Several chromosomes involved in translocations with chromosome 5 shown with fluorescence in situ hybridization in patients with malignant myeloid disorders

In many patients with myelodysplastic syndromes or acute myeloid leukemia, complex chromosome aberrations can be seen, among which aberrations of chromosome 5 constitute a substantial part. With conventional cytogenetic technique, these aberrations are often identified as deletions or monosomy 5. We analyzed nine patients who, under conventional cytogenetic analysis, showed deletion or monosomy 5. We used fluorescence in situ hybridization with whole-chromosome painting probes to identify the counterpart chromosome and locus-specific identifiers for 5q31 and 5q33 approximately q34. A deletion of 5q was found concomitant with unbalanced translocations. Our results and cases from the literature showed that material from chromosome 5 could be translocated to almost all chromosomes. All patients but one had short survival; this one patient had a preserved 5q31 and 5q33 approximately q34 but a deletion of the q-arm more centromeric than these bands. In eight of the nine patients, further 14 translocations were revealed, not involving chromosome 5.     

International quality assurance study for characterization of Streptococcus pyogenes

Surveillance of group A streptococcal (GAS) infections was undertaken as a major component of the European Commission-funded project on severe GAS disease in Europe (strep-EURO). One aim of strep-EURO was to improve the quality of GAS characterization by standardization of methods. An external quality assurance study (EQA) was therefore carried out to evaluate current global performance. Eleven strep-EURO and seven other streptococcal reference centers received a panel of 20 coded GAS isolates for typing. Conventional phenotypic typing (based on cell surface T and M protein antigens and opacity factor [OF] production) and molecular methods (emm gene typing) were used either as single or combined approaches to GAS typing. T typing was performed by 16 centers; 12 centers found one or more of the 20 strains nontypeable (typeability, 89%), and 11 centers reported at least one incorrect result (concordance, 93%). The 10 centers that tested for OF production achieved 96% concordance. Limited availability of antisera resulted in poor typeability values from the four centers that performed phenotypic M typing (41%), three of which also performed anti-OF typing (typeability, 63%); however, concordance was high for both M (100%) and anti-OF (94%) typing. In contrast, the 15 centers that performed emm gene sequencing achieved excellent typeability (97%) and concordance (98%), although comparison of the performance between centers yielded typeability rates from 65 to 100% and concordance values from 83 to 100%. With the rapid expansion and use of molecular genotypic methods to characterize GAS, continuation of EQA is essential in order to achieve international standardization and comparison of type distributions.     

Mapping translocation breakpoints by next-generation sequencing

Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterization of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time-consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using "next-generation" (Illumina/Solexa) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows the determination of their exact nucleotide positions within a few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations.     

NH4 + conductance in Xenopus laevis oocytes

 Superfusing Xenopus laevis oocytes with NH₄Cl (10 mmol/l, pH 7.5) resulted in an inward current at a clamp potential of –70 mV. In paired experiments (n=22), the NH₄Cl-induced peak current was –293±94 nA, under control conditions (osmolality: 240 mosmol/kg), and rose to –523±196 nA when osmolality was reduced to 144 mosmol/kg. In parallel with the rise in NH₄Cl-induced inward current, membrane conductance at –70 mV doubled and the zero-current potential changed from +3.3±9.4 mV to –22.0±8.0 mV (n=22) in the presence of NH₄Cl during exposure to a hypoosmolar solution. In the absence of NH₄Cl, oocytes responded to hypoosmolality with a shift in zero-current potential to more negative values and an increased conductance which became partially sensitive to isosorbiddinitrate (ISDN), suggesting the activation of a volume-sensitive K⁺ channel. Membrane conductance in the presence of NH₄Cl was decreased by ISDN to similar extents under isoosmolal and hypoosmolal conditions, indicating that NH₄ ⁺ enters the oocytes through a volume-sensitive conductance separate from the ISDN-sensitive K⁺ channel. Received: 20 July 1998 / Received after revision and accepted: 19 October 1998     

Isolated oligodontia associated with mutations in EDARADD, AXIN2, MSX1, and PAX9 genes

Oligodontia is defined as the congenital lack of six or more permanent teeth, excluding third molars. Oligodontia as well as hypodontia (lack of one or more permanent teeth) are highly heritable conditions associated with mutations in the AXIN2, MSX1, PAX9, EDA, and EDAR genes. Here we define the prevalence of mutations in the AXIN2, MSX1, PAX9, EDA, and EDAR genes, and the novel candidate gene EDARADD in a cohort of 93 Swedish probands with non-syndromic, isolated oligodontia. Mutation screening was performed using denaturing gradient gel electrophoresis and DNA sequence analysis. Analyses of the coding sequences of the six genes showed sequence alterations predicted to be damaging or potentially damaging in ten of 93 probands (10.8%). Mutations were identified in the EDARADD (n = 1), AXIN2 (n = 3), MSX1 (n = 2), and PAX9 (n = 4) genes, respectively. None of the 10 probands with mutations had other self-reported symptoms from ectodermal tissues. The oral parameters were similar when comparing individuals with and without mutations but a family history of oligodontia was three times more frequent for probands with mutations. EDARADD mutations have previously been reported in a few families segregating hypohidrotic ectodermal dysplasia and this is, to our knowledge, the first report of an EDARADD mutation associated with isolated oligodontia.     

High‐resolution genomic screening in mantle cell lymphoma—specific changes correlate with genomic complexity,the proliferation signature and survival

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) and numerous copy number aberrations (CNAs). Recently, gene expression profiling defined a proliferation gene expression signature in MCL where high scores predict shorter survival. We investigated 31 MCL cases using high‐density single nucleotide polymorphism arrays and correlated CNA patterns with the proliferation signature and with clinical data. Many recurrent CNAs typical of MCL were detected, including losses at 1p (55%), 8p (29%), 9q (29%), 11q (55%), 13q (42%) and 17p (32%), and gains at 3q (39%), 8q (26%), 15q (23%) and 18q (23%). A novel deleted region at 20q (16%) contained only one candidate gene, ZFP64, a putative tumor suppressor. Unsupervised clustering identified subgroups with different patterns of CNAs, including a subset (19%) characterized by the presence of 11q loss in all cases and by the absence of 13q loss, and 3q and 7p gains. Losses at 1p, 8p, 13q and 17p were associated with increased genomic complexity. High proliferation signature scores correlated with increased number of large (>15 Mbp) CNAs (P = 0.03) as well as copy number gains at 7p (P = 0.02) and losses at 9q (P = 0.04). Furthermore, large/complex 13q losses were associated with improved survival (P < 0.05) as were losses/copy number neutral LOH at 19p13 (P = 0.01). In summary, this high‐resolution genomic analysis identified novel aberrations and revealed that several CNAs correlated with genomic complexity, the proliferation status and survival. © 2010 Wiley‐Liss,Inc.     

Potential-dependent steady-state kinetics of a dicarboxylate transporter cloned from winter flounder kidney

The two-electrode voltage-clamp technique in combination with tracer uptake experiments was used to investigate the dependence of dicarboxylate transport kinetics on membrane potential in Xenopus laevis oocytes expressing the flounder renal high-affinity-type sodium dicarboxylate cotransporter (fNaDC-3). Steady-state succinate-dependent currents in the presence of Na+ were saturable with an apparent affinity constant for succinate, K0.5,succ, of 60 microM. K0.5,succ was independent of membrane potential, suggesting succinate binding at the surface of the fNaDC-3 protein. The maximal succinate-dependent current, deltaImax, increased with hyperpolarization, suggesting that the empty carrier may translocate net charge. Succinate-induced currents showed sigmoidal dependence on Na+ concentration, and K0.5,Na+ decreased with hyperpolarization, suggesting Na+ binding in an ion well. Lowering the external Na+ concentration to 20 mM increased K0.5,succ approximately threefold. Succinate-induced currents were inhibited by Li+ with an Ki,Li+ of approximately 0.5 mM, and a Hill coefficient of below unity indicating the interaction of one Li+ ion with an inhibitory site at fNaDC-3.     

A CGG‐Repeat Expansion Mutation in ZNF713 Causes FRA7A: Association with Autistic Spectrum Disorder in Two Families

We report de novo occurrence of the 7p11.2 folate‐sensitive fragile site FRA7A in a male with an autistic spectrum disorder (ASD) due to a CGG‐repeat expansion mutation (～450 repeats) in a 5′ intron of ZNF713. This expanded allele showed hypermethylation of the adjacent CpG island with reduced ZNF713 expression observed in a proband‐derived lymphoblastoid cell line (LCL). His unaffected mother carried an unmethylated premutation (85 repeats). This CGG‐repeat showed length polymorphism in control samples (five to 22 repeats). In a second unrelated family, three siblings with ASD and their unaffected father were found to carry FRA7A premutations, which were partially or mosaically methylated. In one of the affected siblings, mitotic instability of the premutation was observed. ZNF713 expression in LCLs in this family was increased in three of these four premutation carriers. A firm link cannot yet be established between ASD and the repeat expansion mutation but plausible pathogenic mechanisms are discussed.     

NH4 + conductance in Xenopus laevis oocytes

 Current-clamp and voltage-clamp techniques were used to study the effects of NH₄ ⁺ on the cell membrane conductance in Xenopus laevis oocytes. Superfusing the oocytes with NH₄Cl resulted in a depolarization of the oocyte’s cell membrane potential and, at a clamp potential of –70 mV, in an inward current. The magnitude of the inward current was proportional to the NH₄Cl concentration in the extracellular solution and on membrane potential. The reversal potential, E _rev , was –35.5 ± 11.6 mV under control conditions and –3.1 ± 11.0 mV (n = 19) in the presence of NH₄Cl (10 mmol/l). Superfusion of the oocytes with nominally Ca²⁺-free solution affected the NH₄Cl-evoked response only marginally. Replacement of extracellular Na⁺ by N-methyl-D-glucamine⁺ markedly reduced, but did not eliminate, the NH₄Cl-sensitive current and shifted the reversal potential to more negative potentials. The NH₄Cl-induced current was substantially inhibited by 0.1 mmol/l flufenamate, and was less affected by blockers of the endogenous K⁺ conductance, Ba²⁺ and isosorbiddinitrate (ISDN). The results are compatible with the activation of a conductance by NH₄Cl for Na⁺ and NH₄ ⁺. The mechanism by which NH₄Cl activates the conductance remains unknown. Received: 9 December 1996 / Received after revision: 10 March 1997 / Accepted: 13 March 1997     

Elastofibroma dorsi has distinct cytomorphologic features, making diagnostic surgical biopsy unnecessary: cytomorphologic study with clinical, radiologic, and electron microscopic correlations

Elastofibroma dorsi (EFD) is a relatively rare soft tissue mass, probably of reactive nature. The lesion is typically located near the inferior margin of the scapula or between the inferior part of scapula and the chest wall in elderly women. Although location of the tumor together with the age/sex of the patients and radiologic findings is often suggestive of the diagnosis, tissue examination has been considered necessary to confirm the diagnosis. Although the histologic features of EFD are well known, there are only four single case reports of the cytologic findings in the English language literature. We describe the cytologic features of EFD in five patients with correlations to clinical, radiologic, histologic, and electron microscopic findings. The current study suggests that the fine-needle aspiration (FNA) features are highly diagnostic, permitting a firm diagnosis of EFD in a typical clinical setting and eliminating the need for preoperative histologic examination.