Phenotype-genotype discordance in congenital malformations with communication disorders resembling trisomy 18 (Edwards syndrome).

PATIENT: Female, 6 FINAL DIAGNOSIS: Phenotype-genotype discordance in congenital malformations with communication disorders resembling trisomy 18 (Edwards syndrome) Symptoms: - MEDICATION: - Clinical Procedure: - Specialty: Otolaryngology. OBJECTIVE: Congenital defects. BACKGROUND: Communication process disorders are very frequent in rare cases of chromosomal aberrations (deletions, insertions, and trisomies) such as Down syndrome (trisomy 21), Turner syndrome, Edwards syndrome (trisomy 18), or Patau syndrome (trisomy 13). Sometimes phenotype may delusively correspond to the characteristic features of a given syndrome, but genotype tests do not confirm its presence. CASE REPORT: We present the case of a 6-year-old girl admitted to the Clinic of Phoniatrics and Audiology for the assessment of communication in the course of congenital malformations with phenotype characteristic for trisomy 18 (Edwards syndrome). Immediately upon birth, dysmorphic changes suggesting trisomy 18 (Edwards syndrome) were observed, but trisomy 18 was excluded after karyotype test results were normal (46, XX). CONCLUSIONS: DISTURBED ARTICULATION WAS DIAGNOSED: deformed linguo-dental and palatal sounds, interdental realization with flat tongue of the /s/, /z/, /c/, /dz/, /s/, /z/, /c/, /dz/ sounds (sigmatismus interdentalis). Hearing loss was confirmed.

Esterase D and gamma 1 actin level might predict results of induction therapy in patients with acute myeloid leukemia without and with maturation.

Development of modern proteomic methods in recent years has opened also new perspectives in the identification of new biomarkers which ensure more effective diagnosis, treatment monitoring and prediction of therapeutic outcome. We evaluated usefulness of comparative proteomics (MALDI-TOF) in two subtypes of acute myeloid leukemia (AML), M1 and M2, according to FAB classification. The bone marrow or blood cell proteomes were examined in 33 newly diagnosed patients before "3 + 7" induction therapy, after treatment and when the disease relapsed. We found that bone marrow and peripheral mononuclear cells from healthy volunteers revealed a number of quantitative and qualitative differences between the two proteomes, reflecting differences in the maturational status of normal cells. Such differences were not detected in our AML M1/M2 patients. Additionally, we found 9 proteins, which are potential biomarkers differentiating between the AML patients and healthy volunteers. Using comparative proteomics, we found that annexin I, glutathione transferase omega, esterase D and gamma 1 actin had prognostic significance. Applying statistical methods, we detected two proteins which might allow to predict results of induction therapy in AML M1/M2. One of them was esterase D, the higher concentration of which was associated with higher complete remission rate, and the other was gamma 1 actin, the higher concentration of which was related to resistance. In the article, we also discussed the role of these two proteins in the biology of AML, and we suggested potential usefulness of modification in induction therapy reflecting the presence of proteins.

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q.

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10 µg/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.

Loss of protein expression and recurrent DNA hypermethylation of the GNG7 gene in squamous cell carcinoma of the head and neck.

Although down-regulation of GNG7 in cancer was reported before, its role in carcinogenesis is poorly understood. It belongs to a family of large G-proteins that may be involved in cell-contact-induced growth arrest and function in tumor suppression. In the present study, we stained immunohistochemically 188 tumors derived from larynx or floor of the mouth for GNG7 protein and confronted it with clinicopathologic data. Moreover, we performed bisulfite pyrosequencing to analyze GNG7 promoter methylation. We identified recurrent loss of GNG7 protein expression in 68/188 (36%) cases and promoter hypermethylation in (42/98; 43%) primary tumors, predominantly in young patients (p < 0.001). Loss of GNG7 expression correlated with hypermethylation of GNG7 promoter region (p < 0.001). Moreover, loss of GNG7 protein expression correlated with tumor size (p = 0.012) and lack of cervical metastasis (p = 0.02) whereas sustained expression correlated with keratinization (p = 0.008). Taken together, loss of GNG7 protein expression is a frequent event in head and neck cancer. Moreover, our data suggest that hypermethylation of the promoter region of GNG7 is probably the mechanism of the observed inactivation.

Pyrosequencing-based DNA methylation profiling of Fanconi anemia/BRCA pathway genes in laryngeal squamous cell carcinoma.

Fanconi anemia (FA) associated genes [FANCA, -B, -C, FANCD1(BRCA2), -D2, -E, -F, -G, -I, -L, -M, FANCN (PALB2), FANCJ(BRIP1) and FA-linked BRCA1] encode proteins of DNA damage response pathways mutated in FA patients. FA is characterized by congenital malformations, chromosomal instability and high cancer susceptibility. FA patients have a 500-700 times higher risk of head and neck squamous cell carcinoma (HNSCC) compared to the non-FA population. As DNA methylation comprises one of the known gene inactivation mechanisms in cancer we have investigated the methylation status of 13 FA and one FA-linked gene in order to assess the role of FA in sporadic laryngeal squamous cell carcinoma (LSCC) tumor samples. Thirteen laryngeal squamous carcinoma cell lines (UT-SCC) and 64 primary laryngeal carcinoma cases were analyzed by bisulfite pyrosequencing. DNA from buccal swabs of 10 healthy volunteers was used as a control group. Promoter regions of FANCA, BRCA1 and BRCA2 displayed recurrent alterations in the methylation levels in cancer samples as compared to buccal swabs controls. For FANCA, hypomethylation was observed in 11/13 cell lines (p<0.0003) and all 64 primary larynx samples (p<0.001) compared to buccal swabs. For BRCA1, 4/13 cell lines (p=0.04) and 3/58 primary laryngeal cases (p=0.22) showed hypomethylation. In BRCA2, all 13 cell lines (p<0.0001) 4/63 primary LSCC (p<0.01) showed hypermethylation as compared to controls. In conclusion, we show recurrent alterations of DNA methylation levels in three Fanconi anemia genes which might contribute to the pathogenesis of LSCC.

High resolution ArrayCGH and expression profiling identifies PTPRD and PCDH17/PCH68 as tumor suppressor gene candidates in laryngeal squamous cell carcinoma.

Many classical tumor suppressor genes (TSG) were identified by delineation of bi-allelic losses called homozygous deletions. To identify systematically homozygous deletions in laryngeal squamous cell carcinoma (LSCC) and to unravel novel putative tumor suppressor genes, we screened 10 LSCC cell lines using high resolution array comparative genomic hybridization (arrayCGH) and array based expression analysis. ArrayCGH identified altogether 113 regions harboring protein coding genes that showed strong reduction in copy number indicating a potential homozygous deletion. Out of the 113 candidate regions, 22 novel homozygous deletions that affected the coding sequences of 15 genes were confirmed by multiplexPCR. Three genes were homozygously lost in two cell lines: PCDH17/PCH68, PRR20, and PTPRD. For the 15 homozygously deleted genes, four showed statistically significant downregulation of expression in LSCC cell lines as compared with normal human laryngeal controls. These were ATG7 (1/10 cell line), ZMYND11 (BS69) (1/10 cell line), PCDH17/PCH68 (9/10 cell lines), and PTPRD (7/10 cell lines). Quantitative real-time PCR was used to confirm the downregulation of the candidate genes in 10 expression array-studied cell lines and an additional cohort of cell lines; statistical significant downregulation of PCDH17/PCH68 and PTPRD was observed. In line with this also Western blot analyses demonstrated a complete absence of the PCDH17 and PTPRD proteins. Thus, expression profiling confirmed recurrent alterations of two genes identified primarily by delineation of homozygous deletions. These were PCDH17/PCH68, the protocadherin gene, and the STAT3 inhibiting receptor protein tyrosine phosphatase gene PTPRD. These genes are good candidates for novel TSG in LSCC.

Frequent gene hypermethylation in laryngeal cancer cell lines and the resistance to demethylation induction by plant polyphenols.

Promoter hypermethylation is one of the mechanisms in the transcriptional inactivation of certain carcinoma - associated genes. In laryngeal cancers hypermethylation of tumor suppressor genes is related to their major risk factors- cigarette smoking and drinking strong alcohols. Since DNA methylation is reversible, modulation of the activity of DNA methyltransferases is an established therapeutic strategy, which can be also applied in cancer chemoprevention. Here, using the MSP procedure, we evaluated the frequency of hypermethylation of RARbeta, RASSF1A, HIN-1, GSTP1, MGMT, VHL and DAPK genes in several laryngeal and other head and neck squamous cell carcinoma cell lines and the effect of various polyphenols on the methylation of RARbeta and MGMT genes in the UT-SCC 42B cell line. Most of the cell lines tested were characterized by the hypermethylation of at least one of the genes analyzed. The most frequently hypermethylated genes were RARbeta and MGMT, while GSTP1 and VHL were not methylated in any of the cell lines. None of the tested compounds, including decitabine used as a reference compound, changed the methylation of RARbeta and MGMT genes. These findings suggest that although hypermethylation of RARbeta and MGMT may be considered as potential epigenetic biomarker, their application as therapeutic/chemopreventive targets requires further studies.

Megakaryocytic blast crisis in a chronic myeloid leukemia patient with a rare variant of Philadelphia rearrangement t(9;22;22) and a constitutional translocation t(3;7).

Megakaryocytic blast crisis occurs extremely rarely, accounting for <3% of cases of chronic myelogenous leukemia in blastic transformation. In chronic myeloid leukemia, a variant Philadelphia translocation is reported in 2-10% of cases. We report an unusual case of megakaryocytic blast crisis with the Philadelphia variant rearrangement t(9;22;22) and a constitutional translocation t(3;7). The breakpoint in the 22q13 region was involved in this translocation. The chromosome region 22q13 harbors MKL1 gene, which is engaged in a specific translocation associated with acute megakaryoblastic leukemia. Study of deregulation of these four genes could contribute to better understanding of the effects of the t(9;22;22) rearrangement in a megakaryocytic blast crisis.

Recurrent amplification in the 22q11 region in laryngeal squamous cell carcinoma results in overexpression of the CRKL but not the MAPK1 oncogene.

Thirteen laryngeal squamous cell carcinoma cell lines were recently studied by array comparative genomic hybridization (array-CGH) in order to identify recurrent DNA copy number alterations in the tumor genome. A highly amplified region 22q11.2 was found in two of the thirteen cell lines. Two established oncogenes CRKL and MAPK1 are localized in this region, but only CRKL was amplified in both cell lines. Therefore, to check if amplification of either CRKL or MAPK1 genes may be important in the pathogenesis of laryngeal squamous cell carcinoma, the DNA copy number and mRNA expression were measured in a cohort of 17 LSCC cell lines by quantitative real-time PCR (qPCR). For the CRKL gene gains of the copy number were found in 3/17 cell lines, while overexpression was found in 6/17 cell lines. Gains in the copy number for the MAPK1 gene were found in 1/17 cell lines, but overexpression was not detected in any cell line. A highly significant correlation between DNA copy number and expression for CRKL gene, but not for MAPK1 gene was established using the Pearson test. Thereafter, 46 primary samples of laryngeal cancer were tested by qPCR to check for possible gains in copy number of the CRKL gene. Gains were found in 3/46 cases. These results suggest that CRKL, but not MAPK1 is the target oncogene of the rare but recurrent amplification at 22q11.2 in laryngeal squamous cell carcinoma.

Characterization of homozygous deletions in laryngeal squamous cell carcinoma cell lines.

The majority of classical tumor suppressor genes, such as CDKN2A or RB1, were identified by delineation of biallelic losses called homozygous deletions. To systematically identify homozygous deletions in laryngeal squamous cell carcinoma and to unravel novel putative tumor suppressor genes we screened three laryngeal squamous cell carcinoma cell lines (LSCC) using array comparative genomic hybridization (array-CGH). Out of 31 candidate regions for homozygous deletions identified by array-CGH, 5 were verified further by PCR. Among others, these homozygous deletions affected the tumor suppressor gene CDKN2A and the apoptosis-inducing STK17A gene. To assess the frequency of the identified deletions we investigated the affected sites in 9 additional LSCC cell lines. In 5 of the 9 cell lines the CDKN2A gene was homozygously lost. Thus, CDKN2A was homozygously deleted in 7 of the 12 cell lines. No other recurrent homozygous deletions were found. Homozygous deletions was a frequent mechanism of CDKN2A inactivation. Moreover, we identified several other genes, including the putative tumor suppressor gene STK17A, which may be inactivated by homozygous deletions and thus are potentially implicated in laryngeal squamous cell carcinoma development.

[Genes associated with tobacco smoke-associated cancer of head and neck]. / Geny zwiazane z odtytoniowymi nowotworami glowy i szyi.

The article presents the current techniques used for the identification of genes involved in tobacco smoke-associated cancers. The focus is set on the techniques derived from the conventional cytogenetics and includes fluorescence in situ hybridization (FISH), comparative genomes hybridization (CGH) and its further improvement that is array-CGH, and other aspects of microarray DNA technology. The second part deals with the main findings concerning participation of oncogenes and tumor suppressor genes in development and progression of tobacco smoke-associated head and neck cancers.

The Evolution of satellite III DNA subfamilies among primates.

We demonstrate that satellite III (SatIII) DNA subfamilies cloned from human acrocentric chromosomes arose in the Hominoidea superfamily. Two groups, distinguished by sequence composition, evolved nonconcurrently, with group 2 evolving 16-23 million years ago (MYA) and the more recent group 1 sequences emerging approximately 4.5 MYA. We also show the relative order of emergence of each group 2 subfamily in the various primate species. Our results show that each SatIII subfamily is an independent evolutionary unit, that the rate of evolution is not uniform between species, and that the evolution within a species is not uniform between chromosomes.

[Chromosome alterations in tobacco smoke-associated tumors]. / Uszkodzenia chromosomów w tytoniopochodnych nowotworach.

A role of tobacco products in cancer incidence is commonly known and accepted. It is estimated that roughly 1/3 of all the cancers is resulted from previous exposure to tobacco. An impact of tobacco smoke carcinogens in formation of DNA lesions and mutations is well established. Contrary to that, less is known about rearrangements of chromosomes. Nevertheless, there are many indications associating rearrangements of chromosome arms 3p, 3q, 8q, 9p, 17p i 18q with a clastogenic activity of tobacco smoke. An evolution of cytogenetics from conventional techniques to molecular cytogenetics provides an opportunity to find some links between chromosome aberrations and activation of oncogenes as well as deactivation of tumor suppressor genes.

Cytogenetic analysis of cardiovascular disease: karyotyping.

Numerical and structural chromosomal rearrangements, such as aneuploidies, deletions, duplications, and other aberrations have been associated with congenital abnormalities, pregnancy loss, and malignancy. Detection of these genetic changes is possible by cytogenetic analysis. The karyotype is determined by analysis of metaphase or prometaphase chromosomes of peripheral blood lymphocytes after banding procedures. This analysis plays an important role in determining patient diagnosis and care. In this chapter, we describe the basic approach of cytogenetic analysis: arresting the cell in metaphase or prometaphase, the obtaining of metaphase chromosome spreads, and staining and chromosome analysis.

Comparative genomic hybridization by microarray for the detection of cytogenetic imbalance.

Chromosomal abnormalities often result in the improper dosage of genes in a particular chromosome or chromosome segment, which may cause specific and complex clinical phenotypes. Comparative genomic hybridization by microarray (array CGH) is a high-throughput and high-resolution method for the detection of microscopic and submicroscopic chromosome abnormalities, some of which may not be detectable by conventional cytogenetic techniques. In addition, with the human genome sequenced and publicly available, array CGH allows for the direct correlation between chromosomal anomalies and genomic sequence. Properly constructed, microarrays have the potential to be a valuable tool for the detection of chromosomal abnormalities in cancer and genetic disease.

Identification of cryptic imbalance in phenotypically normal and abnormal translocation carriers.

Approximately one in 500 individuals carries a reciprocal translocation. Of the 121 monosomy 1p36 subjects ascertained by our laboratory, three independent cases involved unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34). These derivative chromosomes were inherited from balanced translocation carrier parents. To understand better the causes and consequences of chromosome breakage and rearrangement in the human genome, we characterized each derivative chromosome at the DNA sequence level and identified the junctions between 1p36 and 9q34. The breakpoint regions were unique in all individuals. Insertions and duplications were identified in two balanced translocation carrier parents and their unbalanced offspring. Sequence analyses revealed that the translocation breakpoints disrupted genes. This study demonstrates that apparently balanced reciprocal translocations in phenotypically normal carriers may have cryptic imbalance at the breakpoints. Because disrupted genes were identified in the phenotypically normal translocation carriers, caution should be exercised when interpreting data on phenotypically abnormal carriers with apparently balanced rearrangements that disrupt putative candidate genes.

Identification of sequence motifs at the breakpoint junctions in three t(1;9)(p36.3;q34) and delineation of mechanisms involved in generating balanced translocations.

Although approximately 1 in 500 individuals carries a reciprocal translocation, little is known about the mechanisms that result in their formation. We analyzed the sequences surrounding the breakpoints in three unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34), to investigate the presence of sequence motifs that might mediate nonhomologous end joining (NHEJ). The breakpoint regions were unique in all individuals. Two of three translocations demonstrated insertions and duplications at the junctions, suggesting NHEJ in the formation of the rearrangements. No homology was identified in the breakpoint regions, further supporting NHEJ. We found translin motifs at the breakpoint junctions, suggesting the involvement of translin in the joining of the broken chromosome ends. We propose a model for balanced translocation formation in humans similar to transposition in bacteria, in which staggered nicks are repaired resulting in duplications and insertions at the translocation breakpoints.

Evidence for involvement of TRE-2 (USP6) oncogene, low-copy repeat and acrocentric heterochromatin in two families with chromosomal translocations.

We report clinical findings and molecular cytogenetic analyses for two patients with translocations [t(14;17)(p12;p12) and t(15;17)(p12;p13.2)], in which the chromosome 17 breakpoints map at a large low-copy repeat (LCR) and a breakage-prone TRE-2 (USP6) oncogene, respectively. In family 1, a 6-year-old girl and her 5-year-old brother were diagnosed with mental retardation, short stature, dysmorphic features, and Charcot-Marie-Tooth disease type 1A (CMT1A). G-banding chromosome analysis showed a der(14)t(14;17)(p12;p12) in both siblings, inherited from their father, a carrier of the balanced translocation. Chromosome microarray and FISH analyses revealed that the PMP22 gene was duplicated. The chromosome 17 breakpoint was mapped within an approximately 383 kb LCR17pA that is known to also be the site of several breakpoints of different chromosome aberrations including the evolutionary translocation t(4;19) in Gorilla gorilla. In family two, a patient with developmental delay, subtle dysmorphic features, ventricular enlargement with decreased periventricular white matter, mild findings of bilateral perisylvian polymicrogyria and a very small anterior commissure, a cryptic duplication including the Miller-Dieker syndrome region was identified by chromosome microarray analysis. The chromosome 17 breakpoint was mapped by FISH at the TRE-2 oncogene. Both partner chromosome breakpoints were mapped on the short arm acrocentric heterochromatin within or distal to the rRNA cluster, distal to the region commonly rearranged in Robertsonian translocations. We propose that TRE-2 together with LCR17pA, located approximately 10 Mb apart, also generated the evolutionary gorilla translocation t(4;19). Our results support previous observations that the USP6 oncogene, LCRs, and repetitive DNA sequences play a significant role in the origin of constitutional chromosome aberrations and primate genome evolution.

Aberrations of 11q13 in laryngeal squamous cell lines and their prognostic significance.

Chromosomal aberrations were analyzed in 12 established cell lines derived from laryngeal squamous cell carcinoma. Cytogenetic and fluorescence in situ hybridization studies were used to identify aberrations in the 11q13 region and in some other chromosome regions. Amplification of 11q13 was established only in the cell lines derived from subjects with a survival period of less than 5 years and, together with the 3q gain, were the only chromosomal structural abnormalities connected with short survival. In this group we also found translocations with a breakpoint within 11q13. In three cell lines, 11q13 was observed as a homogenously staining region. The results suggest that amplification of 11q13, as well as re-arrangements potentially involved in up-regulation of the oncogenes mapped in 11q13, should be considered as markers of poor prognosis in laryngeal cancer. A diagnostic significance of 11q13 may be increased by a parallel determination with 3q gain.

[Mutagen sensitivity in patients with multiple primary tumors (MPT) of the head and neck region--quantitative and qualitative assessment based on bleomycin assay]. / Wrazliwosc na mutageny chorych z mnogimi nowotworami pierwotnymi (MPT) glowy i szyi--ilosciowa i jakosciowa ocena zlaman chromosomów w tescie bleomycynowym.

The occurrence of second primary tumors after curative treatment or simultaneous multiple malignancies are current problem in head and neck cancer. The mutagen sensitivity is well known marker to predict patient proneness to develop the second tumor. The frequency and localization of spontaneous and mutagen induced chromatid breaks in peripheral blood lymphocytes (PBLs) in patients with multiple primary tumors (MPT) may help in defining regions involved in cancerogenesis process. The case control study using the bleomycin sensitivity assay (number of chromatid breaks per cell (b/c) was performed in 36 patients with MPT and two control groups: 52 patients with one malignancy and 47 healthy individuals. The differences between examined patients and control groups were estimated using U Mann-Whitney test. The b/c level in PBLs of patients with MPT ranged from 0.26 to 4.12 (mean 1.53) and was significantly higher (p<0.000006) both compared with patients with one malignancy (b/c ranged from 0.02 to 3.08; mean 0.74) and healthy controls (b/c ranged from 0.04 to 1.14; mean 0.41). An increase of b/c index was observed in almost all chromosomal arms. The majority of chromosomal locations with the increased proportion of breaks in the group of patients with multiple tumors were identified as regions where loci involved in DNA repair, cell cycle regulation suppressor genes and oncogenes were found. Statistically higher induced individual susceptibility in MPT patients compared with single tumor and healthy controls was confirmed. Comparable induced mean b/c was found in patients with two smoking-related cancers as well as with not smoking related tumors.