Locked nucleic acid-based in situ hybridisation reveals miR-7a as a hypothalamus-enriched microRNA with a distinct expression pattern.

MicroRNAs (miRNAs) are short (∼22 nucleotides) noncoding RNA molecules that post-transcriptionally repress the expression of protein-coding genes by binding to 3'-untranslated regions of the target mRNAs. To identify miRNAs selectively expressed within the hypothalamus, a part of the brain that controls vital bodily functions, we employed locked nucleic acid (LNA)-fluorescent in situ hybridisation (FISH). The expression pattern of the mature miRNAs miR-7a, miR-7b, miR-137 and miR-153 in mouse brain tissue sections was investigated. Although all studied miRNAs were present in the hypothalamus, miR-7a, was the only miRNA found to be enriched in the hypothalamus, with low or no expression in other parts of the central nervous system (CNS). Within the hypothalamus, strong miR-7a expression was distinct and restricted to some hypothalamic nuclei and adjacent areas. miR-7a expression was particularly prominent in the subfornical organ, as well as the suprachiasmatic, paraventricular, periventricular, supraoptic, dorsomedial and arcuate nuclei. Identical expression patterns for miR-7a were seen in mouse and rat hypothalamus. By combining LNA-FISH with immunohistochemistry, it was shown that miR-7a was preferentially present in small orexigenic neuropeptide Y/agouti-related protein-containing-neurones located in the ventromedial aspect of the arcuate nucleus but not in large pro-opiomelanocortin/cocaine- and amphetamine-regulated transcript-containing anorexigenic neurones of the ventrolateral part of the arcuate nucleus. The limited and distinct expression of miR-7a in the CNS suggests that miR-7a has a role in post-transcriptional regulation in hypothalamic neurones. Particularly strong expression of miR-7a in neurones located in the ventromedial division of the arcuate nucleus, a subregion with a weak blood-brain barrier, raises the possibility that miR-7a is influenced by circulating hormones and is a regulator of the genes involved in body weight control.

RUNX2 analysis of Danish cleidocranial dysplasia families.

Cleidocranial dysplasia (CCD) is an autosomal dominant inherited disease caused by mutations in the Runt gene RUNX2. Screening of 19 Danish CCD families revealed 16 pathogenic mutations (84%) representing 8 missense mutations, 2 nonsense mutations, 4 frame-shift mutations and 2 large deletions in the RUNX2 locus. Eight mutations were novel, two were found twice, and polymorphisms were found in the promoter region and in the conserved polyglutamine/polyalanine repeat. A large duplication downstream of RUNX2 found in one patient suggests a possible regulatory RUNX2 element. The CCD phenotypes and genotypes adhere to the large phenotypic variability reported in previous CCD studies. Identification of large chromosome aberrations in or near the RUNX2 locus in 3 of the 19 cases suggests copy number analyses to be included in future RUNX2 mutation analyses.

Breakpoints around the HOXD cluster result in various limb malformations.

BACKGROUND: Characterisation of disease associated balanced chromosome rearrangements is a promising starting point in the search for candidate genes and regulatory elements. METHODS: We have identified and investigated three patients with limb abnormalities and breakpoints involving chromosome 2q31. Patient 1 with severe brachydactyly and syndactyly, mental retardation, hypoplasia of the cerebellum, scoliosis, and ectopic anus, carries a balanced t(2;10)(q31.1;q26.3) translocation. Patient 2, with translocation t(2;10)(q31.1;q23.33), has aplasia of the ulna, shortening of the radius, finger anomalies, and scoliosis. Patient 3 carries a pericentric inversion of chromosome 2, inv(2)(p15q31). Her phenotype is characterised by bilateral aplasia of the fibula and the radius, bilateral hypoplasia of the ulna, unossified carpal bones, and hypoplasia and dislocation of both tibiae. RESULTS: By fluorescence in situ hybridisation, we have mapped the breakpoints to intervals of approximately 170 kb or less. None of the three 2q31 breakpoints, which all mapped close to the HOXD cluster, disrupted any known genes. CONCLUSIONS: Hoxd gene expression in the mouse is regulated by cis-acting DNA elements acting over distances of several hundred kilobases. Moreover, Hoxd genes play an established role in bone development. It is therefore very likely that the three rearrangements disturb normal HOXD gene regulation by position effects.

LNA-modified oligonucleotides are highly efficient as FISH probes.

Fluorescence in situ hybridization (FISH) is a highly useful technique with a wide range of applications including the delineation of complex karyotypes, prenatal diagnosis of aneuploidies, screening for diagnostic or prognostic markers in cancer cells, gene mapping and gene expression studies. However, it is still a fairly time-consuming method with limitations in both sensitivity and resolution. Locked Nucleic Acids (LNAs) constitute a novel class of RNA analogs that have an exceptionally high affinity towards complementary DNA and RNA. Substitution of DNA oligonucleotide probes with LNA has shown to significantly increase their thermal duplex stability as well as to improve the discrimination between perfectly matched and mismatched target nucleic acids. To exploit the improved hybridization properties of LNA oligonucleotides in FISH, we have designed several LNA substituted oligonucleotide probes specific to different human-specific repetitive elements, such as the classical satellite-2, telomere and alpha-satellite repeats. In the present study we show that LNA modified oligonucleotides are excellent probes in FISH, combining high binding affinity with short hybridization time.

Detection of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies using end sequenced probes.

Translocations involving the immunoglobulin loci are recurring events of B cell oncogenesis. The majority of translocations involve the immunoglobulin heavy chain (IGH) locus, while a minor part involves the immunoglobulin light chain loci consisting of the kappa light chain (IGK) located at 2p11.2 and the lambda light chain (IGL) located at 22q11.2. We characterised BAC clones, spanning the IGK and IGL loci, for detection of illegitimate rearrangements by fluorescence in situ hybridisation (FISH). Within the IGL region we have identified six end sequenced probes (22M5, 1152K19, 2036J16, 3188M21, 3115E23 and 274M7) covering the variable (IGLV) cluster and two probes (165G5 and 31L9) covering the constant (IGLC) cluster. Within the IGK region four probes (969D7, 316G9, 122B6 and 2575M21) have been identified covering the variable (IGKV) cluster, and one probe (1021F11) covering the IGK constant (IGKC) cluster. A series of 24 cell lines of different origin have been analysed for the presence of translocations involving the immunoglobulin light chain loci by dual-colour FISH where the split of the variable cluster and the constant cluster indicated a translocation. Probes established in this study can be used for universal screening of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies.

Trisomy 9p syndrome in two brothers: with new clinical findings and review of the literature.

We report an eleven years old boy and his fourteen years old brother who both have trisomy 9p syndrome. Their cytogenetic analysis using GTL-banding showed 46,XY,der(22)add(22)(p11) karyotype. Cytogenetic analysis of their mother and sister revealed a karyogram designated as 46,XX,t(9;22) (9pter-->9p12::22p11-->22qter). With the help of FISH technique, the derivative chromosome in the proband was further confirmed to be a translocation chromosome 22 carrying the aforementioned segments from chromosome 9 which originated from a segregation event of a mother's balanced translocation. Regarding clinical aspects of our cases, both showed similar findings of 9p trisomy syndrome but low frontal hairline, circular placement of the hair around the face and scarce, inverted eyebrows, findings not previously mentioned in the literature. We conclude that these new clinical findings could be used in the clinical diagnosis of the 9p trisomy syndrome along with the other well-documented symptoms.

Detection of illegitimate rearrangement within the immunoglobulin locus on 14q32.3 in B-cell malignancies using end-sequenced probes.

Translocation involving the immunoglobulin heavy chain (IGH) locus is a recurring event in B-cell oncogenesis. The aim of this study was to characterize clones from bacterial artificial chromosome (BAC) libraries and/or bacteriophage P1 artificial chromosome libraries spanning the IGH locus for detection of illegitimate rearrangement within the region by fluorescence in situ hybridization (FISH). In silico analysis of the IGH variable (IGHV) DNA sequence (NT_001716.v1) was performed to identify BAC probes located within the IGHV cluster. Clones of the constant (IGHC) cluster were found in the literature or at http://www.biologia.uniba.it/rmc/. Validation, orientation, and overlap of these probes were confirmed using interphase-, metaphase-, and fiber-FISH. We have identified seven BAC end-sequenced probes (3087C18, 47P23, 76N15, 12F16, 101G24, 112H5, and 151B17) covering 612 kb of the distal IGHV cluster, which, together with probes covering the IGHC cluster (11771 and 998D24), could be used in interphase nuclei and metaphase chromosome analysis. A visual split of the IGHV and IGHC clusters indicating a translocation was analyzed by dual-color FISH in a series of 21 cell lines of different origins. Translocations were found, as expected, in eight of eight myelomas, four of four lymphomas, none of five leukemias, and none of four Epstein-Barr virus-transformed B-lymphoblastoid cell lines. To summarize, we have established a set of IGHV and IGHC probes that can be used for universal screening of illegitimate rearrangement within the IGH locus in B-cell malignancies. These probes allow for routine FISH analysis to detect this early central oncogenic event.

MECP2 mutations in Danish patients with Rett syndrome: high frequency of mutations but no consistent correlations with clinical severity or with the X chromosome inactivation pattern.

Rett syndrome (RTT) is a neurodevelopmental disorder, which almost exclusively affects girls, who, after an initial period of apparently normal development, display gradual loss of speech and purposeful hand use, gait abnormalities and stereotypical hand movements. In the year 2000, mutations in the gene for the methyl CpG binding protein 2, MECP2, have been identified in 35-80% of the patients in three different studies. We have identified 15 different MECP2 mutations in 26 of 30 Danish RTT patients. The mutations included five novel mutations (one point mutation, three smaller deletions involving identical regions in the gene, and one duplication). In contrast to the point mutations and the duplication, which all affect the methyl binding domain or the transcriptional repressing domain, the three overlapping deletions are clustered in the 3' end of the gene. We found no consistent correlation between the type of mutation and the clinical presentation of the patient or the X-inactivation pattern in peripheral blood. Our high mutation detection rate, compared to two of the previous studies, underscores the importance of the inclusion criteria of the patients and supports that MECP2 is the most important, if not the only, gene responsible for RTT.

Two cases of partial trisomy 10q syndrome due to a familial 10;20 translocation.

We describe an eleven day-old boy and his first degree double cousin who both have distal trisomy 10q syndrome. Their cytogenetic analysis using GTG-banding showed an unbalanced translocation 46, XY, -20, +der(20), t(10;20)(q22.3, p11) mat and 46, XX, -20, +der(20), t(10;20)(q22.3, p11) mat. The translocation was confirmed by FISH. We have found balanced translocation t(10;20)(q22.3; p11) with cytogenetic and FISH studies in the mothers and maternal grandfather of these children. Our cases had typical craniofacial and visceral anomalies of this syndrome. However case 1 had an agenesia of corpus callosum which was not previously described and case 2 had hypertrophied cardiomyopathy and cliteromegaly which were previously described as rare anomalies for this syndrome.

Molecular cytogenetic detection of 9q34 breakpoints associated with nail patella syndrome.

The nail patella syndrome (NPS1) is an autosomal dominant disorder characterised by dysplasia of the finger nails and skeletal abnormalities. NPS1 has been mapped to 9q34, to a 1 cM interval between D9S315 and the adenylate kinase gene (AK1). We have mapped the breakpoints within the candidate NPS1 region in two unrelated patients with balanced translocations. One patient [46,XY,t(1;9)(q32.1;q34)] was detected during a systematic survey of old cytogenetic files in Denmark and southern Sweden. The other patient [46,XY,t(9;17)(q34.1;q25)] was reported previously. D9S315 and AK1 were used to isolate YACs, from which endclones were used to isolate PACs. Two overlapping PAC clones span the 9q34 breakpoints in both patients, suggesting that NPS1 is caused by haploinsufficiency due to truncation or otherwise inactivation of a gene at or in the vicinity of the breakpoints.

Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel.

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated K+ channels exhibiting weak inward rectification (30 and 11 pS at -100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability (PK/PX) sequence K+ = Rb+ (1.0) > Cs+ (10.4) >> Na+, Li+, N-methyl-D-glucamine (>51). NH+4 blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 microM), and cetiedil (IC50 79 microM). Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 of 74 microM.

Not para-, not peri-, but centric inversion of chromosome 12.

A 39 year old male with primary infertility was diagnosed as having Klinefelter syndrome by conventional cytogenetic analysis, which also showed an abnormal chromosome 12. Fluorescence in situ hybridisation (FISH) analysis of the aberrant chromosome using a 12 specific centromeric probe showed a break in the alphoid repeats followed by an inversion within the short arm, resulting in a pseudodicentric chromosome. Further FISH analyses using telomeric and subtelomeric probes showed that the other breakpoint was in the subtelomeric region of the short arm. The karyotype is designated 47,XXY,inv(12)(p10p13.3). To our knowledge this is the first report of a case of "centric inversion".

The human intrinsic factor-vitamin B12 receptor, cubilin: molecular characterization and chromosomal mapping of the gene to 10p within the autosomal recessive megaloblastic anemia (MGA1) region.

Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460-kD receptor, cubilin, present in the epithelium of intestine and kidney. Surface plasmon resonance analysis of ligand-affinity-purified human cubilin demonstrated a high-affinity calcium- and cobalamin-dependent binding of IF-cobalamin. Complete cDNA cloning of the human receptor showed a 3597 amino acid peripheral membrane protein with 69% identity to rat cubilin. Amino-terminal sequencing of the receptor indicates that the cDNA sequence encodes a precursor protein undergoing proteolytic processing due to cleavage at a recognition site (Arg7-Glu8-Lys9-Arg) for the trans-Golgi proteinase furin. Using fluorescence in situ hybridization, radiation hybrid mapping, and screening of YAC clones, the human cubilin gene was mapped between the markers D10S1661 and WI-5445 on the short arm of chromosome 10. This is within the autosomal recessive megaloblastic anemia (MGA1) 6-cM region harboring the unknown recessive-gene locus of juvenile megaloblastic anemia caused by intestinal malabsorption of cobalamin (Imerslund-Gräsbeck's disease). In conclusion, the present molecular and genetic information on human cubilin now provides circumstantial evidence that an impaired synthesis, processing, or ligand binding of cubilin is the molecular background of this hereditary form of megaloblastic anemia.

Characterization of a supernumerary small marker X chromosome in two females with similar phenotypes.

We describe two female patients mosaic for a cell line with an extra marker X chromosome in addition to a normal 46,XX cell line. To our knowledge, these cases are the first reports of females who had a cell line with a supernumerary marker X chromosome in addition to a normal cell line. They also had strikingly similar manifestations, including small hands and feet, minor facial anomalies, obesity, and mental retardation. The DNA content of the mar(X) chromosomes was investigated by fluorescent in situ hybridization using pericentromeric probes. The XIST gene, which is necessary for initiation of X-inactivation, was deleted from both marker chromosomes, suggesting that these chromosomes were not subject to inactivation. The short arm breakpoints of the mar(X)s were between the DNA markers DXS423E on Xp11.21 and UBE1 on Xp11.23. In Patient 1, mar(X) contained the androgen receptor gene and the DNA marker DXS1, both mapping to Xq11.2, whereas in Patient 2 the chromosome breakpoint was proximal to these markers. We suggest that the similar phenotypes of these patients may be due to the overexpression of genes in the common pericentromeric region of the X chromosome.

A small supernumerary marker chromosome X identified by in situ hybridization.

Cytogenetic analysis of a girl with moderate mental retardation and dysmorphic features revealed a 46,XX/47,XX,+mar karyotype. Fluorescence in situ hybridization using chromosome specific alpha satellite probes showed that the supernumerary marker originated from the X chromosome. To our knowledge, this is the first reported case of a female patient mosaic for a supernumerary small marker chromosome derived from X, and hence mosaic for trisomy of the pericentric region of the X chromosome.