Expanding the genotypic spectrum of Perrault syndrome.

Perrault syndrome is a rare autosomal recessive disorder characterized by sensorineural hearing loss (SNHL) in both sexes and primary ovarian insufficiency in 46, XX karyotype females. Biallelic variants in five genes are reported to be causative: HSD17B4, HARS2, LARS2, CLPP and C10orf2. Here we present eight families affected by Perrault syndrome. In five families we identified novel or previously reported variants in HSD17B4, LARS2, CLPP and C10orf2. The proband from each family was whole exome sequenced and variants confirmed by Sanger sequencing. A female was compound heterozygous for a known, p.(Gly16Ser) and novel, p.(Val82Phe) variant in D-bifunctional protein (HSD17B4). A family was homozygous for mitochondrial leucyl aminocyl tRNA synthetase (mtLeuRS) (LARS2) p.(Thr522Asn), previously associated with Perrault syndrome. A further family was compound heterozygous for mtLeuRS, p.(Thr522Asn) and a novel variant, p.(Met117Ile). Affected individuals with LARS2 variants had low frequency SNHL, a feature previously described in Perrault syndrome. A female with significant neurological disability was compound heterozygous for p.(Arg323Gln) and p.(Asn399Ser) variants in Twinkle (C10orf2). A male was homozygous for a novel variant in CLPP, p.(Cys144Arg). In three families there were no putative pathogenic variants in these genes confirming additional disease-causing genes remain unidentified. We have expanded the spectrum of disease-causing variants associated with Perrault syndrome.

DMRTA2 (DMRT5) is mutated in a novel cortical brain malformation.

Lissencephaly is a phenotypically and genetically heterogeneous group of cortical brain malformations due to abnormal neuronal migration. The identification of many causative genes has increased the understanding of normal brain development. A consanguineous family was ascertained with three siblings affected by a severe prenatal neurodevelopmental disorder characterised by fronto-parietal pachygyria, agenesis of the corpus callosum and progressive severe microcephaly. Autozygosity mapping and exome sequencing identified a homozygous novel single base pair deletion, c.1197delT in DMRTA2, predicted to result in a frameshift variant p.(Pro400Leufs*33). DMRTA2 encodes doublesex and mab-3-related transcription factor a2, a transcription factor key to the development of the dorsal telencephalon. Data from murine and zebrafish knockout models are consistent with the variant of DMTRA2 (DMRT5) as responsible for the cortical brain phenotype. Our study suggests that loss of function of DMRTA2 leads to a novel disorder of cortical development.

Unusual cutaneous features associated with a heterozygous gain-of-function mutation in IFIH1: overlap between Aicardi-Goutières and Singleton-Merten syndromes.

Cutaneous lesions described as chilblain lupus occur in the context of familial chilblain lupus or Aicardi-Goutières syndrome. To date, seven genes related to Aicardi-Goutières syndrome have been described. The most recently described encodes the cytosolic double-stranded RNA receptor IFIH1 (also known as MDA5), a key component of the antiviral type I interferon-mediated innate immune response. Enhanced type I interferon signalling secondary to gain-of-function mutations in IFIH1 can result in a range of neuroinflammatory phenotypes including classical Aicardi-Goutières syndrome. It is of note that none of the patients with a neurological phenotype so far described with mutations in this gene was reported to demonstrate cutaneous involvement. We present a family segregating a heterozygous pathogenic mutation in IFIH1 showing dermatological involvement as a prominent feature, variably associated with neurological disturbance and premature tooth loss. All three affected individuals exhibited increased expression of interferon-stimulated genes in whole blood, and the mutant protein resulted in enhanced interferon signalling in vitro, both in the basal state and following ligand stimulation. Our results further extend the phenotypic spectrum associated with mutations in IFIH1, indicating that the disease can be confined predominantly to the skin, while also highlighting phenotypic overlap with both Aicardi-Goutières syndrome and Singleton-Merten syndrome.

The pathophysiology of chronic graft failure in the cardiac transplant patient.

Following cardiac transplantation, many patients develop chronic deterioration of graft function, which may lead to a clinical syndrome similar to native chronic heart failure (CHF). This condition of chronic cardiac graft failure (CGF) may also share pathophysiological processes comparable with that of CHF. However, the unique environment following cardiac transplantation may also contribute with a variety of unique mechanisms, deserved of special attention. This review article discusses the complex pathophysiology of CGF after cardiac transplantation, an important yet neglected condition of transplant medicine.

Potential immunologic effects of statins in cancer following transplantation.

3-hydroxy-3-methyglutaryl CoA reductase inhibitors (statins) are frequently used following organ transplantation and have well reported pleiotropic effects, including immunomodulation, which may be of benefit in preventing graft rejection. However, the immunomodulatory effects of statins on cell transformation and malignancy, combined with the immunologic processes and administration of immunosuppression are almost completely unknown. The administration of immunosuppression is well recognised as the main cause of cancer following transplantation, so the addition of an immunomodulatory agent should be associated with an increased incidence of cancer, as immune surveillance and response may be suppressed, allowing cellular transformation and proliferation combined with lack of recognition to occur. This hypothetical review attempts to delineate the mode of action of statins in terms of pro/anti-carcinogenic mechanisms, while considering graft rejection and the presence of immunosuppression.

BNP directly immunoregulates the innate immune system of cardiac transplant recipients in vitro.

BACKGROUND: The innate immune system plays an important role in cardiac allograft rejection. BNP has frequently been reported to elevate during acute cardiac rejection, yet the explanation behind this phenomenon is unclear. We hypothesized that BNP might interact with the innate immune system in cardiac transplant recipients and devised a series of in vitro culture experiments to explore this phenomena. METHODS: PBMCs were isolated from whole blood of (total n = 40) cardiac transplant recipients. Short (24h, n = 20) and long term (72h, n = 20) co-cultures of innate cells in the presence or absence of BNP were performed. BNP was added at two specific concentrations and compared to placebo control. Innate cells were immunophenotyped using flow cytometry. RESULTS: BNP dose dependently reduced the total number of monocytes, B cells and NK cells. Furthermore, BNP co-culture impaired NK cell cytotoxicity and adhesion of non-classical monocytes (via down-regulation of CD11c). DISCUSSION: BNP has an additional physiological role of moderating components of the innate immune system. Although speculative, this could be beneficial to cardiac transplant recipients as the innate immune system is involved in allograft rejection. Further investigation is required to elucidate the mechanism behind how BNP affects immune cells and whether the same effects are consistent with the adaptive immune system.

Radiation therapy of prostate cancer: rationale, pitfalls and the continuing prospect of success forged by medical physics.

Radiation therapy aimed at curing prostate cancer forms a considerable workload in most contemporary radiation oncology departments. The wide range of currently available therapeutic strategies for this cancer and their increasing complexity further increases the impact these patients have within the treating unit. Grounded in basic anatomy, physiology and pathology, the rationale for the division of prostate cancers into different prognostic and therapeutic groups is discussed, and put into clinical context using the current research evidence base. Weaknesses in this evidence base are highlighted in relation to areas directly impacted on by the work of medical physics.

HMG-CoA reductase inhibitors deplete circulating classical and non-classical monocytes following human heart transplantation.

BACKGROUND: Monocytes mediate immune responses following solid organ transplantation via cytokine secretion and differentiation to macrophage/dendritic cell lineages. To date, the pleiotropic immunomodulatory effect of statins on human monocytes following human heart transplantation has yet to be elucidated. This study was designed to assess the effects of statin administration on the monocyte repertoire. METHODS: 108 patients were recruited into the study. Clinical data were collected from patients' notes. Peripheral blood immunophenotype was determined via flow cytometry (using CD11c, CD14, CD16, CD49d, CD64, CD80 and CD195). RESULTS: There were fewer circulating classical (p=0.0001) and non-classical (p=0.0013) monocytes in patients treated with a statin. CD64 expression was down-regulated (p=0.011 and p=0.049) whereas CD49d expression was up-regulated (p=0.004 and p=0.022) on classical and non-classical monocytes in this group. Patients receiving Atorvastatin had fewer circulating classical monocytes (p=0.001) compared to patients administered Pravastatin. Patients receiving Pravastatin had fewer circulating non-classical monocytes (p=0.029) compared to patients administered Atorvastatin. DISCUSSION: Statin administration alters the circulating monocyte repertoire following heart transplantation, including population size, FcgammaRI and VLA-4 adhesion molecule expression. Furthermore, different statin treatments are associated with a selective depletion of macrophage or DC (re)generating monocytes.

CMV infection is associated with the depletion but lack of activation of peripheral blood natural killer cells in a lung transplant cohort.

INTRODUCTION: Following lung transplantation, cytomegalovirus (CMV) has both direct and indirect adverse effects on the allograft. Natural killer cells mediate immune responses to CMV. This can be both dependent and independent of MHC class I expression. However, their role during CMV infection following lung transplantation is unknown. In this study, the immunophenotypic characteristics of NK cells were correlated with CMV infection following lung transplantation. METHODS: Seventy lung transplant recipients were included in the study. NK cells were characterised via flow cytometric analysis of CD3, CD16, CD56, CD107a, CD107b, and CD161. CMV infection was determined using an established quantitative PCR technique on peripheral blood. RESULTS: The number of peripheral blood NK cells with CD16, CD56 and CD161 phenotypes decreased in patients with CMV infection. However, there were no correlations between CMV infection and NK cell activation determined via LAMP expression. CONCLUSIONS: This study reports comparative differences in the peripheral blood NK cell repertoire in lung transplant recipients with CMV infection versus those without. However, NK cell activity did not alter with CMV infection, suggesting that CMV infection alone does not induce an NK cell response.

Surgical aspects of cardiac resynchronisation therapy.

Despite significant advances in the pharmacological treatment of heart failure, rates of mortality and morbidity from the condition remain a concern. The introduction of cardiac resynchronisation therapy (CRT) has been a welcome addition to the treatment strategy of patients who display ventricular dyssynchrony. Several control studies have shown significant benefits from this intervention in particular improved mortality and reduction in symptom burden. In this short review, we focus on several concepts of CRT and discuss the implications of surgical implantation of the left ventricular (LV) lead as compared to the standard transvenous approach.

Long-term study of re-infection following successful eradication of Helicobacter pylori infection.

BACKGROUND: 'Re-infection' with Helicobacter pylori after eradication has been estimated to occur in 0-14% of patients, although most so-called 're-infections' occur within the first year following 'eradication' and many may actually be due to recrudescence of a temporarily suppressed infection. AIM: To study the true re-infection rate, we have studied re-infection rates after eradication therapy by excluding the first year's data, minimizing the possible confounding effect of recrudescence. METHODS: All patients tested for H. pylori infection following eradication therapy between 1987 and 2004 were evaluated. Testing was carried out by urea breath test and gastric biopsy. Patients were included if they were found to be negative for H. pylori infection by testing at least 1 year following eradication and underwent at least one further test for H. pylori. RESULTS: 1162 patients met the inclusion criteria with median post-eradication follow-up of 3 years (1.5-14) including 4668 tests; 3319 years of follow-up were analysed. Thirteen cases of re-infection occurred (re-infection rate 0.4% per year). CONCLUSIONS: This large study of H. pylori re-infection avoided cases of recrudescence by excluding the first post-eradication year. True re-infection is probably less common than previously thought.

Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration.

We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.

Acyl carrier protein interacts with melittin.

Acyl carrier protein (ACP) from Escherichia coli has been shown to form complexes with melittin, a cationic peptide from bee venom. ACP is a small (Mr 8847), acidic, Ca2(+)-binding protein, which possesses some characteristics resembling those of regulatory Ca2(+)-binding proteins including interaction with melittin. Complexing between melittin and ACP which occurred both in the presence and absence of Ca2+ was evident by chemical cross-linking the two peptides, fluorescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Euglena. Also, anti-Apis mellifera antibodies which contained antibodies against melittin specifically inhibited the same enzyme system activity relative to non-immune IgG.

Early catalytic steps of Euglena gracilis chloroplast type II fatty acid synthase.

Euglena gracilis is a very ancient eukaryote whose chloroplast acquisition and evolution has been independent of higher plants. The organism in unique in possessing two de novo fatty acid synthases, a true multienzyme complex of great size in the cytosol and a plastid-localized type II fatty acid synthase composed of discrete enzymes and acyl carrier protein (ACP). The enzymology of the early steps of fatty acid biosynthesis differed in the Euglena type II fatty acid synthase compared to those of Escherichia coli and plants. The enzymes of Euglena participating in both priming and elongation reactions to form a new carbon-carbon bond were acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I. The effects of inhibitors on the three different enzymes were noted. All carbon-carbon bond formation was inhibited by cerulenin. Although neither fatty acid biosynthesis nor any of the isolated enzymes were sensitive to diisopropylphosphofluoridate, the three Euglena enzymes studied were sensitive to different sulfhydryl-alkylating agents. Acetyl-ACP supported fatty acid biosynthesis as effectively as did comparable amounts of ACPSH and acetyl-CoA. There was no evidence for a beta-ketoacyl-ACP synthase III for priming such as has been reported in type II fatty acid synthase of higher plants and bacteria. The roles of the acetyl-CoA-ACP transacylase and beta-ketoacyl-ACP synthase I appear to be unique in the type II fatty acid synthase of Euglena. Acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I were separated from one another and shown to have different molecular weights.

Comparison of acyl-carrier protein and other protein structures in aqueous solutions by Fourier-transform infrared spectroscopy.

Protein solution structures were analyzed by horizontal attenuated total reflectance (ATR) FTIR spectroscopy. Secondary structure compositions determined from analyses of amide-I and II region and amide-III region difference spectra were compared. Data for proteins of known solution structure, cytochrome c, concanavalin A and lysozyme, were compared with those reported in the literature. Melittin, a peptide from bee venom whose secondary structural configuration varies depending upon solution conditions was also examined. Acyl-carrier protein (ACP) is a small protein of recognized dynamic structure that in its diverse physiologic roles interacts specifically with numerous different proteins. Horizontal ATR FTIR analysis of ACP's secondary structure indicated a predominantly helical structure best defined as a combination of ordered and disordered helices. The FTIR-derived structural composition agreed with those determined for ACP by other techniques. Comparison of independent analyses of the amide-I and III regions to determine protein configuration compositions was a useful method of verifying the internal consistency of the calculated structural compositions of dynamically-structured proteins.

Complementary roles of simple variables, NYHA and N-BNP, in indicating aerobic capacity and severity of heart failure.

AIMS: The extent of exercise intolerance in patients with chronic heart failure (CHF) is dependent on and representative of the severity of heart failure. However, few primary care physicians have direct access to facilities for formal exercise testing. We have therefore explored whether information readily obtainable in the community can reliably predict the functional capacity of patients. METHODS AND RESULTS: Ninety-six subjects with a wide range of cardiac function (10 healthy controls and 86 CHF patients with NYHA classes I-IV, LVEF 36.9+/-15.2%) were recruited into the study and had resting plasma N-BNP and cardiopulmonary exercise testing to measure peak oxygen consumption (VO2). Significantly higher N-BNP levels were found in the CHF group (299.3 [704.8] fmol/ml, median [IQR]) compared with the healthy control group (7.2 [51.2] fmol/ml), p<0.0001. There were significant correlations between peak VO2 and N-BNP levels (R=0.64, P<0.001), peak VO2 and NYHA class (R=0.76, P=0.001), but no significant correlation was seen between peak VO2 and LVEF (R=0.0788, P=0.33). Multivariate analysis identified plasma N-BNP (P<0.0001) and NYHA class (P<0.0001) as significant independent predictors of peak VO2. Logistic modelling with NYHA class and log N-BNP to predict peak VO2<20 ml/kg/min showed that the area under the curve of receiver-operating-characteristic (ROC) curve was 0.906 (95% CI 0.844-0.968). A nomogram based on the data has been constructed to allow clinicians to estimate the likelihood of peak VO2 to be <20 ml/kg/min for given values of plasma N-BNP and NYHA class. CONCLUSIONS: By combining information from a simple objective blood test (N-BNP) and a simple scoring of functional status (NYHA), a clinician can deduce the aerobic exercise capacity and indirectly the extent of cardiac dysfunction of patients with CHF.

Regulation of tcp genes in classical and El Tor strains of Vibrio cholerae O1.

Expression of genes encoding the toxin-co-regulated pilus (TCP) varies between the two biotypes of Vibrio cholerae O1. Sequence analysis of the tcp locus from the classical and El Tor strains has revealed differences in the intergenic regions between tcpI and tcpP, and tcpH and tcpA, which may be involved in regulation. To investigate this possibility, transcription of tcpA, and the predicted upstream promoters for tcpI and tcpP, has been analysed in the classical and El Tor strains using promoter-cat (chloramphenicol acetyltransferase) fusions. Together with primer extension analyses, these studies indicate that the tcpA and tcpP promoters are toxR-dependent and suggest that TcpP may be involved in activation of both the tcpI and tcpP promoters. We conclude that differences in the level of tcpA expression in a classical and an El Tor strain are likely to be due to the effect of sequence variation on the ability of control factors to act on these regulatory regions.

Physiochemical-activity relations in practice. 1. A rational and self-consistent data bank.

A data bank of substituent constants of 26 ortho and 34 meta and para benzenoid substituents is presented for use in physicochemical-activity relations (PAR) studies. The distributive parameters pri and pri-, a bulk parameter based on molar refraction, and positionally weighted electronic parameters F and R are listed for the three substituent positions. There are no gaps in the table caused by missing values and the interparameter correlation are low.

Forecasting the antimalarial activities of arylamidinoureas from their measured physicochemical properties.

1 The octanol : water partition coefficient (PC), protein binding coefficients to albumin (Ba) and haemoglobin (Bh), proton magnetic resonance (PMR) chemical shifts and pKa of 8 arylamidinoureas have been measured. 2 There were statistically significant correlations for 7 of the 8 compounds between the predicted lipophilicity parameter eta and log PC, Ba and Bh, and also between Hammett's electronic parameter sigma and the PMR and pKa observations. 3 3-Chloro,4-nitrophenylamidinourea showed a larger deviation from these correlations than any other compound. Geometric calculations based on the covalent radii and bond angles of the molecule suggested that the neighbouring chloro and nitro substituents were interacting sterically. 4 Molecular orbital calculations and the observed ultra-violet spectrum provided further evidence for such an interaction, and suggested that the nitro group is twisted out of the plane of the benzene ring in this molecule. 5 Such steric interactions can introduce an additional source of error when attempts are made to correlate the biological and predicted physicochemical properties of compounds. 6 Generalized parameters such as eta and sigma cannot be used with confidence to predict the properties of compounds in which such steric interactions occur.