BACKGROUND:Malignant lymphomas are classified based on morphology, immunophenotype, genetics and clinical features. The pathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in specific entities, their detection is an important adjunct for increasing the reliability of the diagnosis. Recently, split-signal fluorescence in situ hybridization has become available as a robust method to detect chromosomal breaks in paraffin-embedded formalin-fixed tissues. A bright field approach would bring this technology within the reach of every pathology laboratory.DESIGN AND METHODS:Our study was initiated to determine the consistency between chromogenic in situ hybridization and fluorescence in situ hybridization, both using split-signal probes developed for the detection of chromosomal breaks. Five hundred and forty cases of 11 lymphoma entities and reactive, benign lymphoid tissues, collected from eight different pathology laboratories, placed on 15 fluorescence in situ hybridization pre-stained tissue microarray slides, were double stained for the chromogenic hybridization. For each core morphology and actual signal were compared to the original fluorescence hybridization results. In addition, hematoxylin background staining intensity and signal intensity of the double-staining chromogenic in situ hybridization procedure were analyzed.RESULTS:With respect to the presence or absence of chromosomal breaks, 97% concordance was found between the results of the two techniques. Hematoxylin background staining intensity and signal intensity were found to correspond. The overall morphology after double-staining chromogenic in situ hybridization had decreased compared to the initial morphology scored after split-signal fluorescence in situ hybridization staining.CONCLUSIONS:We conclude that double-staining chromogenic in situ hybridization is equally reliable as fluorescence in situ hybridization in detecting chromosomal breaks in lymphoid tissue. Although differences in morphology, hematoxylin staining and chromogenic signal intensity vary between the tumor entities none of the entities appeared more easy or difficult to score.

Uterine leiomyomas are the most common neoplasms in women of reproductive age. Approximately 40% of these neoplasms show recurring structural cytogenetic anomalies, including del(7)(q22), t(12;14)(q15;q24), t(1;2)(p36;p24), and anomalies affecting 6p21 or 10q22. Using positional cloning strategies, we and others had previously identified HMGA1, HMGA2, RAD51L1, and MYST4 (previously referred to as MORF); as primary target (fusion) genes associated with tumor development in three of these distinct cytogenetic subgroups. Here, we report the positional cloning of a single, recurrent, leiomyoma-associated anomaly, t(1;2)(p36;p24). Molecular characterization of the reciprocal breakpoint intervals showed that that AJAP1 (alias SHREW1) and NPHP4 flank the breakpoint on chromosome 1 and that ITSN2 and NCOA1 flank the breakpoint on chromosome 2. Detailed analysis of the breakpoint regions revealed that in this particular case the translocation was associated with a 27-bp deletion on chromosome 1 and a 136-bp duplication on chromosome 2. No breakpoint-spanning (fusion) genes were identified. In silico prediction of transcription factor binding sites, however, indicated the presence of several such sites in the respective breakpoint regions, and major changes therein as a result of the t(1;2)(p36;p24) under investigation. We postulate that transcriptional deregulation of one or more of these breakpoint-flanking genes may contribute to the development of human uterine leiomyomas.

The molecular feature of Burkitt lymphoma (BL) is the translocation that places c‐Myc under the control of immunoglobulin gene regulatory elements. However, there is accumulating evidence that some cases may lack an identifiable MYC translocation. In addition, during the EUROFISH project, aiming at the standardization of FISH procedures in lymphoma diagnosis, we found that five cases out of 35 classic endemic BLs were negative for MYC translocations by using a split‐signal as well as a dual‐fusion probe. Here we investigated the expression pattern of miRNAs predicted to target c‐Myc, in BL cases, to clarify whether alternative pathogenetic mechanisms may be responsible for lymphomagenesis in cases lacking the MYC translocation. miRNAs are a class of small RNAs that are able to regulate gene expression at the post‐transcriptional level. Several studies have reported their involvement in cancer and their association with fragile sites in the genome. They have also been shown to control cell growth, differentiation, and apoptosis, suggesting that these molecules could act as tumour suppressors or oncogenes. Our results demonstrated a modulation of specific miRNAs. In particular, down‐regulation of hsa‐let‐7c was observed in BL cases, compared to normal controls. More interestingly, hsa‐mir‐34b was found to be down‐regulated only in BL cases that were negative for MYC translocation, suggesting that this event might be responsible for c‐Myc deregulation in such cases. This hypothesis was further confirmed by our in vitro experiments, which demonstrated that increasing doses of synthetic hsa‐mir‐34b were able to modulate c‐Myc expression. These results indicate for the first time that hsa‐mir‐34b may influence c‐Myc expression in Burkitt lymphoma as the more common aberrant control exercised by the immunoglobulin enhancer locus. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lymphomas originating from the lymphatic system comprise about 30 entities classified according to the World Health Organization (WHO). The histopathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in different lymphoma entities, their detection will be increasingly important. Hence, a split-signal fluorescence in situ hybridisation (FISH) procedure would be helpful in discriminating the most difficult classifications. The Euro-FISH programme, a concerted action of nine European laboratories, has validated a robust, standardised protocol to improve the diagnostic approach on lymphoma entities. Therefore, 16 fluorescent probes and 10 WHO entities, supplemented with reactive cases, were selected. The results of the Euro-FISH programme show that all probes were correctly cytogenetically located, that the standardised protocol is robust, resulting in reliable results in approximately 90% of cases, and that the procedure could be implemented in every laboratory, bringing the relatively easy interpretation of split-signal probes within the reach of many pathology laboratories.

Illegitimate recombination (IR) is a process that takes place far more often than homologous recombination and is characterized by the recombination between non-homologous or short homologous sequences. The consequences of IR frequently emerge after the introduction of DNA in cell lines because it more frequently integrates in non- homologous than in homologous regions of the host genome. As a result, unexpected truncated or elongated products may be found. By not discarding those products as transfection artifacts, but by studying how they are generated, it might elucidate a possible molecular mechanism of IR. Here we review the current literature describing different mechanisms by which non-homologous DNA recombination can be induced.

AlphaB-Crystallin has for a long time been considered a specific eye lens protein. Later on it appeared that this protein belongs to the family of the small heat shock proteins and that it occurs also extra-lenticularly in many different cell types. AlphaB-Crystallin is mainly present in the cytoplasm, but there are some indications that it might have a function in the nucleus too. However, till now its presence in the nucleus is uncertain. We therefore compared the localization of alphaB-crystallin in nine cell lines cultured under normal conditions using four different antisera. All four antisera gave a diffuse staining for alphaB-crystallin in the cytoplasm, but one of the antibodies consistently showed nuclear staining in eight of the cell types, in the form of distinct speckles. These speckles are equally pronounced in the different cell types, whether or not cytoplasmic alphaB-crystallin is present. Preabsorption of the antiserum with alphaB-crystallin abolished the staining. Furthermore we demonstrate that if only minor amounts of alphaB-crystallin are present, the protein seems to be located exclusively in the nucleus. However, in case of higher amounts of protein, alphaB-crystallin is distributed between cytoplasm and nucleus. The nuclear alphaB-crystallin exists, like the cytoplasmic alphaB-crystallin, in non-phosphorylated and phosphorylated forms, is Triton-insoluble but can be extracted by 2 M NaCl. These data suggest that alphaB-crystallin might be bound to the nuclear matrix per se or to nuclear matrix proteins via other proteins. In agreement with other nuclear matrix proteins, nuclear alphaB-crystallin staining turns diffuse upon mitosis and leaves the chromosomes unstained. Double staining experiments revealed colocalization of alphaB-crystallin with the splicing factor SC35 in nuclear speckles, suggesting a role for alphaB-crystallin in splicing or protection of the splicing machinery.

We recently described a transgenic mouse strain overexpressing hamster alphaA-crystallin, a small heat shock protein, under direction of the hamster vimentin promoter. As a result myelin was degraded and axonal dystrophy in both central nervous system (especially spinal cord) and peripheral nervous system occurred. Homozygous transgenic mice developed hind limb paralysis after 8 weeks of age and displayed progressive loss of myelin and axonal dystrophy in both the central and peripheral nervous system with ongoing age. Pathologically the phenotype resembled, to a certain extent, neuroaxonal dystrophy. The biochemical findings presented in this paper (activity of the enzymes superoxide dismutase, catalase and transglutamase, myelin protein zero expression levels and blood sugar levels) confirm this pathology and exclude other putative pathologies like Amyothrophic Lateral Sclerosis and Hereditary Motor and Sensory Neuropathy. Consequently, an excessive cytoplasmic accumulation of the transgenic protein or a disturbance of the normal metabolism are considered to cause the observed neuropathology. Therefore, extra-ocular alphaA-crystallin-expressing transgenic mice may serve as a useful animal model to study neuroaxonal dystrophy.

Microdeletions and microduplications, not visible by routine chromosome analysis, are a major cause of human malformation and mental retardation. Novel high-resolution, whole-genome technologies can improve the diagnostic detection rate of these small chromosomal abnormalities. Array-based comparative genomic hybridization allows such a high-resolution screening by hybridizing differentially labeled test and reference DNAs to arrays consisting of thousands of genomic clones. In this study, we tested the diagnostic capacity of this technology using approximately 3,500 flourescent in situ hybridization-verified clones selected to cover the genome with an average of 1 clone per megabase (Mb). The sensitivity and specificity of the technology were tested in normal-versus-normal control experiments and through the screening of patients with known microdeletion syndromes. Subsequently, a series of 20 cytogenetically normal patients with mental retardation and dysmorphisms suggestive of a chromosomal abnormality were analyzed. In this series, three microdeletions and two microduplications were identified and validated. Two of these genomic changes were identified also in one of the parents, indicating that these are large-scale genomic polymorphisms. Deletions and duplications as small as 1 Mb could be reliably detected by our approach. The percentage of false-positive results was reduced to a minimum by use of a dye-swap-replicate analysis, all but eliminating the need for laborious validation experiments and facilitating implementation in a routine diagnostic setting. This high-resolution assay will facilitate the identification of novel genes involved in human mental retardation and/or malformation syndromes and will provide insight into the flexibility and plasticity of the human genome.

Homozygous mice transgenic for αA‐crystallin, one of the structural eye lens proteins, developed hindlimb paralysis after 8 weeks of age. To unravel the pathogenesis of this unexpected finding and the possible role of αA‐crystallin in this pathological process, mice were subjected to a histopathological and immunohistochemical investigation. Immunohistochemistry showed large deposits of αA‐crystallin in the astrocytes of the spinal cord, and in the Schwann cells of dorsal roots and sciatic nerves. Additionally, microscopy showed dystrophic axons in the spinal cord and digestion chambers as a sign of ongoing demyelination in dorsal roots and sciatic nerves. Apart from a few areas with slight αA‐crystallin‐immunopositive structures, the brain was normal. Because the αA‐crystallin protein expression appeared in specific cells of the nervous system (astrocytes and Schwann cells), the most plausible explanation for the paralysis is a disturbance of cell function caused by the excessive intracytoplasmic accumulation of the αA‐crystallin protein. This is followed by a sequence of secondary changes (demyelination, axonal dystrophy) and finally arthrosis. In conclusion, αA‐crystallin transgenic mice develop a peripheral and central neuropathy primarily affecting spinal cord areas at the dorsal side, dorsal root and sciatic nerve.

αB-Crystallin, which has homology with the small heat shock proteins, is the basic subunit of α-crystallin, a major component of the vertebrate eye lens. These crystallins have for a long time been thought to be absolutely lens specific. However, about a decade ago αB-crystallin has been detected extralenticularly in many tissues among which the central nervous system. Under pathological conditions the expression level of αB-crystallin frequently increases. For this reason it is considered to be a useful marker in a variety of neurodegenerative diseases. In this mini-review, a number of typical neurodegenerative disorders is dealt with in which αB-crystallin may play a role.

PURPOSE. To determine whether alpha -crystallin is capable of forming filament-like structures with other members of the crystallin family.METHODS. Water-soluble crystallins were isolated from calf lenses and fractionated into alpha-, betaH-, betaL-, and gamma -crystallins according to standard procedures. Chaperone-like activity of alpha -crystallin was determined in control and UV-A-irradiated lenses by the heat-induced aggregation assay of betaL-crystallin. Protein samples from this assay were analyzed by electron microscopy. In vitro filament formation was examined by transmission immunoelectron microscopy using specific antibodies directed against the crystallins. Involvement of intermediate filament constituents was excluded by the results of Western blot analysis, which were all negative. Moreover, the in vitro amyloid fibril interaction test using thioflavin T (ThT) was also performed.RESULTS. At the supramolecular level heating at 60 degreesC has no effect on the morphologic appearance of alpha -crystallin as observed by transmission electron microscopy. Moreover alpha -crystallin obtained from UV-A-irradiated lenses shows a virtually identical shape. However, heating in the presence of betaL-crystallin results in the formation of filament-like alpha beta -hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against alpha- or betaL-crystallin. Parallel experiments with alpha -crystallin derived from UV-A-irradiated lenses showed even more pronounced filamentous structures, compared with the controls. Nonetheless, we were able to show that the UV-light treatment affected the chaperone-like capacity of alpha -crystallin, as revealed by a diminished ability to inhibit in vitro denaturation of betaL-crystallin. To exclude the presence of cytoskeletal contamination in the crystallin preparations, vimentin antibodies were also tested. These latter experiments were negative. The filamentous nature of the hybrids was further confirmed by the results obtained with the ThT assay earlier applied for the detection of amyloid fibrils.CONCLUSIONS. Crystallin hybrids have previously been detected in the water-soluble lens crystallin fraction. Our findings indicate that such endogenous hybrids, formerly called "rods," may result from stress-induced interaction between alpha -crystallin and other lens constituents such as betaL-crystallin. Because the hybrid formation is enhanced when alpha -crystallin from UV-A-irradiated lenses is used as one of the two components of the hybrid, one can only speculate that this formation may be one of the factors leading to UV-A cataract.

alphaA-Crystallin, a small heat shock protein with chaperone-like activity, forms dynamic multimeric complexes. Recently we described the spontaneous generation of a mutant protein (super alphaA-crystallin) by exon duplication arisen via exon shuffling confirming a classic hypothesis by Gilbert [Nature 271 (1978) 501]. Comparison of super alphaA-crystallin, which is viable in a mouse skeletal muscle cell line, with normal alphaA-crystallin shows that it has diminished thermostability, increased exposure of hydrophobic patches, a larger complex size and lost its chaperone activity. However, super alphaA-crystallin subunits exchange as readily between complexes as does normal alphaA-crystallin. These data indicate that chaperone-like activity may vanish independent of subunit hydrophobicity and exchangeability.

Amyloid beta (Abeta) is a 40- to 42-residue peptide that is implicated in the pathogenesis of Alzheimer's Disease (AD). As a result of conformational changes, Abeta assembles into neurotoxic fibrils deposited as 'plaques' in the diseased brain. In AD brains, the small heat shock proteins (sHsps) alphaB-crystallin and Hsp27 occur at increased levels and colocalize with these plaques. In vitro, sHsps act as molecular chaperones that recognize unfolding peptides and prevent their aggregation. The presence of sHsps in AD brains may thus reflect an attempt to prevent amyloid fibril formation and toxicity. Here we report that alphaB-crystallin does indeed prevent in vitro fibril formation of Abeta(1-40). However, rather than protecting cultured neurons against Abeta(1-40) toxicity, alphaB-crystallin actually increases the toxic effect. This indicates that the interaction of alphaB-crystallin with conformationally altering Abeta(1-40) may keep the latter in a nonfibrillar, yet highly toxic form.

Amyloid β (Aβ) is a 40- to 42-residue peptide that is implicated in the pathogenesis of Alzheimer's Disease (AD). As a result of conformational changes, Aβ assembles into neurotoxic fibrils deposited as 'plaques' in the diseased brain. In AD brains, the small heat shock proteins (sHsps) αB-crystallin and Hsp27 occur at increased levels and colocalize with these plaques. In vitro, sHsps act as molecular chaperones that recognize unfolding peptides and prevent their aggregation. The presence of sHsps in AD brains may thus reflect an attempt to prevent amyloid fibril formation and toxicity. Here we report that αB-crystallin does indeed prevent in vitro fibril formation of Aβ1–40. However, rather than protecting cultured neurons against Aβ1–40 toxicity, αB-crystallin actually increases the toxic effect. This indicates that the interaction of αB-crystallin with conformationally altering Aβ1–40 may keep the latter in a nonfibrillar, yet highly toxic form.

Malignant lymphomas are classified based on morphology, immunophenotype, genetics and clinical features. The pathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in specific entities, their detection is an important adjunct for increasing the reliability of the diagnosis. Recently, split-signal fluorescent in situ hybridization (FISH) has become available as a robust method to detect chromosomal breaks in paraffin embedded formalin fixed tissues. A bright field approach would bring this technology within the reach of every laboratory of pathology. Our study was initiated to prove consistency between chromogenic in situ hybridization (DuoCISH) and FISH, both using split signal probes devel- oped for the detection of chromosomal breaks. 540 cases of 11 lymphoma entities and reactive, benign lymphoid tissues, collected from 8 different laboratories of pathology, placed on 15 FISH pre-stained tissue micro array slides, were DuoCISH stained. Of each core morphology and actual signal were compared to the original FISH results. In addition Hematoxylin background staining intensity and signal intensity of the DuoCISH procedure were ana- lyzed. With respect to the presence or absence of a chromosomal breaks, 97% concordance between FISH and DuoCISH, was found. Hematoxylin background staining intensity and signal intensity were found to correspond. The overall morphology after DuoCISH had decreased compared to the initial morphology scored after split-sig- nal FISH staining. We conclude that DuoCISH is equally reliable as FISH in detecting chromosomal breaks in lym- phoid tissue. Although differences in morphology, Hematoxylin staining and CISH signal intensity vary between the tumor entities none of the entities appeared more easy or difficult to score.