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.

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.

Disruption of the ATE1 and SLC12A1 Genes by Balanced Translocation in a Boy with Non-Syndromic Hearing Loss.

We report on a boy with non-syndromic hearing loss and an apparently balanced translocation t(10;15)(q26.13;q21.1). The same translocation was found in the normally hearing brother, father and paternal grandfather; however, this does not exclude its involvement in disease pathogenesis, for example, by unmasking a second mutation. Breakpoint analysis via FISH with BAC clones and long-range PCR products revealed a disruption of the arginyltransferase 1 (ATE1) gene on translocation chromosome 10 and the solute carrier family 12, member 1 gene (SLC12A1) on translocation chromosome 15. SNP array analysis revealed neither loss nor gain of chromosomal regions in the affected child, and a targeted gene enrichment panel consisting of 130 known deafness genes was negative for pathogenic mutations. The expression patterns in zebrafish and humans did not provide evidence for ear-specific functions of the ATE1 and SLC12A1 genes. Sanger sequencing of the 2 genes in the boy and 180 GJB2 mutation-negative hearing-impaired individuals did not detect homozygous or compound heterozygous pathogenic mutations. Our study demonstrates the many difficulties in unraveling the molecular causes of a heterogeneous phenotype. We cannot directly implicate disruption of ATE1 and/or SLC12A1 to the abnormal hearing phenotype; however, mutations in these genes may have a role in polygenic or multifactorial forms of hearing impairment. On the other hand, it is conceivable that our patient carries a disease-causing mutation in a so far unidentified deafness gene. Evidently, disruption of ATE1 and/or SLC12A1 gene function alone does not have adverse effects.

A Boy with an LCR3/4-Flanked 10q22.3q23.2 Microdeletion and Uncommon Phenotypic Features.

The recurrent 10q22.3q23.2 deletion with breakpoints within low copy repeats 3 and 4 is a rare genomic disorder, reported in only 13 patients to date. The phenotype is rather uncharacteristic, which makes a clinical diagnosis difficult. A phenotypic feature described in almost all patients is a delay in speech development, albeit systematic studies are still pending. In this study, we report on a boy with an LCR3/4-flanked 10q22.3q23.2 deletion exhibiting an age-appropriate language development evaluated by a standardized test at an age of 2 years and 3 months. The boy was born with a cleft palate - a feature not present in any of the patients described before. Previously reported cases are reviewed, and the role of the BMPR1A gene is discussed. The phenotype of patients with an LCR3/4-flanked 10q22.3q23.2 deletion can be rather variable, so counseling the families regarding the prognosis of an affected child should be done with caution. Long-term studies of affected children are needed to delineate the natural history of this rare disorder.

Broad DNA methylation changes of spermatogenesis, inflammation and immune response-related genes in a subgroup of sperm samples for assisted reproduction.

Aberrant sperm DNA methylation patterns, mainly in imprinted genes, have been associated with male subfertility and oligospermia. Here, we performed a genome-wide methylation analysis in sperm samples representing a wide range of semen parameters. Sperm DNA samples of 38 males attending a fertility centre were analysed with Illumina HumanMethylation27 BeadChips, which quantify methylation of >27 000 CpG sites in cis-regulatory regions of almost 15 000 genes. In an unsupervised analysis of methylation of all analysed sites, the patient samples clustered into a major and a minor group. The major group clustered with samples from normozoospermic healthy volunteers and, thus, may more closely resemble the normal situation. When correlating the clusters with semen and clinical parameters, the sperm counts were significantly different between groups with the minor group exhibiting sperm counts in the low normal range. A linear model identified almost 3000 CpGs with significant methylation differences between groups. Functional analysis revealed a broad gain of methylation in spermatogenesis-related genes and a loss of methylation in inflammation- and immune response-related genes. Quantitative bisulfite pyrosequencing validated differential methylation in three of five significant candidate genes on the array. Collectively, we identified a subgroup of sperm samples for assisted reproduction with sperm counts in the low normal range and broad methylation changes (affecting approximately 10% of analysed CpG sites) in specific pathways, most importantly spermatogenesis-related genes. We propose that epigenetic analysis can supplement traditional semen parameters and has the potential to provide new insights into the aetiology of male subfertility.

Deep intronic 'mutations' cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA.

BACKGROUND: In a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and non-genetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA. METHODS: We determined FVIII and VWF activity and antigen levels and performed VWF-FVIII binding (VWF:FVIIIB) and VWF-collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long-range PCRs (LR-PCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real-time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively. RESULTS: All VWF tests were normal. The LR-PCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT-PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons. CONCLUSION: By systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.

Live-born trisomy 22: patient report and review.

Trisomy 22 is a common trisomy in spontaneous abortions. In contrast, live-born trisomy 22 is rarely seen due to severe organ malformations associated with this condition. Here, we report on a male infant with complete, non-mosaic trisomy 22 born at 35 + 5 weeks via caesarean section. Peripheral blood lymphocytes and fibroblasts showed an additional chromosome 22 in all metaphases analyzed (47,XY,+22). In addition, array CGH confirmed complete trisomy 22. The patient's clinical features included dolichocephalus, hypertelorism, flattened nasal bridge, dysplastic ears with preauricular sinuses and tags, medial cleft palate, anal atresia, and coronary hypospadias with scrotum bipartitum. Essential treatment was implemented in close coordination with the parents. The child died 29 days after birth due to respiratory insufficiency and deterioration of renal function. Our patient's history complements other reports illustrating that children with complete trisomy 22 may survive until birth and beyond.

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.

Sex determination and sex chromosome evolution: insights from medaka.

Among fish that exhibit a variety of sex-determining systems, medaka (Oryzias latipes) has been used as an ideal model to study sex determination and differentiation. Medaka sex differentiation starts with germ cell migration and proliferation during development, and the earliest sexual difference can be seen in germ cell number at stage 38 (1 day before hatching). Differentiation continues in young larvae. Medaka has an XX-XY genetic system, and the male sex-determining gene dmrt1bY (dmy) has been identified. Along with a sister species O. curvinotus, these species are the only 2 fish species whose sex-determining gene has been identified so far. The medaka sex chromosome is of recent origin, and the Y chromosome was formed through generation of the Y-specific region by duplication and insertion of an autosomal region into the proto-Y chromosome. Thus, the dmrt1bY gene is a duplicate of the autosomal dmrt1a gene. This event is estimated to have occurred about 10 million years ago. Sex-determining systems of other Oryzias species are under investigation, and a variety of sex-determining systems seem to exist. These findings may provide new insights into the sex-determining system of medaka.

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.

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.

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.

Governing sex determination in fish: regulatory putsches and ephemeral dictators.

In contrast to the rather stable regulatory regimes established over more that 100 million years in birds and mammals, sex determination in fish might frequently undergo evolutionary changes bringing the sex-determining cascade under new master sex regulators. This phenomenon, possibly associated with the emergence of new sex chromosomes from autosomes, would explain the frequent switching between sex determination systems observed in fish. In the medaka Oryzias latipes, the Y-specific master sex-determining gene dmrt1bY has been formed through duplication of the autosomal gene dmrt1 onto another autosome, thus generating a new Y chromosome. Dmrt1bY emerged about 10 million years ago and is restricted to several Oryzias species, indicating that the Y chromosome of the medaka is evolutionarily much younger than mammalian and bird sex chromosomes. Fertile males without dmrt1bY have been detected in some medaka populations, and this gene might even have been inactivated in one Oryzias species, indicating the existence of sexual regulators already able to supplant dmrt1bY. Studies on other models have confirmed that fish sex chromosomes are generally young and occurred independently in different fish lineages. The identification of new sex-determining genes in these species will shed new light on the exceptional evolutionary instability governing sex determination in fish.

Cytogenetics of the bleak (Alburnus alburnus), with special emphasis on the B chromosomes.

Some of the largest B chromosomes so far discovered in vertebrates are present in the cyprinid fish Alburnus alburnus. Previous cytogenetic analyses revealed a diploid chromosome number of 2n = 50. In addition, in some individuals one or two unusually large B chromosomes are present. Two morphologically different types of B chromosomes were observed. The frequency of animals bearing a supernumerary chromosome was found to vary considerably between different populations. A more detailed analysis of the A and B chromosomes of A. alburnus by conventional banding techniques, as well as fluorescence in-situ hybridization (FISH) with the telomeric DNA repeats (GGGTTA)7/(TAACCC)7, 18S + 28S rDNA and 5S rDNA were performed in the present study. Furthermore, a B chromosome-specific DNA probe obtained by amplified length polymorphism (AFLP) was hybridized on metaphases of A. alburnus carrying supernumerary B chromosomes. The banding analyses showed that the B chromosomes are completely heterochromatic, consist of GC-rich DNA sequences, replicate their DNA in the very late S-phase of the cell cycle and are composed mainly of a specific retrotransposable DNA element. Finally, blood probes from A. alburnus were collected for DNA-flow cytometric measurements. It could be shown that the huge supernumerary chromosomes represent nearly 10% of the total genome size of A. alburnus.

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.

Fanconi anemia: causes and consequences of genetic instability.

Fanconi anemia (FA) is a rare recessive disease that reflects the cellular and phenotypic consequences of genetic instability: growth retardation, congenital malformations, bone marrow failure, high risk of neoplasia, and premature aging. At the cellular level, manifestations of genetic instability include chromosomal breakage, cell cycle disturbance, and increased somatic mutation rates. FA cells are exquisitely sensitive towards oxygen and alkylating drugs such as mitomycin C or diepoxybutane, pointing to a function of FA genes in the defense against reactive oxygen species and other DNA damaging agents. FA is caused by biallelic mutations in at least 12 different genes which appear to function in the maintenance of genomic stability. Eight of the FA proteins form a nuclear core complex with a catalytic function involving ubiquitination of the central FANCD2 protein. The posttranslational modification of FANCD2 promotes its accumulation in nuclear foci, together with known DNA maintenance proteins such as BRCA1, BRCA2, and the RAD51 recombinase. Biallelic mutations in BRCA2 cause a severe FA-like phenotype, as do biallelic mutations in FANCD2. In fact, only leaky or hypomorphic mutations in this central group of FA genes appear to be compatible with life birth and survival. The newly discovered FANCJ (= BRIP1) and FANCM (= Hef ) genes correspond to known DNA-maintenance genes (helicase resp. helicase-associated endonuclease for fork-structured DNA). These genes provide the most convincing evidence to date of a direct involvement of FA genes in DNA repair functions associated with the resolution of DNA crosslinks and stalled replication forks. Even though genetic instability caused by mutational inactivation of the FANC genes has detrimental effects for the majority of FA patients, around 20% of patients appear to benefit from genetic instability since genetic instability also increases the chance of somatic reversion of their constitutional mutations. Intragenic crossover, gene conversion, back mutation and compensating mutations in cis have all been observed in revertant, and, consequently, mosaic FA-patients, leading to improved bone marrow function. There probably is no other experiment of nature in our species in which causes and consequences of genetic instability, including the role of reactive oxygen species, can be better documented and explored than in FA.