Constitutional Haploinsufficiency of Tumor Suppressor Genes in Mentally Retarded Patients With Microdeletions in 17p13.1

Chromosome microdeletions or duplications are detected in 10-20% of patients with mental impairment and normal karyotypes. A few cases have been reported of mental impairment with microdeletions comprising tumor suppressor genes. By array-CGH we detected 4 mentally impaired individuals carrying de novo microdeletions sharing an overlapping segment of similar to 180 kb in 17p13.1. This segment encompasses 18 genes, including 3 involved in cancer, namely KCTD11/REN, DLG4/PSD95, and GPS2. Furthermore, in 2 of the patients, the deletions also included TP53, the most frequently inactivated gene in human cancers. The 3 tumor suppressor genes KCTD11, DLG4, and GPS2, in addition to the GABARAP gene, have a known or suspected function in neuronal development and are candidates for causing mental impairment in our patients. Among our 4 patients with deletions in 17p13.1, 3 were part of a Brazilian cohort of 300 mentally retarded individuals, suggesting that this segment may be particularly prone to rearrangements and appears to be an important cause (similar to 1%) of mental retardation. Further, the constitutive deletion of tumor suppressor genes in these patients, particularly TP53, probably confers a significantly increased lifetime risk for cancer and warrants careful oncological surveillance of these patients. Constitutional chromosome deletions containing tumor suppressor genes in patients with mental impairment or congenital abnormalities may represent an important mechanism linking abnormal phenotypes with increased risks of cancer. Copyright (C) 2009 S. Karger AG, Basel

Non-homologous sex chromosomes in two species of the genus Eigenmannia (Teleostei : Gymnotiformes)

The Neotropical genus Eigenmannia is a fish group with unknown species diversity where representatives possess a broad range of chromosomal sex determining systems namely XY/XX, X1X2Y/X1X1X2X2, ZZ/ZW as well as homomorphic sex chromosomes. To test the homology of two heteromorphic XY sex chromosome systems present in two sympatric populations, reciprocal cross-species FISH experiments were performed using probes derived by microdissection of X and Y chromosomes present in analyzed specimens of Eigenmannia virescens and Eigenmannia sp.2, respectively. While X and Y paint probes hybridized to species-specific sex chromosomes, in reciprocal cross-FISH both probes hybridized exclusively to autosomes. The result suggests multiple independent origins of the XY systems in the analyzed populations. Copyright (C) 2008 S. Karger AG, Basel.

Copy number variants on the X chromosome in women with primary ovarian insufficiency

Objective: To investigate whether submicroscopic copy number variants (CNVs) on the X chromosome can be identified in women with primary ovarian insufficiency (POI), defined as spontaneous secondary amenorrhea before 40 years of age accompanied by follicle-stimulating hormone levels above 40 IU/L on at least two occasions. Design: Analysis of intensity data of single nucleotide polymorphism (SNP) probes generated by genomewide Illumina 370k CNV BeadChips, followed by the validation of identified loci using a custom designed ultra-high-density comparative genomic hybridization array containing 48,325 probes evenly distributed over the X chromosome. Setting: Multicenter genetic cohort study in the Netherlands. Patient(s): 108 Dutch Caucasian women with POI, 97 of whom passed quality control, who had a normal karyogram and absent fragile X premutation, and 235 healthy Dutch Caucasian women as controls. Intervention(s): None. Main Outcome Measure(s): Amount and locus of X chromosomal microdeletions or duplications. Result(s): Intensity differences between SNP probes identify microdeletions and duplications. The initial analysis identified an overrepresentation of deletions in POI patients. Moreover, CNVs in two genes on the Xq21.3 locus (i.e., PCDH11X and TGIF2LX) were statistically significantly associated with the POI phenotype. Mean size of identified CNVs was 262 kb. However, in the validation study the identified putative Xq21.3 deletions samples did not show deviations in intensities in consecutive probes. Conclusion(s): X chromosomal submicroscopic CNVs do not play a major role in Caucasian POI patients. We provide guidelines on how submicroscopic cytogenetic POI research should be conducted. (Fertil Steril (R) 2011;95:1584-8. (C) 2011 by American Society for Reproductive Medicine.)

Deletion 5q12: Delineation of a Phenotype Including Mental Retardation and Ocular Defects

Array-CGH enables the detection of submicroscopic chromosomal deletions and duplications and leads to an accurate delineation of the imbalances, raising the possibility of correlating genotype to phenotype and mapping minimal critical regions associated with particular patterns of clinical features. We report here on four patients sharing common clinical features (psychomotor retardation, coarse facies and ocular anomalies), with proximal 5q deletions identified by oligo array-CGH. The deletions range from 5.75 to 17.26-Mb in size and occurred de novo. A common 2.63-Mb region between the deletions described here can be defined in 5q12.1 (59,390,122-62,021,754 bp bp from 5pter, hg18) and includes 12 genes. Among them, KIF2A, which encodes a kinesin superfamily protein, is a particularly interesting candidate for the phenotype, as it suppresses the growth of axonal collateral branches and is involved in normal brain development. Ocular defects, albeit unspecific, seem to be common in the 5q12.1 deletion. Identification of additional cases of deletions involving the 5q12.1 region will allow more accurate genotype-phenotype correlations. (C) 2011 Wiley-Liss, Inc.

Comparative Cytogenetics and Molecular Phylogeography in the Group Astyanax altiparanae - Astyanax aff. bimaculatus (Teleostei, Characidae)

The genus Astyanax comprises small characin fish of the neotropical region. The so-called `yellow-tailed characins` compose one of the most widely distributed Astyanax groups. A. altiparanae and A. aff. bimaculatus, are evolutionarily closely related and commonly found in several Brazilian hydrographic basins. In the present work, chromosomal data of specimens of A. altiparanae and A. aff. bimaculatus from 4 hydrographic basins in the states of Sao Paulo (Upper Tiete, Paranapanema, Ribeira de Iguape) and Rio de Janeiro (Guapimirim) are shown. All the populations showed 50 chromosomes, with different karyotypic formula. Although only a single Ag-NOR bearing chromosome pair was observed, all populations possess multiple cistrons of 18S rDNA. FISH with the 5S rDNA probe showed single signals at the interstitial position of one metacentric chromosome pair. C-bands are distributed in the terminal and interstitial regions of several chromosomes. However, the As-51 satDNA are frugally located in a few chromosomes of fishes from Upper Tiete, Paranapanema and Guapimirim Rivers, being absent in individuals of A. aff. bimaculatus from Ribeira de Iguape River basin. Beside these 4 populations, molecular phylogeography studies were also performed in individuals from Middle and Lower Tiete River basin and from 2 additional collection sites in the Paranapanema and Ribeira de Iguape River basins. The phylogeographic analysis using 2 mtDNA regions (totalizing 1.314 bp of ND2 and ATPase6/8 genes) of 8 populations of the group of `yellow-tailed characins` from 3 major hydrographic basins showed structuring of populations, suggesting a correlation between chromosomal (nuclear) and molecular (mitochondrial) data. Copyright (C) 2011 S. Karger AG, Basel

On the Relationship between Zaprionus spinipilus Chassagnard & McEvey and Z. vittiger Coquillett, the Type Species of the Genus Zaprionus (Diptera: Drosophilidae)

Zaprionus vittiger Coquillett is the type species of the genus Zaprionus Coquillett. However, the species is only known from five old museum specimens collected from South Africa and Malawi. It has often been confused with many other Zaprionus species, especially with Z. spinipilus Chassagnard & McEvey, a widespread species in Africa known from Madagascar, Malawi, Ethiopia and Cameroon. We have recently collected flies from the type localities of both species (South Africa and Madagascar, respectively). This has prompted us to test the taxonomic boundaries of these two nominal species using molecular (the mitochondrial COII and the nuclear Amyrel genes), chromosomal, morphological (internal and external genitalia), and reproductive isolation analyses. The results suggest Z. spinipilus to be a junior synonym to Z. vittiger.

Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed ""RaTART"" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.

Chromosome imbalances in syndromic hearing loss

The cause of hearing impairment has not been elucidated in a large proportion of patients. We screened by 1-Mb array-based comparative genomic hybridization (aCGH) 29 individuals with syndromic hearing impairment whose clinical features were not typical of known disorders. Rare chromosomal copy number changes were detected in eight patients, four de novo imbalances and four inherited from a normal parent. The de novo alterations define candidate chromosome segments likely to harbor dosage-sensitive genes related to hearing impairment, namely 1q23.3-q25.2, 2q22q23, 6p25.3 and 11q13.2-q13.4. The rare imbalances also present in normal parents might be casually associated with hearing impairment, but its role as a predisposition gene remains a possibility. Our results show that syndromic deafness is frequently associated with chromosome microimbalances (14-27%), and the use of aCGH for defining disease etiology is recommended.

Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.

Astyanax bockmanni Vari and Castro, 2007: an ambiguous karyotype in the Astyanax genus

Despite the widespread distribution of Astyanax bockmanni in streams from Upper Parana River system in central, southeastern, and southern Brazil, just recently, it has been identified as a distinct Astyanax species. Cytogenetic studies were performed in two populations of this species, revealing conservative features. A. bockmanni shows 2n = 50 chromosomes, a karyotypic formula composed of 10 M + 12SM + 12ST + 16A and multiple Ag-NORs. Eight positive signals in subtelocentric/acrocentric chromosomes were identified by fluorescent in situ hybridization (FISH) with 18S rDNA probes. After FISH with 5S rDNA probes, four sites were detected, comprising the interstitial region of a metacentric pair and the terminal region on long arms of another metracentric pair. Little amounts of constitutive heterochromatin were observed, mainly distributed at distal region in two chromosomal pairs. Additionally, heterochromatin was also located close to the centromeres in some chromosomes. No positive signals were detected in the chromosomes of A. bockmanni by FISH with the As-51 satellite DNA probe. The studied species combines a set of characteristics previously identified in two different Astyanax groups. The chromosomal evolution in the genus Astyanax is discussed.

Transcriptional regulation differs in affected facioscapulohumeral muscular dystrophy patients compared to asymptomatic related carriers

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder that has been associated with a contraction of 3.3-kb repeats on chromosome 4q35. FSHD is characterized by a wide clinical inter- and intrafamilial variability, ranging from wheelchair-bound patients to asymptomatic carriers. Our study is unique in comparing the gene expression profiles from related affected, asymptomatic carrier, and control individuals. Our results suggest that the expression of genes on chromosome 4q is altered in affected and asymptomatic individuals. Remarkably, the changes seen in asymptomatic samples are largely in products of genes encoding several chemokines, whereas the changes seen in affected samples are largely in genes governing the synthesis of GPI-linked proteins and histone acetylation. Besides this, the affected patient and related asymptomatic carrier share the 4qA161 haplotype. Thus, these polymorphisms by themselves do not explain the pathogenicity of the contracted allele. Interestingly, our results also suggest that the miRNAs might mediate the regulatory network in FSHD. Together, our results support the previous evidence that FSHD may be caused by transcriptional dysregulation of multiple genes, in cis and in trans, and suggest some factors potentially important for FSHD pathogenesis. The study of the gene expression profiles from asymptomatic carriers and related affected patients is a unique approach to try to enhance our understanding of the missing link between the contraction in D4Z4 repeats and muscle disease, while minimizing the effects of differences resulting from genetic background.

A Comparative Cytogenetic Analysis of 2 Bothriuridae Species and Overview of the Chromosome Data of Scorpiones

The order Scorpiones is one of the most cytogenetically interesting groups within Arachnida by virtue of the combination of chromosome singularities found in the 59 species analyzed so far. In this work, mitotic and meiotic chromosomes of 2 species of the family Bothriuridae were detailed. This family occupies a basal position within the superfamily Scorpionoidea. Furthermore, review of the cytogenetic data of all previously studied scorpions is presented. Light microscopy chromosome analysis showed that Bothriurus araguayae and Bothriurus rochensis possess low diploid numbers compared with those of species belonging to closely related families. Gonadal cells examined under light and in transmission electron microscopy revealed, for the first time, that the Bothriuridae species possess typical monocentric chromosomes, and male meiosis presented chromosomes with synaptic and achiasmatic behavior. Moreover, in the sample of B. araguayae studied, heterozygous translocations were verified. The use of techniques to highlight specific chromosomal regions also revealed additional differences between the 2 Bothriurus species. The results herein recorded and the overview elaborated using the available cytogenetic information of Scorpiones elucidated current understanding regarding the processes of chromosome evolution that have occurred in Bothriuridae and in Scorpiones as a whole.

Conventional and ultrastructural analyses of the chromosomes of Discocyrtus pectinifemur (Opiliones, Laniatores, Gonyleptidae)

Among the Opiliones, species of the suborders Cyphophthalmi, Eupnoi, Dyspnoi and Laniatores have shown very diverse diploid chromosome numbers. However, only certain Eupnoi species exhibit XY/XX and ZZ/ZW sex chromosome systems. Considering the scarcity of karyotypical information and the absence of structurally identifiable sex chromosomes in the suborder Laniatores, we decided to analyse the chromosomes and bivalents of Discocyrtus pectinifemur (Gonyleptidae) to identify possible sex differences. Testicular cells examined under light microscopy showed it high diploid number, 2n = 88, meta/submetacentric chromosome morphology and a nucleolar organizer region on pair 35. Prophase I microspreading observed in transmission electron microscopy exhibited 44 synaptonemal complexes with similar electron density and thickness. The total and regular synapsis between the chromosomes of the bivalents was also noted in pachytene nuclei. Male mitotic and meiotic chromosomes revealed no distinct characteristic that could be related to the occurrence of heteromorphic sex chromosomes. Evolutionary trends of chromosome differentiation in the four suborders of Opiliones are discussed here.

A novel locus for split-hand/foot malformation associated with tibial hemimelia (SHFLD syndrome) maps to chromosome region 17p13.1-17p13.3

Split-hand/foot malformation (SHFM) associated with aplasia of long bones, SHFLD syndrome or Tibial hemimelia-ectrodactyly syndrome is a rare condition with autosomal dominant inheritance, reduced penetrance and an incidence estimated to be about 1 in 1,000,000 liveborns. To date, three chromosomal regions have been reported as strong candidates for harboring SHFLD syndrome genes: 1q42.2-q43, 6q14.1 and 2q14.2. We characterized the phenotype of nine affected individuals from a large family with the aim of mapping the causative gene. Among the nine affected patients, four had only SHFM of the hands and no tibial defects, three had both defects and two had only unilateral tibial hemimelia. In keeping with previous publications of this and other families, there was clear evidence of both variable expression and incomplete penetrance, the latter bearing hallmarks of anticipation. Segregation analysis and multipoint Lod scores calculations (maximum Lod score of 5.03 using the LINKMAP software) using all potentially informative family members, both affected and unaffected, identified the chromosomal region 17p13.1-17p13.3 as the best and only candidate for harboring a novel mutated gene responsible for the syndrome in this family. The candidate gene CRK located within this region was sequenced but no pathogenic mutation was detected.

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.