Purine Nucleoside Phosphorylase Deficiency: A Mutation Update

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.     

Population differentiation in the context of Holocene climate change for a migratory marine species,the southern elephant seal

Understanding observed patterns of connectivity requires an understanding of the evolutionary processes that determine genetic structure among populations, with the most common models being associated with isolation by distance, allopatry or vicariance. Pinnipeds are annual breeders with the capacity for extensive range overlap during seasonal migrations, establishing the potential for the evolution of isolation by distance. Here, we assess the pattern of differentiation among six breeding colonies of the southern elephant seal, Mirounga leonina, based on mtDNA and 15 neutral microsatellite DNA markers, and consider measures of their demography and connectivity. We show that all breeding colonies are genetically divergent and that connectivity in this highly mobile pinniped is not strongly associated with geographic distance, but more likely linked to Holocene climate change and demographic processes. Estimates of divergence times between populations were all after the last glacial maximum, and there was evidence for directional migration in a clockwise pattern (with the prevailing current) around the Antarctic. We discuss the mechanisms by which climate change may have contributed to the contemporary genetic structure of southern elephant seal populations and the broader implications.     

The development of a method for functional assessment of dentures

Aims: To design and validate a method of assessing complete dentures from a functional standpoint. Subjects: A random sample of 40 complete denture wearers took part in the study. Setting: A university clinical department of prosthetic dentistry. Intervention: We undertook a pilot study to refine the protocol and criteria. All participants and their dentures were examined by two authors independently, with no prior knowledge of the patients'complaints. Design: We defined nine clinical factors of functional quality and applied criteria with binary scoring. We analysed the scores for these factors for inter‐rater reliability. Results: The method proved simple to apply and took less than 5 minutes to complete. The inter‐examiner agreement for all factors was 86% to 100% giving Kappa scores of 0.64 to 1.00 (all Good or Very Good). Conclusions: This study successfully demonstrates that the technique, which we call the Functional Assessment of Dentures (FAD), can give good inter‐examiner reliability. It can therefore be used separately as a routine diagnostic tool and to investigate the relationship between denture qualities and functional ‘outcome’ such as difficulty eating or dietary selection.     

Analysis of a Panel of 48 Cytokines in BAL Fluids Specifically Identifies IL-8 Levels as the Only Cytokine that Distinguishes Controlled Asthma from Uncontrolled Asthma,and Correlates Inversely with FEV1

We sought to identify cells and cytokines in bronchoalveolar lavage (BAL) fluids that distinguish asthma from healthy control subjects and those that distinguish controlled asthma from uncontrolled asthma. Following informed consent, 36 human subjects were recruited for this study. These included 11 healthy control subjects, 15 subjects with controlled asthma with FEV₁≥80% predicted and 10 subjects with uncontrolled asthma with FEV₁ <80% predicted. BAL fluid was obtained from all subjects. The numbers of different cell types and the levels of 48 cytokines were measured in these fluids. Compared to healthy control subjects, patients with asthma had significantly more percentages of eosinophils and neutrophils, IL-1RA, IL-1α, IL-1β, IL-2Rα, IL-5, IL-6, IL-7, IL-8, G-CSF, GROα (CXCL1), MIP-1β (CCL4), MIG (CXCL9), RANTES (CCL5) and TRAIL in their BAL fluids. The only inflammatory markers that distinguished controlled asthma from uncontrolled asthma were neutrophil percentage and IL-8 levels, and both were inversely correlated with FEV₁. We examined whether grouping asthma subjects on the basis of BAL eosinophil % or neutrophil % could identify specific cytokine profiles. The only differences between neutrophil-normal asthma (neutrophil≤2.4%) and neutrophil-high asthma (neutrophils%>2.4%) were a higher BAL fluid IL-8 levels, and a lower FEV₁ in the latter group. By contrast, compared to eosinophil-normal asthma (eosinophils≤0.3%), eosinophil-high asthma (eosinophils>0.3%) had higher levels of IL-5, IL-13, IL-16, and PDGF-bb, but same neutrophil percentage, IL-8, and FEV₁. Our results identify neutrophils and IL-8 are the only inflammatory components in BAL fluids that distinguish controlled asthma from uncontrolled asthma, and both correlate inversely with FEV₁.     

HPRT deficiency: identification of twenty-four novel variants including an unusual deep intronic mutation

Hypoxanthine phosphoribosyltranferase (HPRT) deficiency is an X-linked disorder of purine salvage that ranges phenotypically from hyperuricaemia to Lesch-Nyhan Syndrome. Molecular testing is necessary to identify female carriers within families as a prelude to prenatal diagnosis. During the period 1999-2010 the Purine Research Laboratory studied 106 patients from 68 different families. Genomic sequencing revealed mutations in 88% of these families, 24 of which were novel. In eight patients, exon sequencing was not informative. Copy-DNA analysis in one patient revealed an insertion derived from a deep intronic sequence with a genomic mutation flanking this region, resulting in the creation of a false exon. Carrier testing was performed in 21 mothers of affected patients, out of these, 81% (17) were found to be carriers of the disease-associated mutation. Our results confirm the extraordinary variety and complexity of mutations in HPRT deficiency. A combination of genomic and cDNA sequencing may be necessary to define mutations.     

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.     

Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.