DNA Diagnostics of Hereditary Hearing Loss: A Targeted Resequencing Approach Combined with a Mutation Classification System

Although there are nearly 100 different causative genes identified for nonsyndromic hearing loss (NSHL), Sanger sequencing‐based DNA diagnostics usually only analyses three, namely, GJB2, SLC26A4, and OTOF. As this is seen as inadequate, there is a need for high‐throughput diagnostic methods to detect disease‐causing variations, including single‐nucleotide variations (SNVs), insertions/deletions (Indels), and copy‐number variations (CNVs). In this study, a targeted resequencing panel for hearing loss was developed including 79 genes for NSHL and selected forms of syndromic hearing loss. One‐hundred thirty one presumed autosomal‐recessive NSHL (arNSHL) patients of Western‐European ethnicity were analyzed for SNVs, Indels, and CNVs. In addition, we established a straightforward variant classification system to deal with the large number of variants encountered. We estimate that combining prescreening of GJB2 with our panel leads to a diagnosis in 25%–30% of patients. Our data show that after GJB2, the most commonly mutated genes in a Western‐European population are TMC1, MYO15A, and MYO7A (3.1%). CNV analysis resulted in the identification of causative variants in two patients in OTOA and STRC. One of the major challenges for diagnostic gene panels is assigning pathogenicity for variants. A collaborative database collecting all identified variants from multiple centers could be a valuable resource for hearing loss diagnostics.     

Use of a curved-array transducer to reduce interobserver variation in sonographic measurement of thyroid volume in healthy adults

PURPOSE: Sonographic calculation of thyroid volume is used in the diagnosis and follow-up of thyroid diseases. Since the calculated volume of thyroid lobes is highly influenced by the longest (ie, craniocaudal) diameter, we examined whether using a curved-array transducer as opposed to a linear-array transducer to measure the craniocaudal diameter would reduce interobserver variation. METHODS: Three sonographers with different levels of expertise each used a 5-12-MHz linear-array transducer and a 2-5-MHz curved-array transducer to measure the craniocaudal diameter of both thyroid lobes of 25 healthy volunteers. On the basis of these measurements, thyroid lobe volumes were calculated. Single-factor analysis of variance was used to evaluate the interobserver variations between the measurements made by all 3 observers as well as between measurements taken by pairs of observers. A p value of less than 0.05 was considered significant. RESULTS: Using the linear-array transducer to measure the craniocaudal diameter resulted in significant interobserver variation in thyroid volume calculation (p = 0.02), whereas using the convex-array transducer did not. Using either transducer resulted in a highly significant interobserver variation in measurements of the craniocaudal diameter, although the variation was far more pronounced for measurements made with the linear-array transducer (p = 0.0005) than for those made with the curved-array transducer (p = 0.04). For both transducers, the interobserver variations were most pronounced between the most and the least experienced sonographers. CONCLUSIONS: To avoid significant interobserver variation in calculating thyroid lobe volume, we recommend using a curved-array transducer to measure the craniocaudal diameter of the thyroid lobes.     

Impact of several histopathological prognosticators and local tumour extension on oncological outcome in pT3b/c N0M0 renal cell carcinoma

OBJECTIVE

To investigate the prognostic relevance of different histopathological features and local tumour extension in patients with pT3b/c N0M0 renal cell carcinoma (RCC), as recently new proposals of reclassifying tumour fat invasion in pT3b/c RCC have been made but the effect of other histopathological tumour characteristics and combinations thereof with tumour invasion has yet to be determined in these patients.

PATIENTS AND METHODS

Between 1990 and 2006, 1943 patients underwent surgical treatment for renal tumours in our institution, of which 175 patients (8.7%) had pT3b/c RCC. After exclusion of 57 patients (32.6%) with lymph node and/or distant metastases at the time of diagnosis, 118 (67.4%) remained for retrospective analysis. Different histopathological features and local tumour extension were studied for their association with cancer‐specific‐survival (CSS) and progression‐free‐survival (PFS) by univariate and multivariate analyses. Histopathology was reviewed and revised according to the 2002 Tumour‐Nodes‐Metastasis (TNM) classification system by one pathologist (S.B.). CSS and PFS were estimated by the Kaplan–Meier method.

RESULTS

Follow‐up data were obtained from 110 patients at a median (range) of 3.2 (0.3–16.1) years. In univariate analysis, microvascular invasion (MVI) and capsular invasion increased the risk of tumour progression by 2.05‐ and 2.72‐times (P = 0.037 and P < 0.001). Overall, tumour fat invasion (TFI) and the presence of areas composed by cells with eosinophilic cytoplasm were associated with a higher risk of progression (P = 0.001 and P = 0.011) and reduced CSS (P = 0.037 and P = 0.017). In multivariate analysis, MVI and capsular invasion were associated with a two‐fold increased risk of dying from cancer (hazard risk ratio, HR 2.22, P = 0.045 and HR 2.31, P = 0.011). TFI in general (P = 0.004) and specifically coexistent perirenal fat invasion (PFI) and renal sinus fat invasion (RSFI) were associated with a three‐fold increased risk of developing tumour progression (HR 3.36, P = 0.001). The 10‐year CSS and PFS rates were 39% and 36% for all patients, 47% and 45% for pT3b/c RCC with no PFI or RSFI, and 25% and 10% for PFI + RSFI.

CONCLUSION

Patients with pT3b/c RCC with MVI, capsular invasion, TFI and especially PFI + RSFI, have a markedly reduced prognosis compared with patients with pT3b/c RCC without these features. When these results are corroborated by additional studies and external validation, modification of the TNM classification system would be a sensible consequence.     

Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (−/−) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (−/−) mice]. In spite of the host of functions speculatively attributed to the mdr1-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum–chemical, and immunological parameters were not abnormal in mdr1a/1b (−/−) mice. The high level of mdr1b P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (−/−) mice behaved similarly to the previously analyzed mdr1a (−/−) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (−/−) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (−/−) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.     

Metabolic and secretory properties of peripheral and synovial granulocytes in rheumatoid arthritis

The metabolic and secretory properties of peripheral and synovial granulocytes of patients with rheumatoid arthritis were investigated with serum- or immunoglobulin-treated zymosan as activators of cell metabolism. During isolation of the synovial cells precautions were taken to prevent in vitro phagocytosis of immune materials present in the synovial fluids. Oxygen uptake, extracellular release of lysosomal enzymes under resting and activated conditions, yield of the isolated granulocytes, and the granule enzyme content of peripheral and synovial cells did not differ significantly from those of peripheral granulocytes from healthy volunteers. In agreement with the biochemical results, intracellular inclusions could be detected in only a few synovial cells with a direct immunofluorescence technique. The possibility that formation of “ragocytes” may be an in vitro phenomenon is discussed.     

Heterogeneity of proteoglycans extracted before and after collagenase treatment of human articular cartilage: I. Physical properties related to age

Proteoglycans were isolated from young and mature human articular cartilage 4 different ways: by direct extraction with 4M guanidine hydrochloride (GuHCl); after digestion of the residue from this first extraction with collagenase, by extraction with 4M GuHCl; associatively with 0.5M GuHCl after digestion of the cartilage with collagenase; and dissociatively with 4M GuHCl after digestion of the cartilage with collagenase. The structural properties of these proteoglycans were compared. Proteoglycan aggregates and monomers isolated from second extractions and from young cartilage were of larger hydrodynamic size than proteoglycans isolated from first extractions and mature cartilage, respectively. The same applied to the chondroitin sulfate chain lengths of these proteoglycans. The proteoglycan fraction from second extractions of cartilage contained a larger proportion of monomers than the fraction from first extractions. Associative extraction of mature collagenase-digested cartilage yielded mainly proteoglycan monomers, whereas an appreciable amount of proteoglycan aggregate was also liberated from young collagenase-digested cartilage. Our results indicate that, because of their larger size, proteoglycans from second extractions of cartilage are more entrapped in the collagen network. These large proteoglycans can only be liberated from the matrix after extraction of the smaller proteoglycans, followed by digestion of the residue with collagenase. This indicates that proteoglycans overlap and entangle with the collagen and protect it from degradation by collagenase.     

Epidemiology and risk factors for tinnitus after leisure noise exposure in Flemish young adults

Objective: Young people regularly expose themselves to leisure noise and are at risk of acquiring tinnitus. This study examined the prevalence of leisure noise-induced tinnitus among Flemish young adults as well as the relation with sociodemographic factors, health-related variables and attitudes and beliefs towards noise. Design: A self-administered questionnaire was used to evaluate the presence of noise-induced tinnitus, the amount of leisure noise and attitudes towards noise and hearing protection. Study sample: 517 subjects between 18 and 30 years were included. Results: Temporary and chronic tinnitus occurred in 68.5% and 6.4% of the sample, respectively. Chronic tinnitus was more prevalent in male subjects and associated with more hearing-related symptoms. Furthermore, subjects with chronic tinnitus were more aware of the risks of noise and the importance of hearing protection. Finally, higher levels of leisure noise were independently associated with chronic tinnitus. Conclusions: Tinnitus is observed frequently in young adults. Results also indicate that persons with chronic tinnitus were exposed to a higher noise dose during their lives. Longitudinal studies may be useful to evaluate whether the experience of chronic tinnitus has led to behavioural changes. These findings further underpin the importance of educating youth about the risks of leisure noise exposure.     

Novel amorphous microporous silica spheres for controlled release applications

A new synthesis route for large spherical particles of amorphous microporous silica (AMS) was developed. These novel spherical porous particles with diameter of around 100 µm and pore diameter smaller than 2 nm were prepared using an oil drop method. Molten ibuprofen was successfully loaded into the AMS pores. In vitro release experiments revealed that ibuprofen was released from the spheres via a slow pore diffusion process lasting up to 14 days. Experimentally determined diffusion coefficients of ibuprofen in the porous spheres were of the order of 10^?15–10^?16 m² s^?1. The present findings reveal the potential of AMS spheres for controlled release of small drug molecules over long time periods. The spherical shape of the AMS particles is advantageous in formulation processes as it provides adequate powder flow properties and reliable release profiles. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4295–4301, 2011     

Learning by observation requires an early sleep window

Numerous studies have shown that sleep enhances memory for motor skills learned through practice. Motor skills can, however, also be learned through observation, a process possibly involving the mirror neuron system. We investigated whether motor skill enhancement through prior observation requires sleep to follow the observation, either immediately or after a delay, to consolidate the procedural memory. Sequence-specific fingertapping performance was tested in 64 healthy subjects in a balanced design. Electromyography verified absence of overt or subliminal hand muscle activations during observation. The results show that immediate sleep is necessary for the enhancement of a motor skill through prior observation. Immediate sleep improved the speed of subsequent performance by 22 ± 11% (mean ± SEM) (P = 0.04) and reduced the error rate by 42 ± 19% (P = 0.02). In contrast, no performance gains occurred if sleep was initiated more than 12 h after observation. A second study on 64 subjects ruled out explicit familiarity with the sequence or the spatiotemporal rhythm of the sequence to underlie performance improvements. The sleep-dependent observational motor learning enhancement is at least similar to that previously reported for implicit and declarative memory. The apparent prerequisite of observing real movements indicates that subjects transfer experience obtained through observation of movements to subsequent self-initiated movements, in the absence of practice. Moreover, the consolidation of this transfer requires an early sleep window. These findings could improve learning new motor skills in athletes and children, but also in patients having to remaster skills following stroke or injury.     

Humans possess two mitochondrial ferredoxins,Fdx1 and Fdx2, with distinct roles in steroidogenesis,heme, and Fe/S cluster biosynthesis

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.