DFNB16 is a frequent cause of congenital hearing impairment: implementation of STRC mutation analysis in routine diagnostics.

Increasing attention has been directed toward assessing mutational fallout of stereocilin (STRC), the gene underlying DFNB16. A major challenge is due to a closely linked pseudogene with 99.6% coding sequence identity. In 94 GJB2/GJB6-mutation negative individuals with non-syndromic sensorineural hearing loss (NSHL), we identified two homozygous and six heterozygous deletions, encompassing the STRC region by microarray and/or quantitative polymerase chain reaction (qPCR) analysis. To detect smaller mutations, we developed a Sanger sequencing method for pseudogene exclusion. Three heterozygous deletion carriers exhibited hemizygous mutations predicted as negatively impacting the protein. In 30 NSHL individuals without deletion, we detected one with compound heterozygous and two with heterozygous pathogenic mutations. Of 36 total patients undergoing STRC sequencing, two showed the c.3893A>G variant in conjunction with a heterozygous deletion or mutation and three exhibited the variant in a heterozygous state. Although this variant affects a highly conserved amino acid and is predicted as deleterious, comparable minor allele frequencies (MAFs) (around 10%) in NSHL individuals and controls and homozygous variant carriers without NSHL argue against its pathogenicity. Collectively, six (6%) of 94 NSHL individuals were diagnosed with homozygous or compound heterozygous mutations causing DFNB16 and five (5%) as heterozygous mutation carriers. Besides GJB2/GJB6 (DFNB1), STRC is a major contributor to congenital hearing impairment.

Synteny conservation of chicken macrochromosomes 1-10 in different avian lineages revealed by cross-species chromosome painting.

Cross-species chromosome painting can directly visualize syntenies between diverged karyotypes and, thus, increase our knowledge on avian genome evolution. DNA libraries of chicken (Gallus gallus, GGA) macrochromosomes 1 to 10 were hybridized to metaphase spreads of 9 different species from 3 different orders (Anseriformes, Gruiformes and Passeriformes). Depending on the analyzed species, GGA1-10 delineated 11 to 13 syntenic chromosome regions, indicating a high degree of synteny conservation. No exchange between the GGA macrochromosome complement and microchromosomes of the analyzed species was observed. GGA1 and GGA4 were distributed on 2 or 3 chromosomes each in some of the analyzed species, indicating rare evolutionary rearrangements between macrochromosomes. In all 6 analyzed species of Passeriformes, GGA1 was diverged on 2 macrochromosomes, representing a synapomorphic marker for this order. GGA4 was split on 2 chromosomes in most karyotypes, but syntenic to a single chromosome in blackcap (Passeriformes). GGA5/10 and also GGA8/9 associations on chromosomes were found to be important cytogenetic features of the Eurasian nuthatch (Passeriformes) karyotype. Fusion of GGA4 and GGA5 segments and of entire GGA6 and GGA7, respectively, was seen in the 2 analyzed species of Gruiformes. Consistent with the literature, our inter-species chromosome painting demonstrates remarkable conservation of macrochromosomal synteny over 100 million years of avian evolution. The low rate of rearrangements between macrochromosomes and the absence of detectable macrochromosome-microchromosome exchanges suggests a predominant role for rearrangements within the gene-dense microchromosome complement in karyotypic diversification.

Cross-species chromosome painting corroborates microchromosome fusion during karyotype evolution of birds.

The stone curlew, also known as thick-knee (Burhinus oedicnemus, BOE), represents a phylogenetically young species of the shorebirds (Charadriiformes) that exhibits one of the most atypical genome organizations known within the class of Aves, due to an extremely low diploid number (2n = 42) and only 6 pairs of microchromosomes in its complement. This distinct deviation from the 'typical' avian karyotype is attributed to repeated fusions of ancestral microchromosomes. In order to compare different species with this atypical avian karyotype and to investigate the chromosome rearrangement patterns, chromosome-specific painting probes representing the whole genome of the stone curlew were used to delineate chromosome homology between BOE and 5 species belonging to 5 different avian orders: herring gull (Charadriiformes), cockatiel (Psittaciformes), rock pigeon (Columbiformes), great gray owl (Strigiformes) and Eurasian coot (Gruiformes). Paints derived from the 20 BOE autosomes delimited 28 to 33 evolutionarily conserved segments in the karyotypes of the 5 species, similar to the number recognized by BOE paints in such a basal lineage as the chicken (28 conserved segments). This suggests a high degree of conservation in genome organization in birds. BOE paints also revealed some species-specific rearrangements. In particular, chromosomes BOE1-4 and 14, as well as to a large extent BOE5 and 6, showed conserved synteny with macrochromosomes, whereas homologous regions for BOE7-13 are found to be largely distributed on microchromosomes in the species investigated. Interestingly, the 6 pairs of BOE microchromosomes 15-20 appear to have undergone very few rearrangements in the 5 lineages investigated. Although the arrangements of BOE homologous segments on some chromosomes can be explained by complex fusions and inversions, the occurrence of homologous regions at multiple sites may point to fission of ancestral chromosomes in the karyotypes of the species investigated. However, the present results demonstrate that the ancestral microchromosomes most likely experienced fusion in the stone curlew lineage forming the medium-sized BOE chromosomes, while they have been conserved as microchromosomes in the other neoavian lineages.

Synteny conservation of the Z chromosome in 14 avian species (11 families) supports a role for Z dosage in avian sex determination.

In order to determine synteny conservation of the avian Z chromosome, a chicken (Gallus gallus, GGA) Z chromosome painting probe was hybridized to the chromosomes of 14 bird species belonging to 11 different families. The GGAZ painted the Z chromosomes in all species analyzed, suggesting strong conservation of its gene content among the different avian lineages. This was confirmed by the mapping of five GGAZ-orthologous genes (DMRT1, GHR, CHRNB3, ALDOB, B4GALT1) to the Z chromosomes of eight other species. The shuffled order of these genes on different Z chromosomes can be explained by the prevalence of intrachromosomal rearrangements during avian evolution. Synteny conservation of the mammalian X is generally thought to be the result of X chromosome inactivation. The absence of Z chromosome inactivation implies sex-specific dosage differences of a highly conserved array of Z-linked genes in birds. The evolutionary conservation of the entire Z chromosome among avian lineages supports the idea that avian sex determination and/or sex-specific functions are largely based on sex chromosome dosage. We propose that the accumulation of male-specific genes on the Z chromosome confers selective pressure on the Z to conserve its synteny.

Distribution of (TTAGGG)n telomeric sequences in karyotypes of the Xenopus species complex.

The chromosomal distribution of the conserved vertebrate telomeric (TTAGGG)(n) sequence was studied by fluorescence in situ hybridization (FISH) in four Xenopus species and the triploid Silurana tropicalis. As expected, hybridization signals were observed at the distal ends of every chromosome in all species. In addition, the hybridization pattern demonstrates varied organization of (TTAGGG)(n) sequences in the different karyotypes. In X. borealis and X. muelleri hybridization signals intensely labeled one end of a homologous chromosome pair that coincides with the sites containing ribosomal RNA gene clusters. The karyotype of X. clivii remarkably differs from other Xenopus karyotypes in displaying numerous interstitial telomeric sites (ITS). C-banding analysis shows that the non-telomeric sites appear to correspond to the interstitially located constitutive heterochromatin. This suggests that interstitial telomeric sites in X. clivii do not necessarily represent the relic of ancestral telomeres resulting from the fusion of chromosomes, but their occurrence is due to the fact that (TTAGGG)(n) repeat arrays may be a constituent of highly repetitive DNA.

Chromosome repatterning in three representative parrots (Psittaciformes) inferred from comparative chromosome painting.

Parrots (order: Psittaciformes) are the most common captive birds and have attracted human fascination since ancient times because of their remarkable intelligence and ability to imitate human speech. However, their genome organization, evolution and genomic relation with other birds are poorly understood. Chromosome painting with DNA probes derived from the flow-sorted macrochromosomes (1-10) of chicken (Gallus gallus, GGA) has been used to identify and distinguish the homoeologous chromosomal segments in three species of parrots, i.e., Agapornis roseicollis (peach-faced lovebird); Nymphicus hollandicus (cockatiel) and Melopsittacus undulatus (budgerigar). The ten GGA macrochromosome paints unequivocally recognize 14 to 16 hybridizing regions delineating the conserved chromosomal segments for the respective chicken macrochromosomes in these representative parrot species. The cross-species chromosome painting results show that, unlike in many other avian karyotypes with high homology to chicken chromosomes, dramatic rearrangements of the macrochromosomes have occurred in parrot lineages. Among the larger GGA macrochromosomes (1-5), chromosomes 1 and 4 are conserved on two chromosomes in all three species. However, the hybridization pattern for GGA 4 in A. roseicollis and M. undulatus is in sharp contrast to the most common pattern known from hybridization of chicken macrochromosome 4 in other avian karyotypes. With the exception of A. roseicollis, chicken chromosomes 2, 3 and 5 hybridized either completely or partially to a single chromosome. In contrast, the smaller GGA macrochromosomes 6, 7 and 8 displayed a complex hybridization pattern: two or three of these macrochromosomes were found to be contiguously arranged on a single chromosome in all three parrot species. Overall, the study shows that translocations and fusions in conjunction with intragenomic rearrangements have played a major role in the karyotype evolution of parrots. Our inter-species chromosome painting results unequivocally illustrate the dynamic reshuffling of ancestral chromosomes among the karyotypes of Psittaciformes.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

Extensive gross genomic rearrangements between chicken and Old World vultures (Falconiformes: Accipitridae).

The karyotypes of most birds consist of a small number of macrochromosomes and numerous microchromosomes. Intriguingly, most accipitrids which include hawks, eagles, kites, and Old World vultures (Falconiformes) show a sharp contrast to this basic avian karyotype. They exhibit strikingly few microchromosomes and appear to have been drastically restructured during evolution. Chromosome paints specific to the chicken (GGA) macrochromosomes 1-10 were hybridized to metaphase spreads of three species of Old World vultures (Gyps rueppelli, Gyps fulvus, Gypaetus barbatus). Paints of GGA chromosomes 6-10 hybridize only to single chromosomes or large chromosome segments, illustrating the existence of high chromosome homology. In contrast, paints of the large macrochromosomes 1-5 show split hybridization signals on the chromosomes of the accipitrids, disclosing excessive chromosome rearrangements which is in clear contrast to the high degree of chromosome conservation substantiated from comparative chromosome painting in other birds. Furthermore, the GGA chromosome paint hybridization patterns reveal remarkable interchromosomal conservation among the two species of the genus Gyps.

Susceptibility to the development of pigment cell tumors in a clone of the Amazon molly, Poecilia formosa, introduced through a microchromosome.

The Amazon molly Poecilia formosa is a gynogenetic fish that reproduces through the development of ameiotic diploid eggs triggered by insemination by males of related species without following karyogamie. This leads to clonal offspring. In rare cases, however, this gynogenesis is leaky, and paternal DNA in the form of small supernumerary chromosomes is included into the maternal genome. We have obtained a clone where one such microchromosome contains a pigmentary locus, resulting in macromelanophore pigmentation of the carrier. Approximately 5% of these fish spontaneously develop exophytic nodular or papillomatous pigment cell tumors. The tumors display considerable differences with respect to growth characteristics and invasiveness, despite the genetic uniformity of the affected animals. Following transplantation to syngeneic hosts, a remarkable clonal variability was observed. Oncogenes that are involved in tumorigenesis in hereditary melanoma of the closely related fish Xiphophorus appear not to be instrumental for induction of the P. formosa pigment cell tumors. Moreover, a new genetic locus is defined that mediates susceptibility to pigment cell tumor development and leads to transformation of chromatoblasts.

Comparative mapping of human claudin-1 (CLDN1) in great apes.

The gene encoding claudin-1 (CLDN1) has been mapped to human chromosome 3 (HSA3; 3q28-->q29) using a radiation hybrid panel. Employing fluorescence in situ hybridization (FISH) we here show that a human P1-derived artificial chromosome (PAC) containing CLDN1 detects the orthologous sites in chromosomes of the great apes, chimpanzee, gorilla, and orangutan. Furthermore, the chromosomal position of CLDN1 was determined in mouse chromosomes by FISH. The position of fluorescent signals is confined to a single chromosomal site in both great apes and mouse and in each case maps to the chromosomal region that has conserved synteny with HSA3 (PTR2q28, GGO2q28, PPY2q38 and MMU16B1). Using a gene-specific probe our results are consistent with reports of the striking similarity of great ape and human genomes as illustrated previously by chromosome painting.

In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells.

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS(61K) in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation.

The intron-containing ribosomal protein-encoding genes L5, L7a and L37a are unlinked in chicken.

Chicken ribosomal protein (rp)-encoding genes are currently being studied at the nucleotide level and three independent recombinant phages have been isolated from chicken genomic libraries using cloned cDNA probes. Each of these was shown to include an intron-containing rp gene of chicken (L5, L7a, L37a). In this study the chromosomal location of these three intron-containing rp from the large subunit of the chicken ribosome was determined by fluorescence in situ hybridization. L7a mapped to a microchromosome, whereas L5 and L37a mapped to macrochromosomes 8 and 7, respectively. The results demonstrate that these functionally related genes are widely dispersed in the genome. Furthermore, as in the case of many other evolutionarily advanced eukaryotes, there is no apparent linkage of rp and rRNA genes.

The genomic structure of the chicken ICSBP gene and its transcriptional regulation by chicken interferon.

The chicken interferon consensus sequence binding protein (ChICSBP) gene spans over 9 kb of DNA and consists, as its murine homolog, of nine exons. The first untranslated exon was identified by 5'-RACE technology. The second exon contains the translation initiation codon. Canonical consensus splice sites are found on every exon/intron junction. The introns are generally smaller than their mammalian counterparts. The ChICSBP and ChIRF-1 genes have been mapped by fluorescence in situ hybridization to different microchromosomes. The transcription start site has been mapped by primer extension. Inspection of the DNA sequence of a genomic clone containing the first exon and the region 1700-bp upstream revealed several potential cisregulatory elements of transcription. The ChICSBP mRNA is induced by recombinant ChIFN type I and ChIFN-gamma. A palindromic IFN regulatory element (pIRE) with high sequence homology to gamma activation site (GAS) sequences was functionally required in transient transfection assays for the induction of transcription by ChIFN-gamma.

Cloning of chicken interferon regulatory factor-2 (IRF-2) cDNA: expression and mapping of the IRF-2 gene.

A cDNA clone encoding a member of the avian interferon regulatory factor (IRF) family homologous to mammalian IRF-2 was isolated from cDNA library from poly[rI:rC]-induced chicken embryo fibroblasts (CEF). The deduced amino acid sequence shows a characteristic DNA binding domain of 124 amino acids at the amino-terminal end with 96.8% identity to human and 96% to mouse IRF-2. Identities in the C-terminal part are 77.5% and 77%, respectively. Identity to all other known members of the chicken IRF (Ch-IRF) family is distinctly lower. In C32 cells, an IRF-2 mRNA of 2.4 kb is constitutively expressed in very low amounts but is inducible by Ch-IFN in the absence or presence of cycloheximide. The Ch-IRF-2 gene is a single copy gene and was mapped by fluorescence in situ hybridization to the long arm of chromosome 4.

Sequence comparison of avian interferon regulatory factors and identification of the avian CEC-32 cell as a quail cell line.

Interferon (IFN) regulatory factor-1 (IRF-1) is a well-characterized member of the IRF family. Previously, we have cloned cDNA of several members of the chicken IRF (ChIRF) family and studied the function of ChIRF-1 in the avian cell line CEC-32. The IRF-1 proteins from primary chicken embryo fibroblasts (CEF) and CEC-32 cells differed in their electrophoretic mobility. To characterize the different forms of IRF-1 in avian cells, we compared the sequences of IRF-1 cDNA from CEC-32 cells, primary CEF, and quail fibroblasts (QEF). The deduced amino acid sequences of IRF-1 cDNA from chicken and quail show high similarity. Comparison of genomic sequences of IRF-1 and IFN consensus sequence binding protein (ICSBP) also confirm the relatedness of the members of the IRF family in quail and chicken. Based on these data, it is concluded that the avian fibroblast cell line CEC-32 is derived from quail. This conclusion is further supported by deoxynucleotide sequence comparison of a DNA fragment in an avian MHC class II gene and by fluorescence in situ hybridization (FISH) using the vertebrate telomeric (TTAGGG) repeat. Chromosome morphology and the lack of interstitial hybridization signals in macrochromosomes suggest that the CEC-32 cell line has probably been derived from Japanese quail.

Comparative mapping of Z-orthologous genes in vertebrates: implications for the evolution of avian sex chromosomes.

Sex chromosomes of birds and mammals are highly differentiated and share several cytological features. However, comparative gene mapping reveals extensive conserved synteny between the chicken Z sex chromosome and human chromosome 9 but not the human X sex chromosome, implying an independent origin of avian and mammalian sex chromosomes. To better understand the evolution of the avian Z chromosome we analysed the synteny of chicken Z-linked genes in zebrafish, which is the best-mapped teleost genome so far. Existing zebrafish maps do not support the existence of an ancestral Z linkage group in the zebrafish genome, whereas mammalian X-linked genes show at least some degree of synteny conservation. This is consistent with in situ hybridisation mapping data in the freshwater pufferfish, Tetraodon nigroviridis where mammalian X-linked genes show a much higher degree of conserved synteny than human chromosome 9 or the avian Z chromosome. Collectively, these data argue in favour of a more recent evolution of the avian Z chromosome, compared with the mammalian X.

Distribution and stability of supernumerary microchromosomes in natural populations of the Amazon molly, Poecilia formosa.

In animals, supernumerary chromosomes and their evolution have mostly been studied in sexual reproducing species. In the present study, for the first time, the natural distribution and stability of supernumerary microchromosomes were investigated in the unisexual fish species Poecilia formosa. Natural habitats throughout the range of P. formosa were screened for the presence of microchromosomes over several years. A high frequency of microchromosomes was found in the Río Purificación river system. Evidence points to the presence of the same microchromosome lineage over many generations. No supernumerary chromosomes were found elsewhere than in the Río Purificación representing a significant difference in the distribution of microchromosome-bearing individuals between the Río Purificación and all other collection sites.