Summary
Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next‐generation‐sequencing (NGS) has been used to ...investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical‐grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically‐reliable diagnostic test and minimize false‐negative results we developed an open‐source tool (CoverMi) to accurately determine base‐coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33‐gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS‐based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.
The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This ...report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5-methyltetrahydrofolate (5-MTHF) due to an increase in K(m) and decrease in V(max) in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K(m) for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K(m) mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.
Primary mitochondrial disorders (PMDs) are known for their pleiotropic manifestations in humans, affecting almost any organ or system at any time. Hematologic manifestations, such as cytopenias and ...sideroblastic anemia, occur in 10% to 30% of patients with confirmed PMDs. These can be the initial presenting features or complications that develop over time. Surveillance for these manifestations allows for prompt identification and treatment. This article provides an overview of the pathophysiology underpinning the hematologic effects of mitochondrial dysfunction, discussing the 3 key roles of the mitochondria in hematopoiesis: providing energy for cell differentiation and function, synthesizing heme, and generating iron-sulfur clusters. Subsequently, the diagnosis and management of mitochondrial disorders are discussed, focusing on hematologic manifestations and the specific conditions commonly associated with them. Through this, we aimed to provide a concise point of reference for those considering a mitochondrial cause for a patient’s hematologic abnormality, or for those considering a hematologic manifestation in a patient with known or suspected mitochondrial disease.
Telomere biology disorders (TBDs), including dyskeratosis congenita (DC), are a group of rare inherited diseases characterized by very short telomeres. Mutations in the components of the enzyme ...telomerase can lead to insufficient telomere maintenance in hematopoietic stem cells, resulting in the bone marrow failure that is characteristic of these disorders. While an increasing number of genes are being linked to TBDs, the causative mutation remains unidentified in 30‐40% of patients with DC. There is therefore a need for whole genome sequencing (WGS) in these families to identify novel genes, or mutations in regulatory regions of known disease‐causing genes. Here we describe a family in which a partial deletion of the 3′ untranslated region (3′ UTR) of DKC1, encoding the protein dyskerin, was identified by WGS, despite being missed by whole exome sequencing. The deletion segregated with disease across the family and resulted in reduced levels of DKC1 mRNA in the proband. We demonstrate that the DKC1 3′ UTR contains two polyadenylation signals, both of which were removed by this deletion, likely causing mRNA instability. Consistent with the major function of dyskerin in stabilization of the RNA subunit of telomerase, hTR, the level of hTR was also reduced in the proband, providing a molecular basis for his very short telomeres. This study demonstrates that the terminal region of the 3′ UTR of the DKC1 gene is essential for gene function and illustrates the importance of analyzing regulatory regions of the genome for molecular diagnosis of inherited disease.
Unclassified inherited bone marrow failure syndromes are a heterogeneous group of genetic disorders that represent either new syndromes or atypical clinical courses of known inherited bone marrow ...failure syndromes. The relative prevalence of the unclassified inherited bone marrow failure syndromes and their characteristics and the clinical and economic challenges that they create have never been studied.
We analyzed cases of inherited bone marrow failure syndrome in the Canadian Inherited Marrow Failure Registry that were deemed unclassifiable at study entry.
From October 2001 to March 2006, 39 of the 162 patients enrolled in the Canadian Inherited Marrow Failure Registry were registered as having unclassified inherited bone marrow failure syndromes. These patients presented at a significantly older age (median: 9 months) than the patients with classified inherited bone marrow failure syndrome (median: 1 month) and had substantial variation in the clinical presentations. The hematologic phenotype, however, was similar to the classified inherited bone marrow failure syndromes and included single- or multiple-lineage cytopenia, severe aplastic anemia, myelodysplasia, and malignancy. Grouping patients according to the affected blood cell lineage(s) and to the presence of associated physical malformations was not always sufficient to characterize a condition, because affected members from several families fit into different phenotypic groups. Compared with the classified inherited bone marrow failure syndromes, the patients with unclassified inherited bone marrow failure syndromes had 3.2 more specific diagnostic tests at 4.5 times higher cost per evaluated patient to attempt to categorize their syndrome. At last follow-up, only 20% of the unclassified inherited bone marrow failure syndromes were ultimately diagnosed with a specific syndrome on the basis of the development of new clinical findings or positive genetic tests.
Unclassified inherited bone marrow failure syndromes are relatively common among the inherited bone marrow failure syndromes and present a major diagnostic and therapeutic dilemma.
Telomerase is a ribonucleoprotein enzyme that is necessary for overcoming telomere shortening in human germ and stem cells. Mutations in telomerase or other telomere-maintenance proteins can lead to ...diseases characterized by depletion of hematopoietic stem cells and bone marrow failure (BMF). Telomerase localization to telomeres requires an interaction with a region on the surface of the telomere-binding protein TPP1 known as the TEL patch. Here, we identify a family with aplastic anemia and other related hematopoietic disorders in which a 1-amino-acid deletion in the TEL patch of TPP1 (ΔK170) segregates with disease. All family members carrying this mutation, but not those with wild-type TPP1, have short telomeres. When introduced into 293T cells, TPP1 with the ΔK170 mutation is able to localize to telomeres but fails to recruit telomerase to telomeres, supporting a causal relationship between this TPP1 mutation and bone marrow disorders. ACD/TPP1 is thus a newly identified telomere-related gene in which mutations cause aplastic anemia and related BMF disorders.
•Bone marrow failure can derive from germline mutation of the telomere protein TPP1 that is involved in recruiting telomerase to telomeres.•The observed mutation disrupts recruitment of telomerase to telomeres and segregates with short telomere length and disease phenotype.
Aim
Intravenous immunoglobulin (IVIG) is an expensive therapy used in immunodeficiency and autoimmune disorders. Increasing demands and consequent shortages result in a need for usage to conform to ...guidelines.
Method
We retrospectively evaluated IVIG use for neuroimmunological indications and adherence to existing guidelines in a major Australian paediatric hospital between 2000 and 2014.
Results
One‐hundred and ninety‐six children (96 male, 100 female; mean age at disease onset 6y 5mo range 3mo–15y 10mo, mean age at first IVIG dose 7y 2mo range 3mo–16y 5mo) received IVIG for neuroimmunological indications during the study period (28.1% had Guillain–Barré syndrome), representing 15.5% of all hospital indications. In total, 1669 IVIG courses were administered (total 57 221g, median 78g/patient, range 12–5748g). The highest median numbers of courses were in chronic inflammatory demyelinating polyneuropathies, opsoclonus‐myoclonus ataxia syndrome, suspected immune‐mediated epilepsies, and Rasmussen's encephalitis. Adverse reactions occurred in 25.5% of patients, but these were mostly minor. Outcome at follow‐up was best in anti‐N‐methyl‐d‐aspartate receptor (anti‐NMDAR) encephalitis, Guillain–Barré syndrome, and myasthenia gravis, and worst in Rasmussen's encephalitis and epilepsies. The total cost of IVIG was US$2 595 907 (median $3538/patient, range $544–260 766). Of patients receiving IVIG, 45.4% to 57.1% were given the therapy for ‘weak’ indications or indications ‘not listed’ in international guidelines. Some entities commonly treated with IVIG in current practice, such as anti‐NMDAR encephalitis and transverse myelitis, are not listed in most guidelines.
Interpretation
Our study demonstrates that IVIG is generally well tolerated but expensive, and discloses discrepancies between guidelines and clinical practice in paediatric neurology, suggesting both the need for greater adherence to current recommendations, and for recommendations to be updated to accommodate emerging indications.
What this paper adds
Intravenous immunoglobulin (IVIG) is an expensive but relatively well tolerated treatment commmonly used in paediatric neurology.
Some indications for IVIG administration seem to respond poorly to treatment.
Other conditions commonly treated with IVIG are not listed in most guidelines.
Greater adherence to current recommendations is required, and recommendations need to be updated.
This article is commented on by Absoud and Lim on pages 1105–1106 of this issue.
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Background
Sickle cell disease (SCD) is the most common monogenic disorder worldwide. In deoxygenated conditions, the altered beta chain (haemoglobin S HbS) polymerises and distorts the erythrocyte, ...resulting in pain crises, vasculopathy and end‐organ damage. Clinical complications of SCD cause substantial morbidity, and therapy demands expertise and resources. Optimising care for patients and planning resource allocation for the future requires an understanding of the disease in the Australian population. The Australian Haemoglobinopathy Registry (HbR) is a collaborative initiative of specialist centres collating and analysing data on patients with haemoglobin disorders.
Aims
To provide a snapshot of SCD in Australia over a 12‐month period based on data from the HbR.
Methods
Patients with a clinically significant sickling disorder across 12 clinical sites were included for analysis. Data include demographic and diagnostic details, as well as details of the clinical management of the condition over a 12‐month period.
Results
Data on 359 SCD patients demonstrate a shift in the demographic of patients in Australia, with a growing proportion of sub‐Saharan African ethnicities associated with the HbSS genotype. Acute and chronic complications are common, and patients require significant outpatient and inpatient support. Prevalence of disease complications and therapeutic trends are in keeping with other high‐income countries.
Conclusions
This study provides the first national picture of SCD in Australia, describing the characteristics and needs of SCD patients, elucidating demand for current and novel therapy and facilitating the planning of services for this vulnerable population.
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