Chromosomal breakpoints in a cohort of head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma (HNSCC) presents complex chromosomal rearrangements, however, the molecular mechanisms behind HNSCC development remain elusive. The identification of the recurrent chromosomal breakpoints could help to understand these mechanisms. Array-CGH was performed in HNSCC patients and the chromosomal breakpoints involved in gene amplification/loss were analyzed. Frequent breakpoints were clustered in chromosomes 12p, 8p, 3q, 14q, 6p, 4q, Xq and 8q. Chromosomes 6, 14, 3, 8 and X exhibited higher susceptibility to have breaks than other chromosomes. We observed that low copy repeat DNA sequences are localized at or flanking breakpoint sites, ranging from 0 to 200 bp. LINES, SINES and Simple Repeats were the most frequent repeat elements identified in these regions. We conclude that in our cohort specific peri-centromeric and telomeric regions were frequently involved in breakpoints, being the presence of low copy repeats elements one of the explanations for the common rearrangement events observed.

Next generation phenotyping in Emanuel and Pallister-Killian syndrome using computer-aided facial dysmorphology analysis of 2D photos.

High throughput approaches are continuously progressing and have become a major part of clinical diagnostics. Still, the critical process of detailed phenotyping and gathering clinical information has not changed much in the last decades. Forms of next generation phenotyping (NGP) are needed to increase further the value of any kind of genetic approaches, including timely consideration of (molecular) cytogenetics during the diagnostic quest. As NGP we used in this study the facial dysmorphology novel analysis (FDNA) technology to automatically identify facial phenotypes associated with Emanuel (ES) and Pallister-Killian Syndrome (PKS) from 2D facial photos. The comparison between ES or PKS and normal individuals expressed a full separation between the cohorts. Our results show that NPG is able to help in the clinic, and could reduce the time patients spend in diagnostic odyssey. It also helps to differentiate ES or PKS from each other and other patients with small supernumerary marker chromosomes, especially in countries with no access to more sophisticated genetic approaches apart from banding cytogenetics. Inclusion of more facial pictures of patient with sSMC, like isochromosome-18p-, cat-eye-syndrome or others may contribute to higher detection rates in future.

7q11.23 microduplication syndrome: neurophysiological and neuroradiological insights into a rare chromosomal disorder.

BACKGROUND: The phenotypical consequence of the heterozygous chromosome 7q11.23 interstitial microdeletion is the Williams-Beuren syndrome, a very well-known genetic multi-systemic disorder. Much less is known about the reverse condition, the heterozygous interstitial microduplication of 7q11.23 region. The first molecular cytogenetic description was published in 2005, and only after several years were the reported patients numerous enough to attempt a description of a common phenotype. METHOD: By using a broad multidisciplinary approach, we investigated 12 patients with this rare genetic anomaly. Ten of them harboured the duplication of the classical Williams-Beuren syndrome region and two a slightly larger duplication. Upon a detailed description of the clinical and psychological features, we used electroencephalography and magnetic resonance imaging to explore neurophysiological function and brain structures. RESULTS: We analysed the clinical, psychological, neuroradiological and neurophysiological features of 12 yet-unpublished individuals affected by this rare genetic anomaly, focusing specifically on the last two aspects. Several structural abnormalities of the central nervous system were detected, like ventriculomegaly, hypotrophic cerebellum, hypotrophic corpus callosum and hypoplastic temporal lobes. Although only one of 12 individuals suffered from seizures during childhood, three others had abnormal electroencephalography findings prominent in the anterior brain regions, without any visible seizures to date. CONCLUSION: Taken together, we enlarged the yet-underrepresented cohort in the literature of patients affected by 7q11.23 microduplication syndrome and shed further light on neuroradiological and neurophysiological aspects of this rare genetic syndrome.

Highly conserved Z and molecularly diverged W chromosomes in the fish genus Triportheus (Characiformes, Triportheidae).

The main objectives of this study were to test: (1) whether the W-chromosome differentiation matches to species' evolutionary divergence (phylogenetic concordance) and (2) whether sex chromosomes share a common ancestor within a congeneric group. The monophyletic genus Triportheus (Characiformes, Triportheidae) was the model group for this study. All species in this genus so far analyzed have ZW sex chromosome system, where the Z is always the largest chromosome of the karyotype, whereas the W chromosome is highly variable ranging from almost homomorphic to highly heteromorphic. We applied conventional and molecular cytogenetic approaches including C-banding, ribosomal DNA mapping, comparative genomic hybridization (CGH) and cross-species whole chromosome painting (WCP) to test our questions. We developed Z- and W-chromosome paints from T. auritus for cross-species WCP and performed CGH in a representative species (T. signatus) to decipher level of homologies and rates of differentiation of W chromosomes. Our study revealed that the ZW sex chromosome system had a common origin, showing highly conserved Z chromosomes and remarkably divergent W chromosomes. Notably, the W chromosomes have evolved to different shapes and sequence contents within ~15-25 Myr of divergence time. Such differentiation highlights a dynamic process of W-chromosome evolution within congeneric species of Triportheus.

X-autosome and X-Y Translocations in Female Carriers: X-chromosome Inactivation Easily Detectable by 5-ethynyl-2-deoxyuridine (EdU).

Here we report one new case each of an X-autosome translocation (maternally derived), and an X-Y-chromosome translocation. Besides characterizing the involved breakpoints and/or imbalances in detail by molecular cyto-genetics, also skewed X-chromosome inactivation was determined on single cell level using 5-ethynyl-2-deoxyuridine (EdU). Thus, we confirmed that the recently suggested EdU approach can be simply adapted for routine diagnostic use. The latter is important, as only by knowing the real pattern of the skewed X-chromosome inactivation, correct interpretation of obtained results and subsequent reliable genetic counseling, can be done.

BENIGN AND PATHOLOGICAL GAIN OR LOSS OF GENETIC MATERIAL ­ ABOUT MICROSCOPIC AND SUBMICROSCOPIC COPY NUMBER VARIATIONS (CNVs) IN HUMAN GENETICS.

Submicroscopic gains or losses of chromosomal material are known as copy number variations (CNVs). Such CNVs are either connected with a disease or can also be (much more frequently) just a manifestation of human's genetic range of variation. Besides cytogenetic visible copy number variations (CG-CNVs) first discovered as chromosomal heteromorphisms, and later e. g. as euchromatic variants (EVs), there are also submicroscopic CNVs (MG-CNVs). Especially the latter may be a headache for diagnostics as the same MG-CNV may be found in clinically healthy and diseased persons. A so-called two-hit model has been introduced to solve this puzzle. As this considers the number of CNVs present overall in a genome the question arises if CG-CNVs are considered enough in routine cytogenetic as well as MG-CNVs in array-comparative genomic hybridization analysis.

Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes.

Approximately 90 species in the genus Leporinus (Characiformes, Anostomidae) are known, and most of them do not present differentiated sex chromosomes. However, there is a group of 7 species that share a heteromorphic ZW sex system. In all of these species, the W chromosome is the largest one in the karyotype and is mostly heterochromatic. We investigated the distribution of several microsatellites in the genome of 4 Leporinus species that possess ZW chromosomes. Our results showed a very large accumulation of mostly microsatellites on the W chromosomes. This finding supports the presence of an interconnection between heterochromatinization and the accumulation of repetitive sequences, which has been proposed for sex chromosome evolution, and suggests that heterochromatinization is the earlier of the 2 processes. In spite of the common origin that has been proposed for W chromosomes in all of the studied species, the microsatellites followed different evolutionary trajectories in each species, which indicates a high plasticity for sex chromosome differentiation.

Branchio-otic syndrome caused by a genomic rearrangement: clinical findings and molecular cytogenetic studies in a patient with a pericentric inversion of chromosome 8.

Branchio-oto-renal (BOR) syndrome is an autosomal dominantly inherited developmental disorder, which is characterized by anomalies of the ears, the branchial arches and the kidneys. It is caused by mutations in the genes EYA1,SIX1 and SIX5. Genomic rearrangements of chromosome 8 affecting the EYA1 gene have also been described. Owing to this fact, methods for the identification of abnormal copy numbers such as multiplex ligation-dependent probe amplification (MLPA) have been introduced as routine laboratory techniques for molecular diagnostics of BOR syndrome. The advantages of these techniques are clear compared to standard cytogenetic and array approaches as well as Southern blot. MLPA detects deletions or duplications of a part or the entire gene of interest, but not balanced structural aberrations such as inversions and translocations. Consequently, disruption of a gene by a genomic rearrangement may escape detection by a molecular genetic analysis, although this gene interruption results in haploinsufficiency and, therefore, causes the disease. In a patient with clinical features of BOR syndrome, such as hearing loss, preauricular fistulas and facial dysmorphisms, but no renal anomalies, neither sequencing of the 3 genes linked to BOR syndrome nor array comparative genomic hybridization and MLPA were able to uncover a causative mutation. By routine cytogenetic analysis, we finally identified a pericentric inversion of chromosome 8 in the affected female. High-resolution multicolor banding confirmed the chromosome 8 inversion and narrowed down the karyotype to 46,XX,inv(8)(p22q13). By applying fluorescence in situ hybridization, we narrowed down both breakpoints on chromosome 8 and found the EYA1 gene in q13.3 to be directly disrupted. We conclude that standard karyotyping should not be neglected in the genetic diagnostics of BOR syndrome or other Mendelian disorders, particularly when molecular testing failed to detect any causative alteration in patients with a convincing phenotype.

Comparative cytogenetic mapping of rRNA genes among naked catfishes: implications for genomic evolution in the Bagridae family.

In the present study, the karyotype and chromosomal characteristics of 9 species of the Bagridae fish family were investigated using conventional Giemsa staining as well as dual-color fluorescence in situ hybridization to detect the 18S and 5S rDNA sites. In addition to describing the karyotype of several Bagridae catfishes, we established molecular cytogenetic techniques to study this group. The 9 species contained a diploid chromosomal number, varying from 50 (Pseudomystus siamensis) to 62 (Hemibagrus wyckii), while none contained heteromorphic sex chromosomes. 18S rDNA sites were detected in only 1 chromosomal pair among all species evaluated. However, 3 different patterns were observed for the distribution of the 5S rDNA: 2 sites were found in the genus Mystus and in P. siamensis, multiple sites were observed in the genus Hemibagrus, and a syntenic condition for the 18S and 5S rDNA sites was identified in H. wyckii. The extensive variation in the number and chromosomal position of rDNA clusters observed among these Bagridae species may be related to the intense evolutionary dynamics of rDNA-repeated units, which generates divergent chromosomal distribution patterns even among closely related species. In summary, the distribution of repetitive DNA sequences provided novel, useful information regarding the evolutionary relationships between Bagridae fishes.

A family with an inverted tandem duplication 5q22.1q23.2.

Here, we report a 3-year-old boy with short stature, developmental delay and mild facial dysmorphic signs. Karyotype analysis and array-CGH revealed a pure duplication 5q22.1q23.2 with a length of 14.25 Mb. As demonstrated by multicolor-fluorescence in situ hybridization, the duplicated segment was orientated in an inverted tandem manner. One of the 2 older half-brothers of the index patient was intellectually disabled and showed short stature as well. The mother of the siblings was only 149 cm in height. The affected half-brother as well as the mother of the siblings were tested positive for the same duplication. Duplications of the long arm of chromosome 5 are rare. There are 16 reported cases of different 5q segments with a pure duplication and no additional chromosomal imbalance. In order to refine the 5q-duplication phenotype, reported cases were recently classified in 3 groups on the basis of clinical findings and the involved chromosome segments. However, our case does not fit in any of these groups but is placed in the interjacent chromosomal area between 2 of these groups. Overall, this is the second reported family with a duplication of 5q22.1q23.2 and both families share phenotypic features like short stature, facial dysmorphic signs and speech delay. The reported family provides further information for delineating phenotype-genotype correlations of pure duplications of the 5q region.

Independent sex chromosome evolution in lower vertebrates: a molecular cytogenetic overview in the Erythrinidae fish family.

The Erythrinidae fish family is an excellent model for analyzing the evolution of sex chromosomes. Different stages of sex chromosome differentiation from homomorphic to highly differentiated ones can be found among the species of this family. Here, whole chromosome painting, together with the cytogenetic mapping of repetitive DNAs, highlighted the evolutionary relationships of the sex chromosomes among different erythrinid species and genera. It was demonstrated that the sex chromosomes can follow distinct evolutionary pathways inside this family. Reciprocal hybridizations with whole sex chromosome probes revealed that different autosomal pairs have evolved as the sex pair, even among closely related species. In addition, distinct origins and different patterns of differentiation were found for the same type of sex chromosome system. These features expose the high plasticity of the sex chromosome evolution in lower vertebrates, in contrast to that occurring in higher ones. A possible role of this sex chromosome turnover in the speciation processes is also discussed.

Clinical impact of somatic mosaicism in cases with small supernumerary marker chromosomes.

Somatic mosaicism is present in slightly more than 50% of small supernumerary marker chromosome (sSMC) carriers. Interestingly, non-acrocentric derived sSMC show mosaicism much more frequently than acrocentric ones. sSMC can be present in different mosaic rates, which may go below 5% of the studied cells. Also cryptic mosaicism can be present and mosaics may be differently expressed in different tissues of the body. Even though in the overwhelming majority of the cases somatic sSMC mosaicism has no direct clinical effect, there are also cases with altered clinical outcomes due to mosaicism. Also clinically important is the fact that a de novo sSMC, even present in mosaic, may be a hint of uniparental disomy (UPD). As it is under discussion to possibly replace standard karyotyping by methods like array-CGH, the impracticality of the latter to detect low-level sSMC mosaics and/or UPD has to be considered as well. Overall, sSMC mosaicism has to be studied carefully in each individual case, as it can be extremely informative and of importance, especially for prenatal genetic counseling.

Partial trisomy 2p and partial monosomy 2q arising from a paternal intrachromosomal 2q-into-2p between-arm insertion and paracentric inversion: molecular cytogenetic characterization of a four-break rearrangement.

We report on a 26-month-old boy with an interstitial duplication of 2p22.3p22.2 and an interstitial deletion of 2q14.1q21.2. The abnormality was derived from his father having a balanced paracentric inversion and pericentric insertion. The deletion in the child was identified by cytogenetic analysis and characterized in more detail by molecular cytogenetics and array comparative genomic hybridization. The latter revealed a 20-Mb deletion in the long arm and a 5.6-Mb duplication in the short arm of chromosome 2. Fluorescence in situ hybridization in paternal chromosomes characterized an intrachromosomal insertion of 2q14.1q21.2 into 2p23; additionally a paracentric inversion of 2p13p23 was observed. The boy with the unbalanced karyotype suffered from severe psychomotor retardation, thrombophilia due to protein C deficiency, and hypertrophic cardiomyopathy and also had phenotypic abnormalities. Most of these features have previously been described in individuals with interstitial deletion of 2q14.1.

A complex chromosome rearrangement involving four chromosomes, nine breakpoints and a cryptic 0.6-Mb deletion in a boy with cerebellar hypoplasia and defects in skull ossification.

Constitutional complex chromosomal rearrangements (CCRs) are considered rare cytogenetic events. Most apparently balanced CCRs are de novo and are usually found in patients with abnormal phenotypes. High-resolution techniques are unveiling genomic imbalances in a great percentage of these cases. In this paper, we report a patient with growth and developmental delay, dysmorphic features, nervous system anomalies (pachygyria, hypoplasia of the corpus callosum and cerebellum), a marked reduction in the ossification of the cranial vault, skull base sclerosis, and cardiopathy who presents a CCR with 9 breakpoints involving 4 chromosomes (3, 6, 8 and 14) and a 0.6-Mb deletion in 14q24.1. Although the only genomic imbalance revealed by the array technique was a deletion, the clinical phenotype of the patient most likely cannot be attributed exclusively to haploinsufficiency. Other events must also be considered, including the disruption of critical genes and position effects. A combination of several different investigative approaches (G-banding, FISH with different probes and SNP array techniques) was required to describe this CCR in full, suggesting that CCRs may be more frequent than initially thought. Additionally, we propose that a chain chromosome breakage mechanism may have occurred as a single rearrangement event resulting in this CCR. This study demonstrates the importance of applying different cytogenetic and molecular techniques to detect subtle rearrangements and to delineate the rearrangements at a more accurate level, providing a better understanding of the mechanisms involved in CCR formation and a better correlation with phenotype.

First report of a small supernumerary der(8;14) marker chromosome.

Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes, generally equal in size or smaller than a chromosome 20 of the same metaphase spread. Most of them are unexpectedly detected in routine karyotype analyses, and it is usually not easy to correlate them with a specific clinical picture. A small group of sSMCs is derived from more than one chromosome, called complex sSMCs. Here, we report on a patient with a de novo complex sSMC, derived from chromosomes 8 and 14. Banding karyotype analysis, multiplex ligation-dependent probe amplification (MLPA), single nucleotide polymorphism (SNP)-based array, and fluorescence in situ hybridization (FISH) were performed to investigate its origin. Array and FISH analyses revealed a der(14)t(8;14)(p23.2;q22.1)dn. The propositus presents some clinical features commonly found in patients with partial duplication or triplication of 8p and 14q. This is the first report describing a patient with a congenital der(14)t(8;14)(p23.2;q22.1)dn sSMC.

An unusual cytogenetic rearrangement originating from two different abnormalities in chromosome 6 in a child with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is usually associated with a favorable outcome, but about 10% of patients tend to relapse. The genetic hallmark of APL is a balanced translocation involving chromosomes 15 and 17, and the PML-RARa gene fusion is found in more than 90% of these cases. Other chromosomal abnormalities are commonly found in APL, but their clinical significance has yet to be determined. Here we report a case of childhood APL that was studied by conventional cytogenetics along with molecular cytogenetic techniques. The patient showed a complex karyotype with an unusual cytogenetic rearrangement originating from two different abnormalities in a single chromosome 6. Our case is an exceptional example of a cryptic cytogenetic anomaly in APL and underscores the importance of detailed genetic characterization.

Numerical aberrations of chromosome 17 and TP53 in brain metastases derived from breast cancer.

Breast cancer is the second most common origin of brain metastases, after lung cancer, and represents 14-20% of all cases. Abnormalities of chromosome 17 are important molecular genetic events in human breast cancer, and several oncogenes and tumor suppressor genes are located on this chromosome. In about half of all human cancers, the tumor suppressor gene TP53, located at 17p13.1, is either lost or mutated. Loss of p53 protein function influences not only cell cycle checkpoint controls and apoptosis, but also the regulation of other important stages of metastatic progression, such as cell migration and tissue invasion. The aim of our study was to identify numerical aberrations of chromosome 17 and TP53 in 5 subjects with brain metastasis from breast cancer using dual-color fluorescence in situ hybridization experiments. Deletion of TP53 was the most frequent alteration observed, suggesting that if this alteration is present in the primary tumors, breast tumors with loss of TP53 copies have a poorer prognosis and a higher chance for metastasis. If this is true, the analyses of chromosome 17 and TP53 in primary breast cancer could be important for predicting its metastatic potential.

Murine multicolor banding.

Multicolor banding approach, first introduced for human chromosomes only, was established as an optimal approach for karyotyping of murine chromosomes. Here we present the established mcb probe sets for all murine autosomes and the X-chromosome and review their potential application.

Molecular Cytogenetic Study of the NF2 Gene Deletion in Meningioma in Sudanese Patients.

Meningioma is the second most common adult central nervous system tumor. Mutations and/or deletions within the tumor suppressor gene neurofibromatosis type 2 (NF2) are associated with meningioma development and progression. We studied 29 meningioma samples by cytogenetic analysis and interphase fluorescence in situ hybridization (I-FISH) using a locus-specific probe for the NF2 gene region. We detected loss of the NF2 gene in all samples except for one. In 10 of the 29 samples, karyotypic analyses confirmed the I-FISH results and revealed additional numerical and/or structural rearrangements in nine of them. Our study confirmed: i) the limited role of banding cytogenetics in assessing chromosomal rearrangements in meningioma, as this tumor is hard to be grown in cell culture; ii) we could show that two-color I-FISH is well-suited for NF2-deletion screening. Our results were in accordance with those of comparable studies, even though the frequency of 97.0% of meningiomas with NF2 deletions is exceptionally high in the studied Sudanese patients.

Molecular Cytogenetic Characterization of an inv(Y)(p11.2q11.221∼q11.222) in a Syrian Family.

Constitutional chromosomal abnormalities are an important cause of miscarriage, infertility, congenital anomalies and mental retardation in humans. Pericentric inversions of the human Y-chromosome [inv(Y)] are rather common and show an estimated incidence of 0.6-1:1,000 in males in the general population. Most of the reported cases with inv(Y) are familial. For carriers of pericentric inversions the risk of mental retardation or multiple abortions is not apparently increased and there is no relation with abnormal phenotypic features. Polymerase chain reaction (PCR) analysis to detect microdeletions along the Y-chromosome as well as cytogenetic and fluorescence in situ hybridization (FISH) analysis were done to delineate the characteristics of an inv(Y) in a Syrian family. Thus, we present a detailed molecular-cytogenetic characterization of a father and his two sons having an inv(Y)(p11. 2q11.221∼q11.222) with varying mental retardation features but otherwise normal phenotype.