Paternal mosaicism for a novel PBX1 mutation associated with recurrent perinatal death: Phenotypic expansion of the PBX1‐related syndrome

Autosomal dominant (de novo) mutations in PBX1 are known to cause congenital abnormalities of the kidney and urinary tract (CAKUT), with or without extra‐renal abnormalities. Using trio exome sequencing, we identified a PBX1 p.(Arg107Trp) mutation in a deceased one‐day‐old neonate presenting with CAKUT, asplenia, and severe bilateral diaphragmatic thinning and eventration. Further investigation by droplet digital PCR revealed that the mutation had occurred post‐zygotically in the father, with different variant allele frequencies of the mosaic PBX1 mutation in blood (10%) and sperm (20%). Interestingly, the father had subclinical hydronephrosis in childhood. With an expected recurrence risk of one in five, chorionic villus sampling and prenatal diagnosis for the PBX1 mutation identified recurrence in a subsequent pregnancy. The family opted to continue the pregnancy and the second affected sibling was stillborn at 35 weeks, presenting with similar severe bilateral diaphragmatic eventration, microsplenia, and complete sex reversal (46, XY female). This study highlights the importance of follow‐up studies for presumed de novo and low‐level mosaic variants and broadens the phenotypic spectrum of developmental abnormalities caused by PBX1 mutations.     

Muscular fatigue during repeated isokinetic shoulder forward flexions in young females

Summary Peak torque, work, mean power and electromyographic (EMG) activity were recorded for each of 150 repeated isokinetic maximal shoulder flexions (45°–90°) in 23 healthy females. From the EMG signals of trapezius, deltoid, infraspinatus and biceps brachii the mean power frequency and the signal amplitude were determined in real time. The mechanical output showed a steep decrease during the first 40 contractions, followed by a plateau maintained until the end. In all muscles, except the biceps brachii, significant decreases in mean power frequency occurred during the first 40 contractions, showing a tendency to stabilize around the same absolute frequency value. Signal amplitude increased in the trapezius, the deltoid and the infraspinatus, but was constant in the biceps brachii. For some individuals rather high EMG activity was recorded in the muscles during the time the arm was supposed to be passively extended to the starting position, and this was found to be associated with lower strength and endurance levels. Longitudinal analyses showed that the mean power frequencies correlated better than the signal amplitudes with the three mechanical variables. The results suggest that the initial steep decrease in mechanical performance and mean power frequency is caused by fatiguing of type 2 motor units.     

Optimization of peptide linker length in production of MHC class II/peptide tetrameric complexes increases yield and stability,and allows identification of antigen-specific CD4+T cells in peripheral blood mononuclear cells

Reliable, efficient systems for producing soluble HLA-DR molecules, suitable for multimerization and use as staining reagents, have proved elusive. We found that the addition of a flexible linker between peptide and N terminus of the DRB1*0101-chain (Crawford, F., Kozono, H., White, J., Marrack, P. and Kappler, J., Immunity 1998. 8: 675-682.), results in greater in vitro folding efficiency of Escherichia coli-expressed alpha- and beta-chains, and increases both the yield and stability of the DRA1*0101/DRB1*0101/peptide complexes. Although a 10-amino acid linker functioned efficiently for a 20mer epitope from HIV p24, a longer linker was required to produce a DR1 MHC class II tetramer with the influenza hemagglutinin epitope (HA(306-318)). The DR1-HA tetramer was able to stain positively over 98% of a specific clone (HA 1.7) with only a brief 30-min incubation. The tetrameric complexes detected clone cells diluted into PBMC, with high sensitivity, coupled with low background staining in CD4(+) cells. It was possible to detect antigen-specific CD4(+) T cells within a population of PBMC stimulated with the HA peptide. This demonstrates the potential to monitor CD4(+) T cell responses in peripheral blood in a number of clinical scenarios.     

Transcription Factor PU.1 Promotes Conventional Dendritic Cell Identity and Function via Induction of Transcriptional Regulator DC-SCRIPT

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Structure and evolution of the Smith-Magenis syndrome repeat gene clusters, SMS-REPs

An approximately 4-Mb genomic segment on chromosome 17p11.2, commonly deleted in patients with the Smith-Magenis syndrome (SMS) and duplicated in patients with dup(17)(p11.2p11.2) syndrome, is flanked by large, complex low-copy repeats (LCRs), termed proximal and distal SMS-REP. A third copy, the middle SMS-REP, is located between them. SMS-REPs are believed to mediate nonallelic homologous recombination, resulting in both SMS deletions and reciprocal duplications. To delineate the genomic structure and evolutionary origin of SMS-REPs, we constructed a bacterial artificial chromosome/P1 artificial chromosome contig spanning the entire SMS region, including the SMS-REPs, determined its genomic sequence, and used fluorescence in situ hybridization to study the evolution of SMS-REP in several primate species. Our analysis shows that both the proximal SMS-REP (approximately 256 kb) and the distal copy (approximately 176 kb) are located in the same orientation and derived from a progenitor copy, whereas the middle SMS-REP (approximately 241 kb) is inverted and appears to have been derived from the proximal copy. The SMS-REP LCRs are highly homologous (>98%) and contain at least 14 genes/pseudogenes each. SMS-REPs are not present in mice and were duplicated after the divergence of New World monkeys from pre-monkeys approximately 40-65 million years ago. Our findings potentially explain why the vast majority of SMS deletions and dup(17)(p11.2p11.2) occur at proximal and distal SMS-REPs and further support previous observations that higher-order genomic architecture involving LCRs arose recently during primate speciation and may predispose the human genome to both meiotic and mitotic rearrangements.     

Design, testing, and clinical studies of a handheld polarized light camera

Polarized light imaging has been used to detect the borders of skin cancer and facilitate assessment of cancer boundaries. A design for an inexpensive handheld polarized camera is presented and clinical images acquired with this prototype are shown. The camera is built with two universal serial bus (USB) color video cameras, a polarizing beamsplitter cube, and a 4x objective lens. Illumination is provided by three white LEDs and a sheet polarizer. Horizontal and vertical linearly polarized reflected images are processed at 7 frames/s and a resulting polarized image is displayed on screen. We compare the performances of cheap USB camera and a 16-bit electronically cooled camera. Dark noise and image repeatability are compared. In both cases, the 16-bit camera outperforms the USB cameras. Despite these limitations, the results obtained with this USB prototype are very satisfactory. Examples of polarized images of lesions taken prior to surgery are presented.     

Evidence for the involvement of more than one metal cation in the Schiff base deprotonation process during the photocycle of bacteriorhodopsin

The removal of metal cations inhibits the deprotonation process of the protonated Schiff base during the photocycle of bacteriorhodopsin. To understand the nature of the involvement of these cations, a spectroscopic and kinetic study was carried out on bacteriorhodopsin samples in which the native Ca²⁺ and Mg²⁺ were replaced by Eu³⁺, a luminescent cation. The decay of Eu³⁺ emission in bacteriorhodopsin can be fitted to a minimum of three decay components, which are assigned to Eu³⁺ emission from three different sites. This is supported by the response of the decay components to the presence of ²H₂O and to the changes in the Eu³⁺/bR molar ratio. The number of water molecules coordinated to Eu³⁺ in each site is determined from the change in its emission lifetime when ²H₂O replaces H₂O. Most of the emission originates from two “wet” sites of low crystal-field symmetry—e.g., surface sites. Protonated Schiff base deprotonation has no discernable effect on the emission decay of protein-bound Eu³⁺, suggesting an indirect involvement of metal cations in the deprotonation process. Adding Eu³⁺ to deionized bacteriorhodopsin increases the emission intensity of each Eu³⁺ site linearly, but the extent of the deprotonation (and color) changes sigmoidally. This suggests that if only the emitting Eu³⁺ ions cause the deprotonation and bacteriorhodopsin color change, ions in more than one site must be involved—e.g., by inducing protein conformation changes. The latter could allow deprotonation by the interaction between the protonated Schiff base and a positive field of cations either on the surface or within the protein.     

Phaeohyphomycosis caused by the fungal genera Bipolaris and Exserohilum. A report of 9 cases and review of the literature

We have reported 7 new cases of Bipolaris infection and 2 of Exserohilum infection, which demonstrate the capability of these 2 genera to cause invasive as well as "allergic" disease. As noted previously, it is likely that all of the cases of "Helminthosporium" and Drechslera infections reported in the literature were caused by Bipolaris or Exserohilum. Infections due to these 2 genera are probably more common than previously recognized. They should be included in the differential diagnosis of central nervous system and disseminated fungal disease, sinusitis, keratitis, peritonitis associated with continuous ambulatory peritoneal dialysis, and allergic bronchopulmonary disease. These various entities have distinct histopathologic characteristics. With disseminated disease in the immunocompromised patient, the most frequent findings are acute inflammation with prominent vascular invasion, thrombosis, and infarction. In contrast, granulomatous inflammation and leukocytoclastic vasculitis are seen in meningoencephalitis caused by these fungi. The histologic features of allergic bronchopulmonary disease and sinusitis are similar. A chronic inflammatory infiltrate of lymphocytes, plasma cells and eosinophils within edematous granulation tissue is found in addition to squamous metaplasia and thickening of the basement membrane. Infections caused by Bipolaris/Exserohilum and Aspergillus show many clinical and pathologic similarities despite the lack of taxonomic relationship between these fungi. Both cause disseminated disease in immunocompromised patients that is characterized by tissue necrosis and vascular invasion. Both cause central nervous system disease, osteomyelitis, and sinusitis and are associated with allergic bronchopulmonary disease. Sinusitis, the most common form of disease caused by Bipolaris and Exserohilum, occurs in otherwise healthy patients with nasal polyposis and allergic rhinitis. Although pathologic evidence of bone invasion may not be found, there frequently is radiographic evidence of invasive disease. Most patients who are treated initially with surgical debridement and amphotericin B have apparently been cured. However, longer follow-up will be necessary in these patients. Amphotericin B appears to be the treatment of choice for invasive infections caused by Bipolaris/Exserohilum species. Ketoconazole and other imidazole derivatives may also be effective in certain of the disease entities caused by these black moulds; however, their role has yet to be defined.(ABSTRACT TRUNCATED AT 400 WORDS)