The impact of initiating posaconazole on tacrolimus pharmacokinetics in allogeneic stem cell transplantation.

BACKGROUND: Posaconazole reduces the risk of invasive Aspergillus in transplant patients, but significantly inhibits tacrolimus metabolism. One study demonstrated that a three-fold dose reduction of tacrolimus was required to obtain therapeutic concentrations when used with posaconazole. However, with empiric dose reduction, there is a risk of subtherapeutic tacrolimus levels and subsequent graft failure or graft-versus-host disease. Overall, the existing data on the impact of posaconazole on tacrolimus pharmacokinetics is limited. OBJECTIVE: The purpose of this study is to determine whether tacrolimus doses should be decreased upon initiation of posaconazole in patients receiving an allogeneic stem cell transplant. METHODS: This is a retrospective chart review at an academic medical center. All allogeneic stem cell transplant adults who received concomitant posaconazole and tacrolimus from February 2016 through December 2017 were included. RESULTS: Seventy-nine patients identified using an internal electronic database were analyzed. The median time to therapeutic tacrolimus concentration was significantly longer in patients who did not receive an empiric dose reduction (0% DR, 10d; 1-30% DR, 4d; 31-65% DR, 5d; >65% DR, 4d; p = 0.0395). The rate of supratherapeutic levels was highest amongst patients who did not receive an empiric DR, and was noted to be significant compared to the group that had 31-65% DR (p < 0.001). CONCLUSION: This study validates our current practice of instituting an empiric 50% dose reduction of oral tacrolimus to 0.03 mg/kg/day when used concomitantly with posaconazole to achieve therapeutic levels in allogeneic stem cell transplant patients.

Moxifloxacin versus levofloxacin for antibacterial prophylaxis in acute leukemia patients.

OBJECTIVE: The primary endpoint of this study was to determine the incidence of febrile neutropenia among patients receiving either moxifloxacin or levofloxacin for antibacterial prophylaxis. Secondary endpoints were number of documented infections and in-hospital mortality in patients who develop febrile neutropenia. METHODS: A single-center retrospective cohort analysis at a large tertiary care academic medical center was conducted. This study included adult acute leukemia patients (age ≥18 years old) who received inpatient antibacterial prophylaxis (moxifloxacin or levofloxacin) from 1 July 2012 to 1 October 2014. Patients were excluded from the study if they were treated with antimicrobial therapy in the preceding five days or admitted to the hospital with neutropenic fever. Fisher's exact test was used for categorical data and Mann-Whitney test for continuous data. Logistic regression analysis was used to determine risk factors for febrile neutropenia. RESULTS: Eighty-five patients were included in the final analysis with 40 patients who received moxifloxacin and 45 patients who received levofloxacin. Baseline characteristics were similar between the two groups. Twenty-two patients experienced febrile neutropenia requiring intravenous antibiotics in the moxifloxacin group and 30 patients in the levofloxacin group (P = 0.190). Age and duration of neutropenia appeared to predict for febrile neutropenia; however, after multivariate analysis, longer duration of neutropenia was shown to be the best predictor for febrile neutropenia with an odds ratio of 4.69 (95% CI, 1.697-12.968). Both groups had similar rates of documented infections and in-hospital morality. CONCLUSION: Moxifloxacin and levofloxacin showed similar rates of febrile neutropenia when used for neutropenic antibacterial prophylaxis in acute leukemia patients.

Genetic analysis of 10 pedigrees with inherited retinal degeneration by exome sequencing and phenotype-genotype association.

Our purpose was to identify causative mutations and characterize the phenotype associated with the genotype in 10 unrelated families with autosomal recessive retinal degeneration. Ophthalmic evaluation and DNA isolation were carried out in 10 pedigrees with inherited retinal degenerations (IRD). Exomes of probands from eight pedigrees were captured using Nimblegen V2/V3 or Agilent V5+UTR kits, and sequencing was performed on Illumina HiSeq. The DHDDS gene was screened for mutations in the remaining two pedigrees with Ashkenazi Jewish ancestry. Exome variants were filtered to detect candidate causal variants using exomeSuite software. Segregation and ethnicity-matched control sample analysis were performed by dideoxy sequencing. Retinal histology of a patient with DHDDS mutation was studied by microscopy. Genetic analysis identified six known mutations in ABCA4 (p.Gly1961Glu, p.Ala1773Val, c.5461-10T>C), RPE65 (p.Tyr249Cys, p.Gly484Asp), PDE6B (p.Lys706Ter) and DHDDS (p.Lys42Glu) and ten novel potentially pathogenic variants in CERKL (p.Met323Val fsX20), RPE65 (p.Phe252Ser, Thr454Leu fsX31), ARL6 (p.Arg121His), USH2A (p.Gly3142Ter, p.Cys3294Trp), PDE6B (p.Gln652Ter), and DHDDS (p.Thr206Ala) genes. Among these, variants/mutations in two separate genes were observed to segregate with IRD in two pedigrees. Retinal histopathology of a patient with a DHDDS mutation showed severe degeneration of retinal layers with relative preservation of the retinal pigment epithelium. Analysis of exome variants in ten pedigrees revealed nine novel potential disease-causing variants and nine previously reported homozygous or compound heterozygous mutations in the CERKL, ABCA4, RPE65, ARL6, USH2A, PDE6B, and DHDDS genes. Mutations that could be sufficient to cause pathology were observed in more than one gene in one pedigree.

The Tameside and Glossop Early Attachment Service: Meeting the emotional needs of parents and their babies.

Parent-infant emotional health is probably one of the most complex arenas in which mental health, maternity and health visiting services operate. This critical period can be highly emotionally charged, not only for the infant but also be for the parent. While most parents essentially get it right, severe ruptures in the parent-infant relationship can occur, and can have serious consequences. This paper describes a comprehensive and cost-effective parent infant mental health service based on a psychodynamic model. The service aims to meet the needs of all parents from those with a high level of need through to a universal provision. Strategic and theoretical underpinnings of the service model are described.

Application of microdroplet PCR for large-scale targeted bisulfite sequencing.

Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons.

Iron overload secondary to cirrhosis: a mimic of hereditary haemochromatosis?

AIMS: Hepatic iron deposition unrelated to hereditary haemochromatosis is common in cirrhosis. The aim of this study was to determine whether hepatic haemosiderosis secondary to cirrhosis is associated with iron deposition in extrahepatic organs. METHODS AND RESULTS: Records of consecutive adult patients with cirrhosis who underwent autopsy were reviewed. Storage iron was assessed by histochemical staining of sections of liver, heart, pancreas and spleen. HFE genotyping was performed on subjects with significant liver, cardiac and/or pancreatic iron. The 104 individuals were predominantly male (63%), with a mean age of 55 years. About half (46%) had stainable hepatocyte iron, 2+ or less in most cases. In six subjects, there was heavy iron deposition (4+) in hepatocytes and biliary epithelium. All six of these cases had pancreatic iron and five also had cardiac iron. None of these subjects had an explanatory HFE genotype. CONCLUSIONS: In this series, heavy hepatocyte iron deposition secondary to cirrhosis was commonly associated with pancreatic and cardiac iron. Although this phenomenon appears to be relatively uncommon, the resulting pattern of iron deposition is similar to haemochromatosis. Patients with marked hepatic haemosiderosis secondary to cirrhosis may be at risk of developing extrahepatic complications of iron overload.

Getting it right from the start: evaluation of a DVD and booklet for new parents.

Early universal preventive interventions have the potential to improve the parent-infant relationship. Getting it Right from the Start is a DVD and booklet given free to new parents to promote sensitive and responsive early parenting and infant communication. Parents in the local area were canvassed for their views and opinions as to what their needs and feelings were about infant mental health. The resource is based on evidence from research and clinical studies in infant development and infant mental health. A matched sample design evaluation study was conducted. A 'Baby Quiz' testing parents' knowledge of different areas covered in the DVD and booklet was given to parents in receipt of the materials and also to parents not yet in receipt of the resource. Results indicated that the mean number of correct responses to the quiz items was higher in the group receiving the DVD and booklet. The percentage of poor scorers in the group who received the DVD and booklet was almost half that of the control group, showing the effectiveness of this universal intervention. Getting it Right from the Start represents a simple, low-cost method of reaching parents during the perinatal period.

Severe microcytic anemia but increased erythropoiesis in mice lacking Hfe or Tfr2 and Tmprss6.

Cell surface proteins Hfe, Tfr2, hemojuvelin and Tmprss6 play key roles in iron homeostasis. Hfe and Tfr2 induce transcription of hepcidin, a small peptide that promotes the degradation of the iron transporter ferroportin. Hemojuvelin, a co-receptor for bone morphogenic proteins, induces hepcidin transcription through a Smad signaling pathway. Tmprss6 (also known as matriptase-2), a membrane serine protease that has been found to bind and degrade hemojuvelin in vitro, is a potent suppressor of hepcidin expression. In order to examine if Hfe and Tfr2 are substrates for Tmprss6, we generated mice lacking functional Hfe or Tfr2 and Tmprss6. We found that double mutant mice lacking functional Hfe or Tfr2 and Tmprss6 exhibited a severe iron deficiency microcytic anemia phenotype mimicking the phenotype of single mutant mice lacking functional Tmprss6 (Tmprss6msk/msk mutant) demonstrating that Hfe and Tfr2 are not substrates for Tmprss6. Nevertheless, the phenotype of the mice lacking Hfe or Tfr2 and Tmprss6 differed from Tmprss6 deficient mice alone, in that the double mutant mice exhibited much greater erythropoiesis. Hfe and Tfr2 have been shown to play important roles in the erythron, independent of their role in regulating liver hepcidin transcription. We demonstrate that lack of functional Tfr2 and Hfe allows for increased erythropoiesis even in the presence of high hepcidin expression, but the high levels of hepcidin levels significantly limit the availability of iron to the erythron, resulting in ineffective erythropoiesis. Furthermore, repression of hepcidin expression by hypoxia was unaffected by the loss of functional Hfe, Tfr2 and Tmprss6.

Common TMPRSS6 mutations and iron, erythrocyte, and pica phenotypes in 48 women with iron deficiency or depletion.

BACKGROUND: TMPRSS6 A736V is associated with lower transferrin saturation (TS), hemoglobin (Hb), and mean corpuscular volume (MCV) levels in general adult populations. We sought to identify relationships of TMPRSS6 K253E, A736V, and Y739Y to iron, erythrocyte, and pica phenotypes in women with iron deficiency or depletion. METHODS: We tabulated observations on 48 outpatient non-pregnant women who had iron deficiency (serum ferritin (SF) <14pmol/L and TS <10%) or iron depletion (SF<112pmol/L). We performed direct sequencing of TMPRSS6 exons 7 and 17 in each patient. We used age, TS, SF, Hb, MCV, pica, and TMPRSS6 allele positivity (dichotomous) or mutation genotypes (trichotomous) as variables for analyses. RESULTS: Forty-six women were white; two were black. 58.3% had iron deficiency. 45.8% had pica (pagophagia, each case). Allele frequencies were 41.7% (K253E), 36.5% (A736V), and 39.6% (Y739Y). K253E frequency was greater in women with TS ≥10% (p=0.0001). Y739Y was more frequent in women with TS <10% (p=0.0135). Mean TS was also lower in women positive for Y739Y (6±4% vs. 13±16%, respectively; p=0.0021). In multiple regressions, neither K253E, A736V, nor Y739Y genotypes were significantly associated with other variables. CONCLUSIONS: TMPRSS6 K253E frequency was greater in women with TS ≥10%. Frequency of Y739 was greater in women with TS <10%. Mean TS was lower in women with Y739Y. We observed no other significant relationship of TMPRSS6 K253E, A736V, or Y739Y with iron, erythrocyte, or pica phenotypes.

Mild iron overload in an African American man with SLC40A1 D270V.

We report on a 46-year-old black man who resided in Alabama with normal transferrin saturation, mild hyperferritinemia, chronic hepatitis C, and 3+ iron in hepatocytes and Kupffer cells. Exome sequencing revealed heterozygosity for SLC40A1 D270V (exon 7, c.809A→T), a mutation previously reported only in 1 black patient with iron overload who resided in the Republic of South Africa. The present patient was also heterozygous for: heme transporter FLVCR1 novel allele P542S (exon 10, 1624C→T); FLVCR1 T544M (rs3207090); hemopexin (HPX) R371W (rs75307540); ferritin scavenger receptor (SCARA5) R471H (rs61737287); and transferrin receptor (TFRC) G420S (rs41295879). He had no HFE, TFR2,HJV, or HAMP mutations. D270V was not detected in 19 other African Americans with iron overload who resided in Alabama. The allele frequency of SLC40A1 D270V in 258 African American adults who participated in a health appraisal clinic was 0.0019 (95% confidence interval 0-0.0057). D270V could explain 'classical' ferroportin hemochromatosis phenotypes in some African Americans.

Two BMP responsive elements, STAT, and bZIP/HNF4/COUP motifs of the hepcidin promoter are critical for BMP, SMAD1, and HJV responsiveness.

Hepcidin plays a major role in the regulation of iron homeostasis. Several bone morphogenetic proteins (BMPs) are strong inducers of hepcidin (Hamp1, HAMP) expression. Hemojuvelin, a protein critical for maintaining appropriate levels of hepcidin, acts as a coreceptor for BMP2 and BMP4, thereby providing a link between iron homeostasis and the BMP-signaling pathway. We show that a robust BMP, hemojuvelin, and SMAD1 response by murine Hamp1 is dependent on a distal BMP responsive element (BMP-RE2), the adjacent bZIP, HNF4alpha/COUP binding sites, and plus or minus 50 bp of the flanking area within -1.6 to -1.7 kb of the Hamp1 promoter. Furthermore, the STAT site and the BMP responsive element (BMP-RE1) located in the proximal 260-bp region of the Hamp1 promoter are also indispensable for maximal activation of hepcidin transcription. The homologous motifs in the distal and proximal regions of the human HAMP promoter act in a manner similar to the murine Hamp1 promoter. Therefore, we propose that the regulation of hepcidin by the BMP pathway involves the formation of a complex of liver-specific and response-specific transcription factors bound to the distal BMP-RE2 /bZIP/HNF4alpha/COUP region and to the proximal BMP-RE1/STAT region possibly by physical association of the 2 regions.

Erythropoietin, GDF15, IL6, hepcidin and testosterone levels in a large cohort of elderly individuals with anaemia of known and unknown cause.

Epidemiologic studies have documented an increasing frequency of anaemia in individuals 65 yrs and older. Elderly individuals with anaemia have been categorised into the following: those with chronic disease, those with iron, B12 or folate deficiency and those with anaemia of unknown aetiology (AUE). There is considerable interest and debate as to whether AUE has an inflammatory component, is caused by cytokine dysregulation affecting production or response to erythropoietin (EPO) or iron availability or represents a novel pathologic process. Here, we compare a large cohort of AUE cases with a matched, non-anaemic control group and with individuals who have anaemia of defined cause. IL-6, hepcidin, GDF15, EPO and testosterone levels were compared. IL6 and hepcidin levels did not differ significantly between AUE and control groups, indicating that inflammation or iron restriction is not central feature of anaemia in this group. GDF15 levels were significantly elevated when comparing AUE with controls and were markedly elevated in patients with renal disease. Testosterone levels were lower in men from the AUE group compared with non-anaemic controls. EPO levels in the AUE group were increased relative to controls but were inappropriately low for the degree of anaemia. Our data indicate that an impaired EPO response, in the absence of evidence for iron restriction or inflammation, is characteristic of AUE.

Suppression of the hepcidin-encoding gene Hamp permits iron overload in mice lacking both hemojuvelin and matriptase-2/TMPRSS6.

Hepcidin, the master regulator of enteric iron absorption, is controlled by the opposing effects of pathways activated in response to iron excess or iron attenuation. Iron excess is regulated through a pathway involving the cell surface receptor hemojuvelin (HFE2) that stimulates expression of the hepcidin encoding gene (HAMP). Iron attenuation is countered through a pathway involving the hepatocyte-specific plasma membrane protease matriptase-2 encoded by TMPRSS6, leading to suppression of HAMP expression. The non-redundant function of hemojuvelin and matriptase-2 has been deduced from the phenotype imparted by mutations of HFE2 and TMPRSS6, which cause iron excess and iron deficiency respectively. Hemojuvelin is positioned to be the ideal substrate for matriptase-2. To examine the relationship between hemojuvelin and matriptase-2 in vivo, we crossed mice lacking the protease domain of matriptase-2 with mice lacking hemojuvelin. Mice lacking functional matriptase-2 and hemojuvelin exhibited low Hamp (Hamp1) expression, high serum and liver iron, and high transferrin saturation. Surprisingly, the double mutant mice also exhibited lower levels of iron in the heart compared to hemojuvelin-deficient mice, demonstrating a possible cardioprotective effect resulting from the loss of matriptase-2. This phenotype is consistent with hemojuvelin being a major substrate for matriptase-2/TMPRSS6 protease activity.

Severe iron overload with a novel aminolevulinate synthase mutation and hepatitis C infection. A case report.

A 55 year old man with a history of chronic hepatitis C infection was found to have severe hemochromatosis: hepatic cirrhosis, cardiomyopathy, arrhythmia, hypogonadism, diabetes and bronzed skin color. After 50 phlebotomies, he underwent a combined heart and liver transplant. Genetic analyses identified a novel mutation in the iron responsive element of the ALAS2 gene. No mutations were found in other genes associated with adult or juvenile hemochromatosis including HFE, transferrin receptor-2 (TFR2), ferroportin (SLC40A1), hepcidin (HAMP) and hemojuvelin (HJV). We suggest that the ALAS2 mutation together with chronic hepatitis C infection may have caused the severe iron overload phenotype.

Role of matriptase-2 (TMPRSS6) in iron metabolism.

Iron, an essential element for life, is regulated primarily at the level of uptake, storage, and transport in order to maintain sufficient availability for normal physiology. The key protein in iron homeostasis is a 25-amino-acid peptide, hepcidin, which modulates the amount of iron in the circulation by binding and promoting the degradation of the iron exporter ferroportin. Given the central importance of hepcidin, recent studies have focused on how iron is sensed and how the iron signal is transmitted to hepcidin. Mutations in a type II serine protease, matriptase-2/TMPRSS6, were recently identified to be associated with severe iron deficiency caused by inappropriately high levels of hepcidin expression. A key biologically relevant substrate for the proteolytic activity of matriptase-2/TMPRSS6 was found to be hemojuvelin, a cell surface protein that regulates hepcidin expression through a BMP/SMAD pathway. In this review, we discuss the putative role of matriptase-2/TMPRSS6 in iron homeostasis.

The anemia of ageing is not associated with increased plasma hepcidin levels.

It has been proposed that the anemia of ageing may be caused, at least in part, by elevated hepcidin levels, representing a response to increased IL-6 levels. Using a recently developed immunoassay, we have measured the plasma hepcidin levels of eight patients with the anemia of ageing and sex- and age-matched controls, and found that the levels of hepcidin were not increased in patients with the anemia of ageing. In contrast, patients with the anemia of inflammation have higher hepcidin levels than sex- and age-matched controls. In the overall group there was a strong correlation between serum ferritin levels and hepcidin levels, as has been found previously.

Multi-organ iron overload in an African-American man with ALAS2 R452S and SLC40A1 R561G.

BACKGROUND: X-linked sideroblastic anemia (XLSA) is associated with iron overload and mutations in ALAS2, which encodes 5-aminolevulinate synthase. There are few reports of XLSA in persons of sub-Saharan African descent. METHODS: A 47-year-old African-American man had microcytic anemia, elevated iron measures, cardiomyopathy, hepatic cirrhosis, diabetes mellitus, a history of cocaine use and hepatitis C. We amplified and directly sequenced his genomic DNA to detect mutations of SLC40A1, HFE, TFR2, HAMP, HJV and ALAS2. RESULTS: The subject's transferrin saturation was 100% and his serum ferritin was 2,960 ng/ml. An MRI scan revealed diffusely decreased T(2) signals of the heart, liver and pancreas. Transjugular right endomyocardial and liver biopsy specimens revealed marked iron deposition in cardiac myocytes and hepatocytes, and cirrhosis. He died of progressive cardiomyopathy. He was hemizygous for ALAS2 R452S (exon 9; c.1354C-->A) and heterozygous for SLC40A1 R561G (exon 8; c.1681A-->G). He did not have coding region mutations in HFE, TFR2, HAMP or HJV. CONCLUSIONS: ALAS2 R452S largely explains this patient's microcytic anemia and multi-organ iron overload and dysfunction. SLC40A1 R561G may have increased his iron absorption and overload further. Acquired factors, especially cocaine use and hepatitis C, may have contributed to his clinical phenotype.

SLC40A1 c.1402G-->a results in aberrant splicing, ferroportin truncation after glycine 330, and an autosomal dominant hemochromatosis phenotype.

BACKGROUND/AIMS: To determine the molecular basis of a mild hemochromatosis phenotype in a man of Scottish-Irish descent. METHODS: We sequenced genomic DNA to detect mutations of HFE, SLC40A1, TFR2, HAMP, and HFE2. RNA isolated from blood mononuclear cells was used to make cDNA. RT-PCR was performed to amplify ferroportin from cDNA, and amplified products were visualized by electrophoresis and sequenced. RESULTS: The proband was heterozygous for the novel mutation c.1402G-->A (predicted G468S) in exon 7 of the ferroportin gene (SLC40A1). Located in the last nucleotide before the splice junction, this mutation results in aberrant splicing to a cryptic upstream splice site located at nt 990 within the same exon. This causes truncation of ferroportin after glycine 330 and the addition of 4 irrelevant amino acids before terminating. The truncated ferroportin protein, missing its C-terminal 241 amino acids, would lack all structural motifs beyond transmembrane region 7. The patient was also heterozygous for the common HFE H63D polymorphism, but did not have coding region mutations in TFR2, HAMP, or HFE2. CONCLUSIONS: We conclude that this patient represents a unique example of hemochromatosis due to a single base-pair mutation of SLC40A1 that results in aberrant splicing and truncation of ferroportin.