain. As there is no defect in platelet aggregation, the family are classified as heterozygous carriers of a Bernard-Soulier syndrome-related mutation. The levels of the vWF receptor on platelets are reduced to 50% of the controls, with the presence of large platelets but normal platelet aggregation demonstrating that decreased vWF receptor expression impacts proplatelet formation but not platelet function.

The Mouse Papillomavirus Epigenetic Signature Is Characterised by DNA Hypermethylation after Lesion Regression.

Papillomaviruses (PVs) are double-stranded DNA tumour viruses that can infect cutaneous and mucosal epidermis. Human papillomavirus (HPV) types have been linked to the causality of cutaneous squamous cell carcinoma (cSCC); however, HPV DNA is not always detected in the resultant tumour. DNA methylation is an epigenetic change that can contribute to carcinogenesis. We hypothesise that the DNA methylation pattern in cells is altered following PV infection. We tested if DNA methylation was altered by PV infection in the mouse papillomavirus (MmuPV1) model. Immunosuppressed mice were infected with MmuPV1 on cutaneous tail skin. Immunosuppression was withdrawn for some mice, causing lesions to spontaneously regress. Reduced representation bisulphite sequencing was carried out on DNA from the actively infected lesions, visibly regressed lesions, and mock-infected control mice. DNA methylation libraries were generated and analysed for differentially methylated regions throughout the genome. The presence of MmuPV1 sequences was also assessed. We identified 834 predominantly differentially hypermethylated fragments in regressed lesions, and no methylation differences in actively infected lesions. The promoter regions of genes associated with tumorigenicity, including the tumour suppressor protein DAPK1 and mismatch repair proteins MSH6 and PAPD7, were hypermethylated. Viral DNA was detected in active lesions and in some lesions that had regressed. This is the first description of the genome-wide DNA methylation landscape for active and regressed MmuPV1 lesions. We propose that the DNA hypermethylation in the regressed lesions that we report here may increase the susceptibility of cells to ultraviolet-induced cSCC.

Trends in survival from myeloma, 1990-2015: a competing risks analysis.

BACKGROUND: Myeloma survival has greatly increased over past decades. We investigated trends in survival over time in New Zealand by age, ethnicity, and geography and thus examined potential inequalities among these population subgroups. METHODS: From data supplied by the New Zealand Ministry of Health, all new diagnoses of multiple myeloma (ICD-10 code C90) between 1990 and 2016 were extracted, as well as their matched mortality data. Cox's proportional hazards regression and competing risks regression were used to estimate multivariable survival functions. RESULTS: Between 1 January 1990 and 1 December 2015, 6642 myeloma cases were registered by the New Zealand Cancer Registry. Although survival from myeloma increased substantially from 1990-1994 to 2010-2015, 5-year survival was still only about 60% in 2010-2015. The greatest improvement in survival was for people aged 60-69 years at diagnosis. Using Cox's proportional hazards regression, Maori showed an increased risk of myeloma death but this was predominantly due to differences in competing risks among ethnic groups. Competing risks analysis found the greatest improvement in myeloma survival in Pacific Islanders, and in 2010-2015 Maori had better survival than other ethnicities. Myeloma survival improved significantly over time in all regional health authorities but in all time periods the Central and Southern regions had significantly poorer survival than the Midland region. CONCLUSIONS: Improvements in myeloma survival have been unequal across subgroups and regions in New Zealand. Detailed information about utilization of chemotherapeutic agents and transplantation in New Zealand is not available. This information, as well as more detailed hematological data, is essential to further explore the relationships and reasons for differing myeloma survival in population subgroups of New Zealand.

Identification and validation of DNA methylation changes in pre-eclampsia.

INTRODUCTION: Pre-eclampsia (PE) is a dangerous placental condition that can lead to premature labour, seizures and death of mother and infant. Several studies have identified altered placental DNA methylation in PE; however, there is widespread inconsistency between studies and most findings have not been replicated. This study aimed to identify and validate consistent differences in methylation across multiple PE cohorts. METHODS: Seven publicly available 450K methylation array datasets were analysed to identify consistent differentially methylated positions (DMPs) in PE. DMPs were identified based on methylation difference (≥10%) and significance (p-value ≤ 1 × 10-7). Targeted deep bisulfite sequencing was then performed to validate a subset of DMPs in an additional independent PE cohort. RESULTS: Stringent analysis of the seven 450K datasets identified 25 DMPs (associated with 11 genes) in only one dataset. Using more relaxed criteria confirmed 19 of the stringent 25 DMPs in at least four of the remaining six datasets. Targeted deep bisulfite sequencing of eight DMPs (associated with three genes; CMIP, ST3GAL1 and DAPK3) in an independent PE cohort validated two DMPs in the CMIP gene. Seven additional CpG sites in CMIP were found to be significantly differentially methylated in PE. DISCUSSION: The identification and validation of significant differential methylation in CMIP suggests that the altered DNA methylation of this gene may be associated with the pathogenesis of PE, and may have the potential to serve as diagnostic biomarkers for this dangerous condition of pregnancy.

Regional distribution of myeloma in New Zealand.

AIMS: To investigate regional variation in myeloma incidence in New Zealand in order to inform aetiological investigations. METHODS: All new registrations of myeloma (1991-2016) were extracted from the New Zealand Cancer Registry. Ethnic classifications used prioritised ethnicity. For geographical groupings, 74 Territorial Local Authority (TLA) categories for 2006 and population densities were used. Negative binomial regression was used to estimate incidence rate ratios, 95% confidence intervals and p-values. RESULTS: Between 1 January 1991 and 31 December 2016, 7,083 myelomas were registered. The Clutha TLA had a significantly lower incidence than the New Zealand average. Compared to Clutha, many regions had a significantly higher incidence, but there was no clear spatial pattern. The highest incidence rate was for Maori men in the North Island. Women had significantly lower incidence than men of the same ethnic group and in the same area. CONCLUSIONS: As both extremes of myeloma incidence occurred in rural areas, and as all TLAs (except one, Horowhenua) in the two lowest risk categories were rural, it seems unlikely that farming confers an increased risk. Results suggest that some other factor is driving the differences in myeloma incidence by ethnic group. We have provided a baseline of the geographical burden of myeloma in New Zealand.

Hairpin-bisulfite sequencing of cells exposed to decitabine documents the process of DNA demethylation.

Although the mechanism of DNA demethylating drugs has been understood for many years, the direct effect of these drugs on methylation of the complementary strands of DNA has not been formally demonstrated. By using hairpin-bisulphite sequencing, we describe the kinetics and pattern of DNA methylation following treatment of cells by the DNA methyltransferase 1 (DNMT1) inhibitor, decitabine. As expected, we demonstrate complete loss of methylation on the daughter strand following S-phase in selected densely methylated genes in synchronized Jurkat cells. Thereafter, cells showed a heterogeneous pattern of methylation reflecting replication of the unmethylated strand and restoration of methylation.

Wilms tumor in patients with osteopathia striata with cranial sclerosis.

Germline pathogenic variants in AMER1 cause osteopathia striata with cranial sclerosis (OSCS: OMIM 300373), an X-linked sclerosing bone disorder. Female heterozygotes exhibit metaphyseal striations in long bones, macrocephaly, cleft palate, and, occasionally, learning disability. Male hemizygotes typically manifest the condition as fetal or neonatal death. Somatically acquired variants in AMER1 are found in neoplastic tissue in 15-30% of patients with Wilms tumor; however, to date, only one individual with OSCS has been reported with a Wilms tumor. Here we present four cases of Wilms tumor in unrelated individuals with OSCS, including the single previously published case. We also report the first case of bilateral Wilms tumor in a patient with OSCS. Tumor tissue analysis showed no clear pattern of histological subtypes. In Beckwith-Wiedemann syndrome, which has a known predisposition to Wilms tumor development, clinical protocols have been developed for tumor surveillance. In the absence of further evidence, we propose a similar protocol for patients with OSCS to be instituted as an initial precautionary approach to tumor surveillance. Further evidence is needed to refine this protocol and to evaluate the possibility of development of other neoplasms later in life, in patients with OSCS.

Silencing of Testin expression is a frequent event in spontaneous lymphomas from Trp53-mutant mice.

The tumour suppressor gene, TES, is frequently methylated in many human tumours. Previously, we demonstrated that TES promoter methylation and transcriptional silencing was the most common molecular abnormality detected in childhood acute lymphoblastic leukaemia (ALL). Trp53-mutant mouse models predominantly develop B- and T-cell lymphomas, which are widely considered equivalent to childhood T and B ALL. In this study, we examined expression of Tes transcript and Testin protein in spontaneous tumours obtained from three Trp53-mutant mouse models. Using immunohistochemistry, we report that 47% of lymphomas lacked Testin protein compared to only 7% of non-lymphoid tumours. Further examination of the lymphomas from Trp53-null and Trp53-mΔpro homozygous mutant mice revealed that 63% and 69% respectively of the isolated lymphomas were Testin negative, which is similar to reported rates in childhood T-ALL. Surprisingly, lymphomas from Trp53-Δ122 mice were frequently Testin positive (> 60%), suggesting that the presence of the Trp53-Δ122 protein appeared to mitigate the requirement for Tes silencing in lymphomagenesis. Quantitative RT-PCR results confirmed that this lack of Testin protein was due to Tes transcriptional silencing, although bisulfite sequencing demonstrated that this was not due to promoter methylation. These results are consistent with the Testin protein having lymphoid tumour suppressor activity in both mice and humans.

A practical guide to laboratory investigations at diagnosis and follow up in Waldenström macroglobulinaemia: recommendations from the Medical and Scientific Advisory Group, Myeloma Australia, the Pathology Sub-committee of the Lymphoma and Related Diseases Registry and the Australasian Association of Clinical Biochemists Monoclonal Gammopathy Working Group.

Waldenström macroglobulinaemia (WM) is an indolent non-Hodgkin lymphoma which usually presents with symptoms related to infiltration of bone marrow or other tissues like lymph nodes, liver or spleen and has certain unusual clinical manifestations, e.g., renal and central nervous system (CNS) involvement. It also has an array of laboratory features including hypersecretion of IgM, cryoglobulinaemia, increased plasma viscosity and identification of mutated MYD88L265P in more than 90% of cases. In this review, we aim to provide a guide to the laboratory investigations recommended for WM at initial diagnosis and at follow-up. A discussion on the nuances of diagnosis and differential diagnoses is followed by bone marrow (BM) assessment, measurement of paraprotein and other ancillary investigations. Recommendations are provided on laboratory work-up at diagnosis, in the asymptomatic follow-up phase, and during and post-treatment. Finally, we briefly discuss the implications of laboratory diagnosis in regard to recruitment and monitoring on clinical trials.

Trends in myeloma incidence, mortality and survival in New Zealand (1985-2016).

BACKGROUND: Myeloma, one of the most common haematological malignancies worldwide arises in the bone marrow. Incidence rates vary by age and ethnicity but reasons behind these trends are unknown. Treatment of myeloma has changed significantly over recent decades, resulting in longer survival and decreased mortality. METHODS: From data supplied by the Ministry of Health, all new registrations of and deaths from myeloma between 1985 and 2016 were extracted. Trends in age-specific rates were assessed using the method of Armitage. Age-standardised rates were calculated, and trends in age-adjusted rates analysed using the Mantel-Haenszel extension chi-square test. Age-adjusted incidence and mortality rate ratios were calculated. Myeloma-specific survival was visualised using Kaplan-Meier curves and multivariable hazard ratios calculated using Cox regression. RESULTS: Between 1985 and 2016, 7826 New Zealanders were registered with myeloma. Over this time the age-specific incidence of myeloma increased significantly for men, who had higher rates than women. Myeloma mortality was highest in Maori men. Men had higher mortality rates than women in all time periods. Since 1995-1999, mortality has decreased in women whereas in men it has declined since about 2000-2004. Survival has increased significantly since 1990 but Maori still have a higher risk of death than non-Maori. CONCLUSION: The patterns of variation in myeloma incidence, mortality and survival, as well as their trends over time may be used to assist research into the causes and management of myeloma in New Zealand.